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Treating Obesity Like a Tumor
Randy J. Seeley1,*
1University of Cincinnati, Cincinnati, OH 45237, USA
*Correspondence: randy.seeley@uc.edu
DOI 10.1016/j.cmet.2011.12.007
Expanding adipose tissue in obesity requires a great deal of angiogenesis to support increasing volumes of
tissue. A growing body of evidence indicates that inhibiting these blood vessels can result in substantial
weight loss, and now this has been demonstrated in nonhuman primates.
Extremely skeptical. That is the best
description of my response to publications indicating that either diet-induced
or genetic forms of obesity could be reversed by giving inhibitors to blood vessel
formation. The first of these reports came
from Maria Rupnick and Judah Folkman,
who used agents that inhibit tumor growth
and found profound weight loss in mice
(Rupnick et al., 2002). The next of these
reports used a more sophisticated approach in an attempt to direct the inhibition of the blood vessels specifically to
adipose tissue. In an effort led by Wadih
Arap and Renata Pasqulini, they used
a technology called phage display that
had been designed to identify the signatures of tumor-associated blood vessels
that might be different than other blood
vessels. However, they determined that
many tissues, including adipose tissue,
had unique properties. They were able to
identify short peptide sequences that
would selectively bind to the vasculature
of white adipose tissue, but not other
tissues they examined. As cancer researchers, they took the next logical step.
They used this peptide and attached a
poison pill that would produce apoptosis
in the targeted blood vessels (Kolonin
et al., 2004). This targeted approach also
led to rapid and substantial weight loss
in mice. In their most recent manuscript
published in Science Translational Medicine, this group has extended these
results to obese nonhuman primates,
showing substantial weight and body fat
loss after 28 days of treatment with this
peptide (Barnhart et al., 2011).
The reason for my skepticism toward
this approach centered on the assumption that, whether untargeted or targeted,
reducing adipose tissue blood vessels
would impair adipose tissue function.
While obesity is a scourge to be fought
and is the direct result of expanding
Cell Metabolism
Previews
environment where few effective treatment strategies short of bariatric surgery are available to help obese patients.
It is clear that more creative strategies
from a wider range of disciplines are
needed. To that end, further understanding of how adipose tissue signaling is altered by various aspects of its
milieu, including the supporting blood
vessels, macrophages, and its extracellular matrix, is necessary if we are to bring
more therapies to the large unmet medical
REFERENCES
Cell and Regenerative Biology Department, Harvard University, Cambridge, MA 02138, USA
Immune Disease Institute and Program in Cellular and Molecular Medicine, Childrens Hospital Boston, Boston, MA 02115, USA
*Correspondence: rossi@idi.harvard.edu
DOI 10.1016/j.cmet.2011.12.008
2The
While age-dependent stem cell decline is widely recognized as being a key component of organismal aging,
the underlying mechanisms remain elusive. In this issue of Cell Metabolism, Suomalainen and colleagues
provide evidence that mitochondrial mutation and associated reactive oxygen species can adversely impact
tissue-specific stem and progenitor cell function.
Physiological aging invariably leads to
a loss in normal tissue maintenance and
reduced regenerative potential. The fact
that these processes are normally under
the functional purview of adult tissuespecific stem cells implicates age-associated stem cell decline as a fundamental
contributor to the aging of tissues and
organisms. Indeed, the importance of
the stem cell compartment in contributing
to age-associated pathophysiology has
been demonstrated in a number of studies (Rossi et al., 2008). Consistent with
the inherent complexity of physiological
aging, the mechanistic basis for agerelated stem cell decline is similarly
complex, with evidence suggesting the
involvement of cellular, genetic, and epigenetic components (Rossi et al., 2008).
However, recent evidence from a number
of papers, including a paper by Suomalainen and colleagues in this issue of Cell
Cell Metabolism
Previews
environment where few effective treatment strategies short of bariatric surgery are available to help obese patients.
It is clear that more creative strategies
from a wider range of disciplines are
needed. To that end, further understanding of how adipose tissue signaling is altered by various aspects of its
milieu, including the supporting blood
vessels, macrophages, and its extracellular matrix, is necessary if we are to bring
more therapies to the large unmet medical
REFERENCES
Cell and Regenerative Biology Department, Harvard University, Cambridge, MA 02138, USA
Immune Disease Institute and Program in Cellular and Molecular Medicine, Childrens Hospital Boston, Boston, MA 02115, USA
*Correspondence: rossi@idi.harvard.edu
DOI 10.1016/j.cmet.2011.12.008
2The
While age-dependent stem cell decline is widely recognized as being a key component of organismal aging,
the underlying mechanisms remain elusive. In this issue of Cell Metabolism, Suomalainen and colleagues
provide evidence that mitochondrial mutation and associated reactive oxygen species can adversely impact
tissue-specific stem and progenitor cell function.
Physiological aging invariably leads to
a loss in normal tissue maintenance and
reduced regenerative potential. The fact
that these processes are normally under
the functional purview of adult tissuespecific stem cells implicates age-associated stem cell decline as a fundamental
contributor to the aging of tissues and
organisms. Indeed, the importance of
the stem cell compartment in contributing
to age-associated pathophysiology has
been demonstrated in a number of studies (Rossi et al., 2008). Consistent with
the inherent complexity of physiological
aging, the mechanistic basis for agerelated stem cell decline is similarly
complex, with evidence suggesting the
involvement of cellular, genetic, and epigenetic components (Rossi et al., 2008).
However, recent evidence from a number
of papers, including a paper by Suomalainen and colleagues in this issue of Cell
Cell Metabolism
Previews
the hematopoietic system and, to a lesser
extent, the brain. In the blood, the hematopoietic stem cell (HSC) compartment
has been well documented to contribute
to pathophysiological conditions associated with aging, which include reduced
regenerative potential, diminished adaptive immune competence, and myelogenous disease predisposition (Rossi et al.,
2008). And while genomic DNA damage
accrual is thought to limit the regenerative
response of HSCs from old mice (Rossi
et al., 2007), the impact of mitochondrial
mutagenesis on hematopoietic aging has
until recently been unexplored. Two
groups have recently shown that the
mtDNA mutator mice exhibited a number
of hematopoietic phenotypes, including
abnormalities in erythroid and lymphocyte
development (Chen et al., 2009; Norddahl
et al., 2011). The fact that these hematologic deficiencies could be transplanted
into wild-type recipients indicated that
they were cell-autonomous defects transmitted by HSCs (Chen et al., 2009; Norddahl et al., 2011). However, although
anemia and lymphoid deficiencies are
commonly associated with aging in both
mice and people, Bryder and colleagues
went on to show that the aging of mutator stem cells was molecularly distinct
from normal physiological stem cell aging
(Norddahl et al., 2011). Using a transcriptional profiling strategy to compare
steady-state mutator HSCs against stem
cells purified from old wild-type mice, little
similarity was noted in their respective
profiles, suggesting that, at least at this
resolution, mitochondrial mutation-driven
stem cell decline could be uncoupled
from normal physiological stem cell aging
(Norddahl et al., 2011). Nonetheless, it remains possible that mitochondrial mutation contributes mechanistically to stem
cell aging despite the lack of evidence
at the transcriptional level, perhaps by
influencing the mobilization of energy
stores that these normally dormant stem
cells must harness when called into
action under conditions of stress or
regeneration.
An important outstanding question was
whether the hematopoietic phenotypes
exhibited by the mutator mice arose only
in self-renewal that could explain the decreased NSC numbers observed in vivo.
Importantly, as the authors had observed
with the hematopoietic defects, the selfrenewal deficits of the mutator NSCs
could also be largely ameliorated through
treatment with NAC.
The findings of Suomalainen and colleagues at once support a growing body
of evidence showing how critical ROS
management is for proper stem and progenitor cell function (Ito et al., 2006; Tothova and Gilliland, 2007) and, at the
same time, identify that a consequence
of mutation accrual in the mitochondrial
genome is deregulation of ROS, which
can then work its mischief by impairing
tissue-specific stem and progenitor cells.
However, the connection of mitochondrial
genome maintenance to physiological
stem cell aging remains unclear.
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Cell Metabolism
Previews
Power Surge: Supporting Cells
Fuel Cancer Cell Mitochondria
Ubaldo E. Martinez-Outschoorn,1,2,3,4 Federica Sotgia,1,2,3,5,* and Michael P. Lisanti1,2,3,4,5,*
1The
An emerging paradigm in tumor metabolism is that catabolism in host cells fuels the anabolic growth of
cancer cells via energy transfer. A study in Nature Medicine (Nieman et al., 2011) supports this; they
show that triglyceride catabolism in adipocytes drives ovarian cancer metastasis by providing fatty acids
as mitochondrial fuels.
Our understanding of tumor metabolism
is evolving. A new central concept in
cancer metabolism is that tumor cells
function as metabolic parasites to extract
energy from supporting host cells, such
as fibroblasts and adipocytes. It has recently been demonstrated that metabolic
coupling exists in human tumors (Sotgia
et al., 2011). In two-compartment tumor
metabolism, the tumor stroma and adjacent host tissues are catabolic and the
cancer cells are anabolic (Figure 1). In
this model, energy is transferred from the
catabolic compartment to the anabolic
compartment via the sharing of nutrients
that promote tumor growth, behaving as
onco-metabolites. Although most studies
on two-compartment tumor metabolism
were first performed on fibroblasts and
breast cancer cells (Martinez-Outschoorn
et al., 2011; Sotgia et al., 2011, 2012;
Whitaker-Menezes et al., 2011), an elegant study in Nature Medicine now
broadens this emerging paradigm to adipocytes and ovarian cancer cells (Nieman
et al., 2011).
The tumor cellular microenvironment
contains supporting host cells, including
fibroblasts, adipocytes, smooth muscle
cells, endothelia, and immune cells, which
functionally promote tumor growth. In
two-compartment tumor metabolism, anabolic cancer cells extract energy from
the surrounding host cells by inducing
catabolic processes, such as autophagy,
mitophagy, and aerobic glycolysis. These
processes provide high-energy mitochondrial fuels (L-lactate, ketones, and gluta-
Cell Metabolism
Previews
leading to mitochondrial biogenesis
and effectively treat advanced
Two-Compartment Tumor Metabolism
and OXPHOS in tumor cells, drivand metastatic cancers. New imaging chemoresistance and distant
ing techniques to visualize twoSupporting Host Cell
Cancer Cell
metastasis.
compartment tumor metabolism in
Catabolism
Anabolic Growth
Thus, the authors demonstrate
real time will allow us to measure
-Autophagy
that it is crucial to include the
the effectiveness of anticancer ther-Mitophagy
supporting microenvironment when
apies and facilitate more personal-Glycolysis
Energy
-Lipolysis
studying cancer cell metabolism
ized cancer treatments.
Mitochondrial
and that simply examining primary
Nutrients
Metabolism
cancer cells alone may not be
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-L-lactate
-OXPHOS
-Ketones
adequate. Unfortunately, most tradi-Beta-OX
-Glutamine
Chen, Z.X., and Pervaiz, S. (2010). Cell
Therapy
tional cancer metabolism studies
-Fatty Acids
Death Differ. 17, 408420.
have been carried out using tumor
Das, S.K., Eder, S., Schauer, S., Diwoky, C.,
cells alone, or using whole tumors,
Temmel, H., Guertl, B., Gorkiewicz, G.,
Figure 1. Two-Compartment Tumor Metabolism:
without modeling the host microenTamilarasan, K.P., Kumari, P., Trauner, M.,
Catabolic Host Cells Fuel Anabolic Cancer Cell
vironment. As such, one might gain
et al. (2011). Science 333, 233238.
Growth and Metastasis via Mitochondrial Metabolism
incomplete or inaccurate informaCatabolic host cells that make up the microenvironment (e.g.,
Fogal, V., Richardson, A.D., Karmali, P.P.,
fibroblasts and adipocytes) generate and transfer high-energy
tion by studying primary cancer cells
Scheffler, I.E., Smith, J.W., and Ruoslahti,
metabolites (L-lactate, ketones, glutamine, and free fatty
or cancer cell lines in the absence of
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acids) to epithelial cancer cells, energetically promoting tumor
supporting host cells.
growth and metastasis. Cancer cells increase their mitochonLe, A., Cooper, C.R., Gouw, A.M., Dinavahi,
drial mass and activity (OXPHOS and b-oxidation) to efficiently
Studies describing the compartR., Maitra, A., Deck, L.M., Royer, R.E., Vanburn
these
energy-rich
mitochondrial
fuels.
Targeted
therader Jagt, D.L., Semenza, G.L., and Dang,
mentalization of tumor metabolism
pies that metabolically uncouple parasitic cancer cells
C.V. (2010). Proc. Natl. Acad. Sci. USA
and energy transfer may pave the
from catabolic host cells (such as mitochondrial inhibitors
107, 20372042.
way toward the development of re[metformin and arsenic trioxide], as well as powerful antiMartinez-Outschoorn, U.E., Goldberg, A.,
oxidants) will starve tumor cells. Effective therapies would
lated predictive biomarkers and
Lin, Z., Ko, Y.H., Flomenberg, N., Wang,
block energy transfer, cutting off the fuel supply to cancer
targeted personalized therapies. It
C., Pavlides, S., Pestell, R.G., Howell, A.,
cells.
Sotgia, F., and Lisanti, M.P. (2011). Cancer
will be important to investigate
Biol. Ther. 12, 924938.
whether metabolically uncoupling
cancer cells from catabolic host cells IL-8 (Sotgia et al., 2012). Most importantly, Nieman, K.M., Kenny, H.A., Penicka, C.V.,
can be used as a new effective anticancer FDA-approved drugs that inhibit mito- Ladanyi, A., Buell-Gutbrod, R., Zillhardt, M.R.,
Romero, I.L., Carey, M.S., Mills, G.B., Hotamisligil,
strategy. Earlier studies have suggested chondrial metabolism (metformin, arsenic G.S., et al. (2011). Nat. Med. 17, 14981503.
that the detection of host-tumor meta- trioxide) or strong antioxidants (catalase)
bolic coupling may be useful for identi- can uncouple two-compartment tumor Sotgia, F., Martinez-Outschoorn, U.E., Pavlides,
S., Howell, A., Pestell, R.G., and Lisanti, M.P.
fying high-risk patients at diagnosis in metabolism and induce apoptosis in (2011). Breast Cancer Res. 13, 213.
human breast cancers. For example, cancer cells (Martinez-Outschoorn et al.,
Sotgia, F., Martinez-Outschoorn, U.E., Howell, A.,
loss of expression of the caveolin-1 pro- 2011; Sotgia et al., 2012) (Figure 1).
Pestell, R.G., Pavlides, S., and Lisanti, M.P.
tein in cancer-associated fibroblasts is
In conclusion, the importance of the (2012). Annu. Rev. Pathol. 7, 423467. Published
a marker for tumor-stroma metabolic host microenvironment and energy trans- online November 7, 2011. 10.1146/annurevpathol-011811-120856.
coupling (Sotgia et al., 2011) and is tightly fer in cancer metabolism is highlighted by
correlated with recurrence, metastasis, Nieman et al. (2011). More studies on two- Vander Heiden, M.G., Li, X.X., Gottleib, E., Hill,
and tamoxifen resistance as well as compartment tumor metabolism will be R.B., Thompson, C.B., and Colombini, M. (2001).
J. Biol. Chem. 276, 1941419419.
poor clinical outcome. Metabolic coupling necessary to understand and therabetween host cells and breast cancer peutically exploit the metabolic coupling Whitaker-Menezes, D., Martinez-Outschoorn,
cells also results in the generation of reac- between parasitic tumor cells and their U.E., Flomenberg, N., Birbe, R.C., Witkiewicz,
A.K., Howell, A., Pavlides, S., Tsirigos, A., Ertel,
tive oxygen species and inflammatory hosts. Uncoupling parasitic cancer A., Pestell, R.G., et al. (2011). Cell Cycle 10,
cytokine production, such as IL-6 and cells should allow us to starve cancer cells 40474064.
Cell Metabolism
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Transgenerational Inheritance of Longevity:
Epigenetic Mysteries Abound
Shelley L. Berger1,2,3,*
1Department
Transgenerational inheritance of epigenetic characteristics in plants has been reported, whereas nongenetic
persistence of complex phenotypes in animals is controversial. A recent report by Anne Brunet and
colleagues describes a fascinating example of persistence across generations of extended life span in
worm and explores whether epigenetic mechanisms account for the longevity.
Cell Metabolism
Previews
would be reduced H3 K4 methylagenerational longevity exist and
SET-2
A
tion in the F3 and F4 generations in
have not yet been tested.
H3K4me
spite of wild-type SET-2; however,
Thus, this report establishes a
the authors tested genome-wide
precedent that longevity can be
gene
methylation in the long-lived but
maintained
transgenerationally;
Aging
wild-type offspring, but found no
however, major challenges remain
lowering of H3 K4 methylation by
to show a direct epigenetic basis
global western analysis. This does
for the transgenerational inheritance.
not rule out possible localized reTaken together with other emerging
duction in methylation, for example,
examples in animals of transB
at specific genes that might regulate
generational inheritance of complex
set-2-/- X WT
Parents
longevity. To indirectly test this
phenotypes with possible underlying
Shorter LS
Longer LS
hypothesis, the authors performed
epigenetic mechanisms (Carone
genome-wide RNA expression miet al., 2010), we can anticipate
F1 (set-2+/-)
croarray analysis. Although the
many new studies exploring this
authors detected certain restricted
fascinating question in the near
F2
gene expression similarities of the
future.
F3 and F4 transgenerational offlonger
F3,F4 SET-2+/+
LS
spring to the actual mutant, the
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overall transcriptional picture unexH3K4me
shorter
pectedly showed expression clusF5
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tering more similar to the actual
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wild-type expression spectrum
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Figure 1. Transgenerational Inheritance of Longevity
than to the actual mutant expression
(A) Aging may lead to decompaction of chromatin, resulting in
Carone, B.R., Fauquier, L., Habib, N., Shea,
spectrum. The authors also pointed
inappropriate gene expression. In the worm C. elegans, deleJ.M., Hart, C.E., Li, R., Bock, C., Li, C., Gu,
tion of the H3 K4 methylase, SET-2, extends life span.
out that certain specific GO cateH., Zamore, P.D., et al. (2010). Cell 143,
(B) Transgenerational inheritance of extended life span (LS) in
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gories that regulate metabolism are
the worm C. elegans results from parental deletion of SET-2
altered like the actual mutant; thus,
(set2 / ) and transmittal to wild-type F3 and F4 generations.
Dang, W., Steffen, K.K., Perry, R., Dorsey,
modest changes at a few genes
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may lead to persistent longevity in
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the F3 and F4 generations. To
address whether this is a direct or indirect mined. One possibility is that, since there Daxinger, L., and Whitelaw, E. (2010). Genome
effect, it will be important to explore is no transitional partial state, a threshold Res. 20, 16231628.
whether H3 K4 methylation is reduced at effect may exist, which would support Feser, J., Truong, D., Das, C., Carson, J.J., Kieft,
these genes in the wild-type offspring.
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the extended life span abruptly returns the methylation level increases to a certain Greer, E.L., Maures, T.J., Hauswirth, A.G., Green,
to normal length in the F5 generation level in the F5, the life span is no longer E.M., Leeman, D.S., Maro, G.S., Han, S., Banko,
M.R., Gozani, O., and Brunet, A. (2010). Nature
(Figure 1B) without passing through any extended.
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The study also reports that several
intermediate longevity state. The authors
E.L., Maures, T.J., Ucar, D., Hauswirth,
show an F5 RNA expression microarray other longevity genes, including deletion Greer,
A.G., Mancini, E., Lim, J.P., Benayoun, B.A., Shi,
with levels similar to the true SET-2 wild- of a few chromatin modulators of tran- Y., and Brunet, A. (2011). Nature 479, 365371.
type, but since the F3 and F4 transcription scription that extend life span (e.g., the
Michishita, E., McCord, R.A., Berber, E., Kioi, M.,
results are not clearly like set-2 mutant, H3 K9 methylase and H3 K27 demethy- Padilla-Nash, H., Damian, M., Cheung, P., Kusuthe interpretation of these findings re- lases) (Greer et al., 2010; Carone et al., moto, R., Kawahara, T.L., Barrett, J.C., et al.
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altered chromatin state was detected in longevity effect. Thus, it is tempting to Ringrose, L., and Paro, R. (2004). Annu. Rev.
the F3 and F4 wild-type-but-extended speculate that K4 methylation plays a Genet. 38, 413443.
offspring, at present the chromatin state key role in transgenerational longevity or Rodrguez-Paredes, M., and Esteller, M. (2011).
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Cell Metabolism
Previews
IL-6 Muscles In on the Gut and Pancreas
to Enhance Insulin Secretion
Tamara L. Allen,1 Martin Whitham,1 and Mark A. Febbraio1,*
1Cellular and Molecular Metabolism Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne 3008, Victoria, Australia
*Correspondence: mark.febbraio@bakeridi.edu.au
DOI 10.1016/j.cmet.2011.12.004
The role of the cytokine interleukin-6 (IL-6) in metabolic homeostasis is the subject of conjecture. Recent
work in Nature Medicine (Ellingsgaard et al., 2011) demonstrates that IL-6 released from skeletal muscle
during exercise mediates crosstalk between insulin-sensitive tissues, intestinal L cells, and pancreatic islets
to adapt to changes in insulin demand.
The debate regarding the pathological
versus beneficial nature of IL-6 in metabolism remains unclear and the subject
of continuing debate (Mooney, 2007; Pedersen and Febbraio, 2007). On one hand,
increased IL-6 in obesity is associated
with the physiopathology of type 2 diabetes (Klover et al., 2003), while on the
other, muscle-derived IL-6 appears to
contribute to improved glycemia following
exercise (Pedersen and Febbraio, 2008).
A recent study published in Nature Medicine (Ellingsgaard et al., 2011) makes
a telling contribution to our understanding
of this apparent paradox. The authors
found that IL-6, released from either contracting skeletal muscle or white adipose
tissue, stimulated glucagon-like peptide-1
(GLP-1) from the gut and the pancreas.
As GLP-1 is a hormone that induces
insulin secretion, this complex organto-organ crosstalk provides strong evidence that IL-6 is a cytokine that has
positive effects on maintaining glucose
homeostasis.
The basic concept that IL-6 is an inflammatory cytokine that leads to insulin resistance was first challenged with the finding
that skeletal muscle releases IL-6 during
contraction (Steensberg et al., 2000) to
act in an endocrine-like manner (Febbraio
et al., 2004). Since this discovery, IL-6 has
been found to increase glucose uptake
and fat oxidation in skeletal muscle and
improve glucose tolerance and insulin
sensitivity (Carey et al., 2006; van Hall
et al., 2003), effects that oppose those
seen in the development of metabolic
syndrome. The recent paper by Donath
and colleagues (Ellingsgaard et al., 2011)
neatly demonstrates that IL-6 mediated
increases in GLP-1 secretion from L cells
in the intestine and a cells in the pancreas
Cell Metabolism
Previews
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Ellingsgaard, H., Hauselmann, I.,
In summary, the work by
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Figure 1. The Pleiotropic Metabolic Effects of the Cytokine IL-6
Pedersen, B.K., and Febbraio, M.A.
IL-6 is released from contracting skeletal muscle and adipose tissue to trigger
linking adipose tissue, skel(2008). Physiol. Rev. 88, 13791406.
GLP-1 release from the gut and the pancreas to improve glucose tolerance.
etal muscle, intestines, and
Steensberg, A., van Hall, G., Osada,
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T., Sacchetti, M., Saltin, B., and
Moreover, the study strengthens the secretory factors that may modulate Klarlund Pedersen, B. (2000). J. Physiol. 529,
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concept that the skeletal muscle is an metabolic processes.
van Hall, G., Steensberg, A., Sacchetti, M., Fischer,
endocrine organ, capable of secreting
C., Keller, C., Schjerling, P., Hiscock, N., Mller, K.,
factors that can affect not only the
Saltin, B., Febbraio, M.A., and Pedersen, B.K.
adipose tissue and the liver, but also the ACKNOWLEDGMENTS
(2003). J. Clin. Endocrinol. Metab. 88, 30053010.
gut, pancreas, and perhaps many other
M.A.F. is a Senior Principal Research Fellow of the
Wojtaszewski, J.F., Hansen, B.F., Gade, Kiens, B.,
organs. The work opens the door to the National Health and Medical Research Council of Markuns, J.F., Goodyear, L.J., and Richter, E.A.
(2000). Diabetes 49, 325331.
identification of other skeletal muscle Australia.
Cell Metabolism
Review
The Inflammasome Puts Obesity in the Danger Zone
Rinke Stienstra,1,2,3 Cees J. Tack,1 Thirumala-Devi Kanneganti,4 Leo A.B. Joosten,1,2 and Mihai G. Netea1,2,*
1Department
of Medicine
Institute for Infection, Inflammation and Immunity (N4I)
Radboud University Nijmegen Medical Centre, Nijmegen 6525 GA, The Netherlands
3Nutrition, Metabolism and Genomics Group, Wageningen University, Wageningen 6703 HD, The Netherlands
4Department of Immunology, St. Jude Childrens Research Hospital, Memphis, TN 38105, USA
*Correspondence: m.netea@aig.umcn.nl
DOI 10.1016/j.cmet.2011.10.011
2Nijmegen
Obesity-induced inflammation is an important contributor to the induction of insulin resistance. Recently, the
cytokine interleukin-1b (IL-1b) has emerged as a prominent instigator of the proinflammatory response in
obesity. Several studies over the last year have subsequently deciphered the molecular mechanisms responsible for IL-1b activation in adipose tissue, liver, and macrophages and demonstrated a central role of the
processing enzyme caspase-1 and of the protein complex leading to its activation called the inflammasome.
These data suggest that activation of the inflammasome represents a crucial step in the road from obesity to
insulin resistance and type 2 diabetes.
Introduction
Accumulating evidence links inflammation to metabolic changes
associated with obesity and type 2 diabetes (Donath and Shoelson, 2011). While several studies suggest that expanding
adipose tissue is an important first step in driving the enhanced
state of inflammation, the underlying molecular mechanisms
modulating this process are less clear. A wide variety of immune
cells, including macrophages, monocytes, T cells, and b cells,
have been shown to infiltrate the adipose tissue and affect its
homeostasis by releasing inflammatory cytokines (Anderson
et al., 2010). Adipocytes themselves are also capable of
releasing inflammatory mediators and contribute to the inflammatory response (McGillicuddy et al., 2011; Stienstra et al.,
2010; Meijer et al., 2011). In addition to adipose tissue, inflammation in liver and pancreatic islets is also evident in obese individuals and participates in the pathogenesis of type 2 diabetes (Gregor and Hotamisligil, 2011). One of the proinflammatory
cytokines mediating obesity-induced inflammation is interleukin
(IL)-1b, which is processed by caspase-1, a cysteine protease
regulated by a protein complex called the inflammasome.
Although growing evidence points to a crucial role for IL-1b in
mediating the development of insulin resistance, it should be
stressed that the inflammatory response driving the development of insulin resistance probably is comprised of a combination of proinflammatory cytokines that jointly effectuate type 2
diabetes progression. For example, involvement of TNFa in
obesity-associated insulin resistance has been frequently reported. Since biological processes are often multifactorial,
involvement of other cytokines like TNFa is plausible.
Although detrimental effects of IL-1b on b cell function have
been well documented, the proinflammatory effects of IL-1b that
mediate the development of tissue dysfunction and peripheral
insulin resistance have only recently received more interest. While
several lines of evidence have shown involvement of IL-1b in the
development of obesity-associated insulin resistance peripherally,
the quantitative contribution remains to be defined in more detail.
In the present review we will discuss recently identified metabolic triggers that may function as potential danger signals,
10 Cell Metabolism 15, January 4, 2012 2012 Elsevier Inc.
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Figure 1. IL-1b Signaling Pathway: Overview of the Intracellular Signaling Pathways Activated by Binding of IL-1b to the IL-1 Receptor
Upon activation of the IL-1 receptor complex, IL-1b induces recruitment of the myeloid differentiation primary response gene 88 (MyD88), which in turn promotes
activation of the interleukin-1 receptor kinase (IRAK) cascade. Via tumor necrosis factor-associated factor 6 (TRAF6) and the c-Jun N terminus (JNK), p38
mitogen-activated protein (p38 MAPK), and the inhibitor of nuclear factor b (IKK) kinases, the IkB cofactor is degraded, which subsequently promotes the nuclear
translocation of NF-kB and AP-1. Both transcription factors have the capacity to induce proinflammatory gene expression of various cytokines and chemokines
that modulate the inflammatory response.
sensitivity in patients diagnosed with rheumatoid arthritis (Gonzalez-Gay et al., 2010; Huvers et al., 2007).
Until recently, the triggers and molecular switches controlling
IL-1b production during the development of obesity and insulin
resistance have remained largely unknown. In as much as
IL-1b elicits a vigorous inflammatory response, activation must
be tightly controlled and requires processing from an inactive
procytokine into the biologically active form by proteolytic
enzymes. Processing of cytokines of the IL-1 family, such as
IL-1b and IL-18, is mainly mediated by the cysteine protease
caspase-1, which in turn is activated by a protein platform
termed the inflammasome.
The Inflammasome
The inflammasome is an important part of our innate immune
system that responds to danger signals that are sensed by
a number of different intracellular NOD-like receptors (NLRs).
Different inflammasomes have been identified, including NLR
family pyrin domain-containing 1 (NLRP1), NLR family pyrin
domain-containing 3 (NLRP3), NLR family pyrin domain-containing 6 (NLRP6), AIM2 (absent in melanoma 2), and IPAF
Cell Metabolism 15, January 4, 2012 2012 Elsevier Inc. 11
Cell Metabolism
Review
(IL-1b-converting enzyme protease-activating factor), which
each have the ability to respond to a wide variety of microbial
products or endogenous danger signals (Dunne, 2011). Hitherto,
NLRP3 is the most extensively studied inflammasome that, upon
its activation, forms a complex with its adaptor molecule PYD
and CARD domain containing protein (ASC) and thereby facilitates caspase-1-dependent processing of pro-IL-1b into its
active form. Normally, this proinflammatory response provides
protection for the host by inducing an acute phase response
(Dinarello, 2011).
Recent studies have identified the inflammasome as an important contributor to the development of insulin resistance by
mediation of processing and release of IL-1b in various tissues
and cell types during the development of obesity. In addition to
IL-1b, caspase-1 is also able to process and activate IL-18 and
IL-33 (Arend et al., 2008). In contrast to IL-1b, IL-18 ameliorates
development of obesity and insulin resistance (Netea et al.,
2006). Interestingly, caspase-1 deficient mice lacking both
IL-1b and IL-18 are characterized by an improvement in insulin
resistance (Stienstra et al., 2010), suggesting that the insulindesensitizing effects of IL-1b override IL-18 action. The fact
that expression and circulating levels of IL-18 are easily detectable in healthy subjects, as compared to IL-1b, suggests that this
cytokine executes different functions, among which are the
opposing effects on insulin resistance.
NLRP3-Mediated Caspase-1 Activity in the Drivers Seat
Several lines of evidence suggest that activation of inflammasome-mediated caspase-1 is one of the culprits behind the
enhanced inflammatory state characteristic of obesity and has
center stage in the pathogenesis of type 2 diabetes by acting
at two different levels.
First, caspase-1 appears to instigate defective insulin secretion by promoting pancreatic dysfunction. Pancreatic b cells itself are capable of producing IL-1b (Boni-Schnetzler et al.,
2008; Maedler et al., 2002) through mechanisms involving the
NLRP3 inflammasome (Zhou et al., 2010). Additionally,
enhanced macrophages infiltration of the pancreas observed
in human type 2 diabetic patients and high-fat diet (HFD)-fed
mice (Ehses et al., 2007) may further potentiate IL-1b production.
The IL-1b-driven inflammation of the islets is proposed as the
central mediator of glucose-, lipid-, and amyloid-induced b cell
failure leading to defective insulin secretion (Masters et al.,
2011; Mandrup-Poulsen, 2010) and ultimately b cell death, two
processes distinctive for the pathogenesis of type 2 diabetes.
Second, activation of caspase-1 can alter the function of
peripheral tissues, including adipose tissue and liver, both critically involved in maintaining glucose homeostasis. Adipose
tissue of animals fed a high-fat diet to induce obesity and insulin
resistance is characterized by enhanced gene expression and
increased protein levels of caspase-1 (Stienstra et al., 2010; Vandanmagsar et al., 2011). Similarly, feeding mice a methioninecholine-deficient diet, which promotes the development of
nonalcoholic steatohepatitis (NASH), has been shown to
promote NLRP3-dependent caspase-1 activation within hepatocytes (Csak et al., 2011). The elevated activity of caspase-1 leads
to increased processing of IL-1b and promotes a proinflammatory environment that drives tissue dysfunction. Indeed, the
absence of caspase-1 provides protection for the host against
12 Cell Metabolism 15, January 4, 2012 2012 Elsevier Inc.
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visceral fat depot, which is a strong determinant of insulin resistance, as compared to subcutaneous adipose tissue of mildly
obese subjects (Koenen et al., 2011b). In liver, expression levels
of NLRP3, caspase-1, and ASC are elevated in patients suffering
from NASH compared to healthy controls (Csak et al., 2011).
Conversely, substantial weight loss in obese subjects with type
2 diabetes was shown to reduce expression levels of IL-1b and
NLRP3 in adipose tissue and positively correlated with changes
in fasting plasma glucose levels (Vandanmagsar et al., 2011).
Overall, these results argue that activation of IL-1b by the inflammasome plays a central role in the pathogenesis of
obesity-induced insulin resistance. Since activation of caspase-1 leading to the cleavage and secretion of IL-1b is under
tight control of the inflammasome, specific danger signals
should exist that arise during the development of obesity and
that are sensed by intracellular NLRs. Indeed, several studies
have identified metabolic danger signals that can efficiently activate the inflammasome, leading to caspase-1-driven cleavage of
IL-1b in a variety of cell types, both from nonmyeloid and myeloid
origin (Vandanmagsar et al., 2011; Wen et al., 2011; Csak et al.,
2011; Zhou et al., 2010; Masters et al., 2010).
Potential Metabolic Danger Signals that Activate IL-1b
Recent studies have revealed that activation of IL-1b requires
two signals (Gong et al., 2010; Joosten et al., 2010; Hornung
et al., 2009). Whereas the first signal primes the cell and acts
as an inducer of IL-1b and NLR mRNA transcription, the second
signal induces conformational changes in the inflammasome
that instigates caspase-1 activation. Classically, the first signal
is provided by invading pathogens driving IL-1b mRNA transcription through pattern recognition receptors such as toll-like
receptors (TLRs) (Lamkanfi et al., 2008). The second signal is
of a more heterogeneous nature, as it can be represented by
microbial components such as microbial DNA (Muruve et al.,
2008), cell-wall components, and toxins (Ali et al., 2011), but
also by endogenous ligands such as adenosine triphosphate
(ATP) or uric acid (Mariathasan et al., 2006). The precise unifying
mechanism through which all these ligands induce caspase-1
activation is not known, although a rapid K+ efflux from cells
seems to be a common denominator that triggers activation of
the inflammasome (Lamkanfi et al., 2008). Notably, not all cell
types need sequential hits to induce IL-1b production. For
example, monocytes only require the first signal to induce inflammasome-dependent IL-1b release, due to the constitutive
activation of caspase-1 in this cell type (Netea et al., 2009).
Moreover, IL-1b can also be processed by caspase-1-independent cleavage through neutrophil-derived serine proteases
(Joosten et al., 2009), yet no information is available concerning
the mechanism that controls this process.
Very recently, fatty acids, glucose, uric acid, and islet amyloid
polypeptide (IAPP) have been put forward as metabolic danger
signals that possess the capacity to activate the inflammasome
and stimulate IL-1b production (Figure 2). Below we will discuss
where exactly these signals might act to promote IL-1b release.
Fatty Acids
Elevated circulating levels of free fatty acids are one of the hallmarks of type 2 diabetes (Krebs and Roden, 2005). Interestingly,
it has been suggested that saturated fatty acids bind and activate members of the TLR-family in vitro (Lee et al., 2001). For
Cell Metabolism
Review
activation of innate immunity, this may not imply that all fatty acids
cause activation of the inflammasome. Since unsaturated fatty
acids have anti-inflammatory effects (Browning, 2003) and lack
the ability to activate TLR4 or evoke ceramide synthesis (Chavez
et al., 2003), one would predict that highly unsaturated species
prevent inflammasome activation. Accordingly, it is important to
characterize the inflammasome-activating potential of various
fatty acids differing in length and degree of unsaturation.
A potential confounder in most of the experiments pinpointing
ceramide or palmitate as activators of the inflammasome (and
other metabolic danger signals) may be the fact that cells were
primed with LPS (Wen et al., 2011; Vandanmagsar et al.,
2011). Notably, both palmitate and ceramide did not induce inflammasome activation and subsequent cleavage and secretion
of IL-1b without prior exposure of macrophages to LPS. Since it
has been reported that serum endotoxin levels are elevated in
obese animals due to a disruption in intestinal integrity (Cani
et al., 2008), LPS may serve as a first signal in obese individuals.
However, other priming signals may exist in vivo that synergize
with ceramide or palmitate to induce both proIL-1b transcription
and inflammasome activation, and these include other fatty
acids, glucose, or adipocytokines.
14 Cell Metabolism 15, January 4, 2012 2012 Elsevier Inc.
Glucose
It has been known for almost a decade that high glucose levels
promote IL-1b mRNA transcription (Shanmugam et al., 2003).
Glucose has been shown to activate PKC-alpha and, via phosphorylation of p38 MAPK and ERK1/2, lead to NF-kB activation
and subsequent IL-1b transcription in monocytes (Dasu et al.,
2007), hence priming cells for inflammasome activation.
Additional pathways may also participate in high glucosemediated activation of IL-1b release by providing the second
signal that is required to activate the inflammasome. Indeed,
glucose has been identified as a direct stimulator of caspase-1
activity (Vincent and Mohr, 2007). Recent evidence has suggested that the NLRP3 inflammasome is activated in response
to high levels of glucose (Zhou et al., 2010). The mechanism of
action involves thioredoxin-interacting protein (TXNIP), a protein
that acts as an endogenous inhibitor of the ROS scavenging
protein thioredoxin (Minn et al., 2005; Parikh et al., 2007). Interestingly, TXNIP levels are elevated in subjects with type 2 diabetes mellitus (Parikh et al., 2007), and its expression is robustly
induced by glucose (Stoltzman et al., 2008). Zhou et al. (2010)
have shown that, upon activation, TXNIP is able to directly
interact with NLRP3 in a ROS-dependent manner, leading to
Cell Metabolism
Review
activation of caspase-1 and processing of IL-1b in pancreatic
islets. However, these observations could not be reproduced
in bone-marrow-derived macrophages lacking TXNIP and
exposed to high glucose levels (Masters et al., 2010). Nevertheless, treatment of the cells with 2-deoxy-D-glucose, which
blocks metabolic processing, prevents IL-1b processing and
release, implying that adequate glucose metabolism is indispensable for inflammasome function (Masters et al., 2010).
It is reasonable to expect that the effects of high levels of
glucose on caspase-1-mediated IL-1b production differ among
various cell types. Indeed, although adipose tissue ex vivo
responds to high levels of glucose by inducing TXNIP and
producing higher levels of IL-1b, TXNIP ablation in adipocytes
did not affect caspase-1 activity levels (Koenen et al., 2011a).
However, knockdown of TXNIP did reduce IL-1b mRNA levels
within adipocytes, indicating that TXNIP regulates IL-1b gene
expression levels during the presence of high levels of glucose.
As TXNIP itself has the capacity to induce chromatin modification (Perrone et al., 2009), it is conceivable that TXNIP may regulate IL-1b promoter availability.
In summary, high glucose levels may provide the priming
signal for transcription of IL-1b by activation of TXNIP, which
subsequently facilitates enhanced IL-1b expression levels. Additionally, high concentrations of glucose also promote the generation of ROS that is sufficient as second signal that promotes inflammasome activation and subsequently induces release of
bioactive IL-1b.
Uric Acid
Hyperuricemia often occurs in obese individuals and frequently
precedes the development of type 2 diabetes. It has been thought
that high levels of uric acid are a result of hyperinsulinemia, since
insulin normally reduces the renal secretion of uric acid. Interestingly, lowering of circulating levels of uric acid improved insulin
sensitivity in fructose-fed rats (Nakagawa et al., 2006).
It has been suggested that uric acid drives inflammatory and
oxidative changes in adipocytes. Uric acid directly affects the
inflammatory status of the adipose tissue by regulating MCP-1
levels in adipocytes (Baldwin et al., 2011), thereby promoting
infiltration of macrophages (Ito et al., 2008). Interestingly, crystals of monosodium urate, frequently observed in patients diagnosed with gout, have been uncovered as a robust inducer of the
NLRP3 inflammasome in macrophages (Martinon et al.,
2006).Can uric acid also drive obesity-induced inflammation by
activating the inflammasome?
Interestingly, uric acid is indispensable for normal adipogenesis, as animals that lack xanthine oxidoreductase (XOR), an
enzyme that generates uric acid from xanthine, exhibit a
decrease in adipose content and are characterized by a reduction in PPARg activity as compared to wild-type mice. More
importantly, uric acid levels within adipose tissue are enhanced
in obese animals as compared to lean mice, and this is accompanied by elevated transcription levels of XOR (Cheung et al.,
2007). Thus, it may be hypothesized that uric acid can directly
promote the inflammasome or modulate IL-1b mRNA transcription. In theory, adipocyte death, which has been suggested to be
the driving force of adipose tissue inflammation by attracting
macrophages (Cinti et al., 2005), may also activate the inflammasome, since dying cells are known to release uric acid to alert the
immune system (Shi et al., 2003).
Although more work is needed, uric acid is an important candidate that may modulate inflammasome activation during the
development of obesity and insulin resistance.
Islet Amyloid Polypeptide
Islet amyloid polypeptide (IAPP), also known as amylin, is a regulatory peptide of 37 amino acids that is produced by pancreatic
b cells and inhibits insulin and glucagon secretion. The ability of
amylin to form protein aggregates that cluster in pancreatic islets
as amyloid deposits is believed to be of critical importance for
the loss of b cell mass and type 2 diabetes progression (Westermark et al., 2011). Recent evidence has uncovered that IAPP has
the ability to energize the NLRP3 inflammasome, subsequently
promoting IL-1b production by the pancreas (Masters et al.,
2010). In contrast to glucose and palmitate, IAPP lacks the ability
to drive IL-1b mRNA expression, yet robustly promotes NLRP3mediated caspase-1 activation, partly by a disturbance in the
phagocytic machinery causing oxidative stress.
Interestingly, expression of IAPP by the pancreatic b cell line
MIN6 cells is activated by palmitate (Westermark et al., 2011),
suggesting that fatty acids may boost pancreatic IL-1b production in type 2 diabetes.
Additional lines of evidence suggest that IAPP may also have
extrapancreatic sites of action, since expression of the peptide
has been detected in the gastrointestinal tract and sensory
neurons (Westermark et al., 2011). Moreover, inasmuch as circulating levels of IAPP are elevated upon high-fat diet feeding of
animals (Westermark et al., 2011), it is plausible to expect that
IAAP-dependent activation of the inflammasome also occurs at
extrapancreatic sites.
Altogether, these data suggest that IAPP functions as a second
signal promoting NLRP3 activation and subsequent caspase-1dependent cleavage of IL-1b.
Therapeutic Interventions
Given the wealth of data that have implicated activation of the inflammasome in the pathogenesis of obesity-induced insulin
resistance, inhibition of IL-1b and caspase-1 may represent an
attractive therapeutic target. So far, only a few studies have
been conducted aimed at limiting IL-1b action, either by using
receptor antagonists or by neutralizing IL-1b. Blockade of
IL-1R-mediated signaling by the recombinant IL-1 receptor
antagonist (IL-1Ra) Anakinra was effective in improving glycemic
control in patients with type 2 diabetes (Larsen et al., 2007).
However, no improvement in insulin sensitivity levels as determined by euglycemic clamp was observed. In addition, no
changes in muscle gene expression or serum adipokine levels
were seen. Additionally, treatment with Anakinra had no direct
beneficial effect on insulin sensitivity in nondiabetic insulin-resistant subjects (van Asseldonk et al., 2011). Although these observations suggest that IL-1 blockade in human subjects does not
reverse insulin resistance, only a limited number of studies
have been conducted so far. Additionally, Anakinra has a short
half-life and requires daily injections that often cause side effects
at the site of the subcutaneous injection, limiting its therapeutic
potential. Inhibition of IL-1 may be of more benefit in reversing
hyperglycemia-associated insulin resistance. It should be also
underlined that one cannot exclude the possibility that effects
on hepatic insulin resistance explain the effects observed in
the study of Larsen et al. (2007).
Cell Metabolism 15, January 4, 2012 2012 Elsevier Inc. 15
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More recently, the development of IL-1b-neutralizing antibodies that have successfully ameliorated insulin resistance
(Donath et al.,2008; Sloan-Lancaster et al.,2011; Rissanen
et al.,2011) has been reported. These preparations have a longer
half-life and require fewer injections and may therefore bear
superior therapeutic potential. Nevertheless, it must be emphasized that, to date, no clinical studies using anti-IL-1b therapies
have reported any improvement in insulin sensitivity levels.
A second potential approach would be direct inhibition of caspase-1. Specific caspase-1 inhibitors have potent beneficial
effects in animal models of insulin resistance and type 2 diabetes
(Stienstra et al., 2010), and such pharmacological agents may
prove useful in humans as well. However, at this moment, no
caspase-1 inhibitors are available for human use. Interestingly,
Glyburide, a frequently used sulfonylurea drug for the treatment
of type 2 diabetes, has been shown to inhibit the NLRP3 inflammasome (Lamkanfi et al., 2009; Masters et al., 2010), and one
may hypothesize that at least part of its effects may be due to
its anti-inflammatory action.
However, one has to keep in mind that caspase-1 cleaves
alternative substrates including caspase-7 (Lamkanfi et al.,
2008), which may mediate its deleterious effects during the
development of obesity and insulin resistance. Therefore, therapies aimed at specifically blocking caspase-1 may prove to be
more successful in attenuating type 2 diabetes, as compared
to strategies aimed solely at blockage of IL-1b.
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Cell Metabolism
Perspective
Metabolic Disease Drug Discovery
Hitting the Target Is Easier Said Than Done
David E. Moller1,*
1Division of Endocrinology and Cardiovascular Discovery Research and Clinical Investigation, Eli Lilly and Company, Lilly Research
Laboratories, Indianapolis, IN 46285, USA
*Correspondence: mollerda@lilly.com
DOI 10.1016/j.cmet.2011.10.012
Despite the advent of new drug classes, the global epidemic of cardiometabolic disease has not abated.
Continuing unmet medical needs remain a major driver for new research. Drug discovery approaches in
this field have mirrored industry trends, leading to a recent increase in the number of molecules entering
development. However, worrisome trends and newer hurdles are also apparent. The history of two newer
drug classesglucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitorsillustrates
both progress and challenges. Future success requires that researchers learn from these experiences and
continue to explore and apply new technology platforms and research paradigms.
Introduction
The global epidemic of obesity and diabetes continues to progress relentlessly. The International Diabetes Federation predicts
an even greater diabetes burden (>430 million people afflicted)
by 2030, which will disproportionately affect developing nations
(International Diabetes Federation, 2011). Yet existing drug
classes for diabetes, obesity, and comorbid cardiovascular
(CV) conditions have substantial limitations.
Currently available prescription drugs for treatment of hyperglycemia in patients with type 2 diabetes (Table 1) have notable
shortcomings. In general, their efficacy profiles fall short of
achieving accepted treatment goals (American Diabetes Association, 2011). Therefore, clinicians must often use combination
therapy, adding additional agents over time. Ultimately many
patients will need to use insulina therapeutic class first introduced in 1922. Most existing agents also have issues around
safety and tolerability as well as dosing convenience (which
can impact patient compliance).
Future therapies must focus on unmet medical needs in the
following ways: (1) they must have mechanisms that can safely
achieve greater glycemic efficacy; (2) they must mitigate the
CV risk associated with diabetes, obesity, and dyslipidemia; (3)
they should help patients with or at-risk of diabetes to achieve
meaningful and safe weight loss; and (4) they should attenuate
the progression of microvascular complications. For diabetes
and obesity in particular, an overriding need exists for true
disease-modifying agents that deliver longer term benefitsto
prevent disease and/or disease progression. In examining the
extent to which existing therapies address these needs, consider
the fact that pharmacotherapy for obesity has been available in
the U.S. since the FDA approval of amphetamine sulfate (Benzedrine) in 1939. Yet, after many intervening episodes of transient
success and spectacular failure, today there are only two
approved (U.S.) weight-loss agents, orlistat and phentermine,
both with limited effectiveness. Phentermine is only indicated
for transient use, whereas treatment of metabolic disease generally requires lifelong intervention! Moreover, improvements in
glucose control over the past 20 years have been generally
modest. Even in a wealthy developed nation like the U.S.,
Cell Metabolism
Perspective
Table 1. Major Existing Drug Classes Approved for Therapy of Hyperglycemia in Patients with Type 2 Diabetes (Excluding Insulin
Products)
Drug Class (Examples)
Molecular Mechanism
Metformin
Unknown
Moderate ( 0.7%1.0%)
Gastrointestinal symptoms;
twice-daily dosing; lactic
acidosis (rare); contraindicated
with reduced renal function
Thiazolidinediones
(pioglitazone, rosiglitazone)
PPARg agonist
(insulin sensitization)
Moderate ( 0.6%1.1%)
a-glucosidase inhibitors
(acarbose)
Inhibition of intestinal
glucose absorption
Fair ( 0.4%0.8%)
Gastrointestinal symptoms
(frequent)
Incretin-mimetics
(exenatide; liraglutide)
Moderate-good ( 0.5%1.3%);
plus modest weight loss
Dipeptidyl-peptidase-4
inhibitors (sitagliptin,
saxagliptin, linagliptin)
Increased endogenous
GLP-1 and GIP levels
Fair ( 0.5%0.8%)
Table information derived from US prescribing and label information for representative products available at http://www.FDA.gov. PPARg = peroxisome proliferator-activated receptor g. * Hb A1c = hemoglobin A1c (glycosylated hemoglobin); D Hb A1c ranges noted represent effects seen as
monotherapy and/or incremental when added to metformin.
Cell Metabolism
Perspective
Cell Metabolism
Perspective
19 9 0
1995
2000
2005
2010
Figure 2. Abbreviated Timeline of the Discovery and Development of GLP-1 Receptor Agonists and DPP-4 Inhibitors
Cell Metabolism
Perspective
generated presents great challenges. Even major fans concede
that systems approaches are still in their infancy (Schadt,
2009).
Importantly, many of the more modern techniques noted
above were not routinely applied before 19952000. Therefore,
their impact on net industry output cannot yet be fully assessed.
2. Better Human Validation
Our understanding of human disease has not kept pace with the
abundance of identified target opportunities. Better understanding of disease pathogenesis remains essential. For type 2
diabetes, obesity, and CV disease, drug target selection and
prioritization should take human genetics into account (McCarthy, 2010; Libby et al., 2011). Moreover, increased POS can be
achieved with selected targets such as PCSK9 where monogenic traits affecting LDL cholesterol strongly predict future
clinical efficacy (reviewed by Libby et al., 2011). The application
of metabolite profiling (metabolomics) to larger clinical
samples also provides new insights. Rather than relying on
cross-sectional studies, it is important to incorporate prospective clinical endpoints. Findings related to diabetes pathogenesis
obtained using mass spectrometry in >2000 blood samples
from the Framingham Offspring cohort illustrate this point
(Wang et al., 2011). Similarly, tissue biobanks with associated
prospective clinical outcomes data can be used to detect critical
pathogenic nodes as exemplified by recent results of osteopontin levels in carotid atherosclerotic lesions (de Kleijn et al., 2010).
3. Apply Translational Medicine Principles
Efficient progression of drug discovery projects toward successful clinical readouts is at the heart of translational medicine
(Milne, 2009). Information from high-quality human experiments
elucidates next steps in research. Biomarkers that inform target
engagement and early efficacy signals are critical to making
succinct go/no-go decisions. This requires exploiting a full
repertoire of imaging, gene expression profiles, and circulating
markers (including metabolomics). Fortunately, many mechanisms targeting cardiometabolic disease lend themselves to
early efficacy signal detection. For example, 24 hr glucose
profiles can predict longer term glycemic efficacy (HbA1c
lowering). Remember, however, that even accepted surrogate
biomarkers for outcomes like HbA1c may have limitations as
illustrated by the unexpected results from ACCORD. Methods
for detecting small changes in human energy balance (intake
or expenditure) that predict future weight loss have also been
perfected (Gaich and Moller, 2011). With better translational
medicine tools and molecular probes in hand, a shift toward
earlier implementation of exploratory human testing (clinical
target assessment) is warranted. For example, a candidate
obesity pathway was examined in humans via continuous infusion of a melanocortin 4 selective agonist peptide over several
days (Greenfield et al., 2009).
Where Do We Go from Here?
Future success requires a closer relationship between industry
and academia as well as active knowledge sharing between
research groups through multiparty partnerships and consortia.
The Innovative Medicines Initiative for Diabetes is an excellent
example. This industry-academic consortium focused on relevant questions pertaining to the regulation of b cell mass and
function (IMIDIA, 2011). Other consortia have the potential for
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Cell Metabolism
Article
Srf-Dependent Paracrine Signals Produced
by Myofibers Control Satellite Cell-Mediated
Skeletal Muscle Hypertrophy
Aline Guerci,1,2,8 Charlotte Lahoute,1,2,4,8 Sophie Hebrard,1,2,4 Laura Collard,1,2,4 Dany Graindorge,1,2,4
Maryline Favier,1,2,4 Nicolas Cagnard,3 Sabrina Batonnet-Pichon,1,2,4 Guillaume Precigout,5,6,7 Luis Garcia,5,6,7
David Tuil,1,2,4 Dominique Daegelen,1,2,4 and Athanassia Sotiropoulos1,2,4,*
1Inserm
SUMMARY
Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is
required for satellite cell-mediated hypertrophic
muscle growth. Deletion of Srf from myofibers and
not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and
recruitment to pre-existing fibers. We reveal a gene
network in which Srf within myofibers modulates
interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of
satellite cell functions. In Srf-deleted muscles, in vivo
overexpression of interleukin-6 is sufficient to restore
satellite cell proliferation but not satellite cell fusion
and overall growth. In contrast cyclooxygenase2/interleukin-4 overexpression rescue satellite cell
recruitment and muscle growth without affecting
satellite cell proliferation, identifying altered fusion
as the limiting cellular event. These findings unravel
a role for Srf in the translation of mechanical cues
applied to myofibers into paracrine signals, which in
turn will modulate satellite cell functions and support
muscle growth.
INTRODUCTION
Adult skeletal muscle is a highly plastic tissue, the mass of which
changes in response to environmental cues and physiological
stimuli. The basic cellular building blocks of adult muscle are
the multinucleated myofibers, which are able to grow during
postnatal growth, during regeneration after injury, and in
response to a functional demand, such as external loads.
Mature myofibers can grow following the addition of new contractile proteins to pre-existing sarcomere units, which involve
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
(Pipes et al., 2006). The Rho family of small GTPases and actin
dynamics has been shown to modulate Srf activity by controlling
the nuclear accumulation of its coactivator, myocardin-related
transcription factor A (Mrtf-A) (Miralles et al., 2003). Data obtained from mouse models with skeletal muscle-specific loss
of Srf or Mrtfs functions emphasize their crucial role in postnatal
muscle growth (Charvet et al., 2006; Li et al., 2005). In adults, Srf
activity could also be important for the control of skeletal muscle
growth, as suggested by increased Srf expression during overload-induced hypertrophy (Fluck et al., 1999). In cardiac muscle,
Srf and Mrtf-A are necessary for mediating cardiomyocyte
hypertrophy (Kuwahara et al., 2010; Parlakian et al., 2005). Taken
together, these data point toward Srf as a good candidate transcription factor in the control of skeletal muscle mass during
hypertrophy.
To examine the role of Srf during overload-induced muscle
hypertrophy, and the underlying cellular and molecular mechanisms, we used a mouse model of conditional and inducible
deletion of Srf within myofibers but not in satellite cells (Lahoute
et al., 2008). We show here that plantaris myofibers lacking Srf
are no longer able to display a hypertrophic response when
subjected to an experimental overload, thus revealing the
requirement of Srf for muscle growth response to increased
26 Cell Metabolism 15, 2537, January 4, 2012 2012 Elsevier Inc.
activity. Using a combination of in vivo and in vitro assays, we demonstrate that this hypertrophic growth defect is due to lack of satellite
cell proliferation and fusion to the pre-existing
myofibers, independent of the IGF1/Akt
pathway. We show that impaired production of
secreted factors by myofibers lacking Srf is
responsible for these defects. We also unraveled a gene network associating Srf, Il6, Il4,
and Cox2 genes, which drive overload-induced
muscle growth. By modulating Il6, Cox2/Il4
expression levels in the myofibers, Srf controls
both satellite cell proliferation and fusion. Altogether, these data identify Srf as a crucial transcription factor
required for the translation of mechanical cues applied to the
myofibers into paracrine signals that support the proliferation
and fusion of satellite cells, leading to muscle growth.
RESULTS
Srf Is Required for Overload-Induced Hypertrophy
To investigate the role of Srf in skeletal muscle hypertrophy,
compensatory hypertrophy (CH) of the plantaris muscle was
performed in HSA-Cre-ERT2:Srfflox/flox mutant mice previously
tamoxifen-injected to induce myofiber-specific Srf loss. In addition, control and mutant mice were injected with tamoxifen
during the CH procedure. Efficient Srf loss was achieved at the
protein (Figure 1A) and transcript levels (Figure 1B). As previously
described, no obvious differences in muscle mass, myofiber
cross-sectional area (CSA), and myofiber number were observed between control and mutant plantaris muscles before
overload (Figures 1C1F, and Lahoute et al., 2008). In this
muscle growth model, we observed a significant increase
in muscle mass (Figure 1D) and the myofibers CSA of control
muscles 21 days after overload (Figure 1E), which was not
accompanied by a modification of fiber number (Figure 1F). In
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
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Control of Skeletal Muscle Hypertrophy by Srf
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
Figure 5. Il4 Overexpression Restores Satellite Cell Fusion and Muscle Growth
(A) Il4 mRNA expression was analyzed by qRT-PCR in myotubes (SRFflox/flox) transduced with AdGFP or AdCreGFP for 72 hr or with AdSRFVP16 for 48 hr.
Data (mean SEM) are normalized by cyclophilin expression and presented as fold-induction relative to AdGFP. *p < 0.05 versus AdGFP.
(BD) Numbers of Pax7+ cells per mm2 (B), Pax7+Ki67+ cells per mm2 (C), and myonuclei per fiber (D) were quantified in control and mutant plantaris muscle
sections injected (+) or not () with AAV-IL4 after 7 days of CH. Data are mean SEM. *p < 0.05 versus controls (); xp < 0.05 versus mutants ().
(E) Muscle sections immunostained for dystrophin and mean CSA ( SEM) from control and mutant muscles injected (+) or not () with AAV-IL4 after 14 days
of CH. *p < 0.05 versus controls (); xp < 0.05 versus mutants ().
2003) and 2) the postnatal muscle-growth defect linked to skeletal muscle-specific loss of Srf has been correlated to decreased
Il4 expression (Charvet et al., 2006). In primary myotubes lacking
Srf (AdCreGFP-transduced for 3 days), Il4 expression is reduced
by 40% when compared to control myotubes (AdGFP) (Figure 5A). To determine whether decreased Il4 expression could
account for defective fusion of control myocytes to Srf-deleted
myotubes, in vitro fusion experiments were conducted using
CM issued from Il4/ myotubes and exogenous Il4. Importantly,
Il4 addition rescued fusion, whereas CM from Il4/ myotubes
did not ameliorate fusion (Figure S4). The implication of
decreased Il4 expression in the lack of growth in Srf-deleted
muscles was then addressed in vivo by injecting control and
mutant plantaris muscles with an AAV driving Il4 expression
(AAV-IL4) prior to CH. A robust Il4 overexpression was measured
in plantaris muscle injected with AAV-IL4 (Figure S5A). When Il4
was overexpressed in control muscles on day 7 post-CH, the
number of Pax7+ cells was unchanged and the number of
proliferating Pax7+Ki67+ satellite cells was lower, suggesting
that Il4 might drive satellite cells toward fusion and accelerate
the growth process. In mutant muscles, we observed that Il4
overexpression had no effect on the total number of Pax7+ or
Pax7+Ki67+ satellite cells (Figures 5B and 5C). Most interestingly, the impaired fusion of satellite cells to mutant myofibers
was fully rescued by Il4 overexpression, as assessed by count-
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
EXPERIMENTAL PROCEDURES
Mouse Protocols
Mice homozygous for Srf floxed alleles (Srfflox/flox) and HSA-Cre-ERT2:
Srfflox/flox tamoxifen-dependent Cre recombinase premutant mice have been
Cell Metabolism
Control of Skeletal Muscle Hypertrophy by Srf
formed using a Light Cycler instrument with SYBR Green I kit (Roche). Primer
sequences are listed in Supplemental Information. The values were normalized
to housekeeping genes 18S rRNA or Cyclophilin.
ChIPs
Myotubes (5.106 cells) were fixed with 1% paraformaldehyde for 11 min. For
each immunoprecipitation, sonicated chromatin was incubated with 3 mg
anti-Srf (G20, Santa Cruz) or IgG and immunocomplexes were recovered
using a Magna ChIP G Kit (Millipore). Quantification of immunoprecipitated
DNA was done by real-time PCR using the appropriated primers (see Supplemental Information) and reported to input chromatin. Results shown are
normalized to the recovery of a non-Srf-regulated gene region (in the intronic
part of Il4).
Reporter Assays
Fifty thousand myoblasts were transfected using Lipofectamine 2000 reagent
(Invitrogen) with 80 ng luciferase plasmid reporter, 100 ng pRL-TK and 200
400 ng of indicated plasmid in a total of 800 ng DNA. Twenty-four hours
post-transfection, cells were cultured in DM and harvested 24 hr later. Firefly
and Renilla luciferase activities were determined using the Dual-Luciferase
Reporter Assay System (Promega). Each experiment was performed in triplicate and repeated three times.
Immunostaining
Plantaris muscles were frozen in cooled isopentane and cut in 7 mm-thick
sections. Fiber size, myonuclei number, and satellite cells were analyzed
by immunostaining using anti-dystrophin (Novocastra), anti-P-Akt (Cell
Signaling), anti-Pax7 (Santa Cruz), and/or anti-Ki67 (Abcam) and DAPI staining. Between 300 and 600 myofibers were analyzed per muscle. The distribution of fiber cross-sectional area was determined using Metamorph.
Western Blot Analysis
Western blotting was performed as described (Lahoute et al., 2008). Immunoblots were done using anti-phospho-Akt Ser473 and anti-Akt (Cell Signaling),
anti-Srf, and anti-Gapdh (Santa Cruz) antibodies.
Statistical Analysis
The significance of differences between means was assessed with a Students
t-test. P values of < 0.05 were considered statistically significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, seven figures, and one table and appears with this article online at
doi:10.1016/j.cmet.2011.12.001.
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Cell Metabolism
Article
PGRN is a Key Adipokine Mediating
High Fat Diet-Induced Insulin Resistance
and Obesity through IL-6 in Adipose Tissue
Toshiya Matsubara,1,3,6,10 Ayako Mita,1,2,10 Kohtaro Minami,1 Tetsuya Hosooka,2 Sohei Kitazawa,4,11 Kenichi Takahashi,9
Yoshikazu Tamori,2,7 Norihide Yokoi,1 Makoto Watanabe,6,8 Ei-ichi Matsuo,6,8 Osamu Nishimura,3,8,*
and Susumu Seino1,2,3,5,*
1Division
SUMMARY
(IL-6), and monocyte chemoattractant protein (MCP)-1 (Shoelson et al., 2007; Waki and Tontonoz, 2007). Defects in adipokine
secretion accompanying adipose tissue dysfunction contribute
to the pathophysiology of insulin resistance and obesity (Kahn
and Flier, 2000). Reduced expression and secretion of adiponectin in obesity promotes the development of systemic insulin
resistance by enhancing hepatic gluconeogenesis and suppressing glucose uptake in skeletal muscle (Guilherme et al.,
2008; Berg et al., 2001). In contrast, resistin, TNF-a, IL-6 and
MCP-1, the levels of which in adipose tissues and blood are
elevated in obesity, have been shown to be mediators in progression of insulin resistance (Waki and Tontonoz, 2007).
The relationship between inflammatory process and insulin
resistance has recently drawn considerable attention. For example, TNF-a, a proinflammatory cytokine, has been shown to
contribute to the development of insulin resistance by altering
insulin signaling mediated by activation of the IKK-NFkB and
JNK-AP1 signaling pathways (Hotamisligil et al., 1994, 1995;
Uysal et al., 1997). On the other hand, glucocorticoids, which
are known to have an anti-inflammatory action, also induce
insulin resistance in human and animals (Caro and Amatruda,
1982). Dexamethasone, a glucocorticoid, has been reported to
impair insulin signaling and insulin-stimulated glucose uptake
in adipose tissue, liver, and skeletal muscle (Qi and Rodrigues,
2007). Since TNF-a and dexamethasone both induce insulin
resistance despite their opposite inflammatory properties (Hotamisligil et al., 1994; Hotamisligil, 2006; Wellen and Hotamisligil,
2005; Qi and Rodrigues, 2007; van Putten et al., 1985; Turnbow
et al., 1994; Sakoda et al., 2000), we reasoned that there might
be a common mediator responsible for the cellular basis of
insulin resistance induced by TNF-a and dexamethasone. In
the present study, we searched for a novel adipokine(s) that
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
A
TNF-
or
Dexamethasone
Control
100
3T3-L1 adipocytes
60
13C
0NBS
60
40
20
0
80
Protein extraction
40
6NBS
100
80
m/z
20
0
m/z
100
80
100
80
m/z = 6
60
40
MS/MS
20
60
40
20
0
m/z
Protein identification
m/z
PGRN (aa297-309) peptide: R.LNTGAW*GCCPFAK.A
D
PGRN
Relative
protein expression
GAPDH
**
-1 0 1 2 3 4 5 6 7 8 9 10
**
2.5
PGRN
2.0
Pref-1
1.5
1.0
PPAR
0.5
FABP4
0
Pio (10 M)
ACTIN
tro
n
Co
F-
TN
m
xa
De
e
on
as
h
et
We then examined Grn expression in ob/ob mice, a well-characterized obese and insulin resistance model (Friedman and
Halaas, 1998). Grn expression in ob/ob mice was upregulated
in both peritoneal and subcutaneous white adipose tissues
(WAT) but not in brown adipose tissue (BAT), as compared
with that of ob/+ mice (Figure 2E, left). Serum PGRN levels of
ob/ob mice were also higher than those of ob/+ mice (Figure 2E,
right). Immunohistochemistry of epididymal fat revealed that
40 Cell Metabolism 15, 3850, January 4, 2012 2012 Elsevier Inc.
PGRN was detected predominantly in macrophages (as assessed by Mac-3 costaining) and also was present in the cytoplasm of adipocytes (Figure 2F).
ob/ob mice treated with pioglitazone exhibited a significant
improvement of glucose tolerance (Figure S2D) and increases
in expressions of TZD/PPARg-dependent genes in adipose
tissues (Table S2) but no significant change in body weight (Figure S2C). Under these conditions, Grn expressions in both
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
peritoneal and subcutaneous WAT but not in BAT were decreased significantly (Figure 2G, left). Pioglitazone also normalized serum PGRN levels (Figure 2G, right). These results indicate
that PGRN levels in both adipose tissues and blood are associated with insulin resistance and obesity in vivo.
To determine whether PGRN causes insulin resistance in vivo
directly, recombinant mouse PGRN (rmPGRN) was administrated intraperitoneally to wild-type (WT) mice under standard
diet (SD) condition. We found that serum PGRN levels increased
about 2.4-fold within 1 hr after administration and were kept
constant for 24 hr onward (Figure S2E); we also found that
PGRN levels increased about 2.02.5-fold after treatment with
rmPGRN for 14 days (Figure S2F). This level of increased serum
PGRN is similar to that observed in obese (ob/ob) mice (Figure 2G, right). Under these conditions, the fasting insulin level
in the mice tended to increase (Figure S2I) despite no change
in either body weight or blood glucose level (Figures S2G,
and S2H). We also found that WT mice treated with rmPGRN
for 14 days exhibited insulin resistance, as assessed by insulin
tolerance test (ITT) (Figure 2H). Thus, PGRN has a causative
role in insulin resistance in vivo. These findings indicate that
PGRN is associated with insulin resistance and obesity and
that PGRN directly causes insulin resistance in vivo.
Ablation of Grn Prevents HFD-Induced Obesity
and Insulin Resistance In Vivo
To clarify the physiological and pathophysiological roles of
PGRN directly, we utilized Grn deficient (Grn / ) mice (Kayasuga
et al., 2007). The body weight of Grn / mice fed SD was similar
to that of WT mice (Figure 3A,top left), whereas the body weight
of Grn / mice fed HFD was significantly lower than that of WT
mice (Figure 3A, top right), despite similar food intake (Figure 3A,
bottom). In addition, Grn / mice fed HFD exhibited a marked
reduction in deposition of peritoneal fat compared to WT mice
(Figure 3C, left) as well as in fat mass of both visceral and subcutaneous fat pad (Figure 3B). Immunohistochemistry revealed that
the size of adipocytes in epididymal fat pads of Grn / mice was
significantly smaller than that of WT mice (Figures 3C, middle,
and 3D) and that infiltration of mac-3 positive inflammatory cells
was significantly less in Grn / mice than that in WT mice under
HFD condition (Figures 3C,right, and 3E). In addition, glucose
intolerance induced by HFD was improved in Grn / mice with
a decrease in serum insulin levels (Figures 3F and S3A), suggesting enhanced insulin sensitivity in Grn / mice. Indeed, insulin
tolerance test confirmed that insulin resistance induced by
HFD, which was seen in WT mice, was prevented in Grn /
mice (Figure 3G). These results indicate that ablation of Grn
prevents HFD-induced obesity and insulin resistance in vivo.
PGRN Impairs Insulin Signaling in Adipocytes
Since PGRN has been shown to be involved in the PI3K/Akt
signaling pathway (He and Bateman, 2003; Youn et al., 2009;
Zanocco-Marani et al., 1999; Lu and Serrero, 2001), we reasoned that PGRN might directly affect insulin signaling in
3T3-L1 adipocytes. Although PGRN treatment did not affect
phosphorylation of insulin receptor (IR) (Figure 4A), it decreased
insulin-stimulated phosphorylation of both insulin receptor
substrate (IRS)-1 (Figure 4A) and Akt in a dose-dependent
manner (Figure 4B). PGRN treatment also suppressed insulin-
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
EF
(Grn/18S)
**
**
**
**
**
N.
D
N. .
D.
**
S
HF D
D
S
HF D
D
5.0
4.0
3.0
2.0
1.0
0
PGRN
GAPDH
MF
S
HF D
D
SD
HF
D
Protein expression
(PGRN/GAPDH)
Liver Skeletal
muscle
Adipocytes
SD
HFD
SVF
SD
HFD
**
**
D
SD HF
D
SD HF
SVF
Adipocytes
mRNA expression
(Grn/Rplp0)
Protein expression
(PGRN/ACTIN)
**
300
250
200
150
100
50
0
**
**
ob/+
ob/ob
1 2 3 4 51 2 3 4 5 1 2 3 4 5 1 2 3 4 5
PGRN
ACTIN
4.0
3.0
ob/+
ob/ob
** **
** ** * ** **
**
2.0
1.0
MF
EF
SF
BAT
**
N.S.
MF
SF
BAT
WAT
EF
WAT
Vehicle
Pio
N.S.
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
**
Pio
ob
/+
Ve
hic
le
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Merged
DAPI
G
Mac-3
PGRN
ob/ob
**
**
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
(H) Induction of insulin resistance by administration of recombinant PGRN in vivo. Recombinant mouse PGRN (rmPGRN, i.p. 20 mg/day) or PBS (vehicle) was
administered to C57BL/6J mice once daily for 14 days (n = 7) under SD condition. Insulin sensitivity was assessed by insulin tolerance test (ITT) (left). Blood
glucose levels were determined at the indicated times after intraperitoneal injection of insulin (0.5 IU/kg). Inverse area under curve (AUC) of ITT was shown (right).
All data are means SEM. *p < 0.05; **p < 0.01 in (A), (B), (D), (E), left of (G), (H) (Students unpaired t-test) and in right (G) (Dunnets method); N.S., not significant.
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
B
Body weight (g)
A
45
40
35
30
25
20
15
10
5
0
SD
WT
Grn -/6
8 10 12 14 16
45
40
35
30
25
20
15
10
5
0
N.S.
HFD
WT
Grn -/-
*
*****
WT
Grn-/6
N.S.
8 10 12 14 16
N.S.
HE
Mac-3
WT
Grn/-
WT
(SD)
WT
(HFD)
250
200
150
Grn-/(SD)
N.S.
100
50
SD
Grn-/(HFD)
Grn-/-
WT
HF
SD
HF
WT
Grn/-
F
N.S.
WT (HFD)
Grn-/- (HFD)
WT (HFD)
Grn-/- (HFD)
N.S.
N.S.
WT (SD)
WT (HFD)
Grn-/- (HFD)
Grn-/- (HFD)
WT
Grn-/-
**
**
Figure 3. Prevention of High Fat Diet-Induced Insulin Resistance, Adipocyte Hypertrophy, and Obesity by Ablation of Grn In Vivo
(A) Changes in body weight and food consumption on SD or HFD. Changes in body weights in WT (SD, n = 8, HFD, n = 8) and Grn / (SD, n = 5, HFD, n = 9) mice
were measured. Food consumptions in WT (n = 11) and Grn / (n = 9) mice fed SD or HFD for 1 week are shown as food intake (g) per day.
(B) Tissue weight of WATs in WT (SD, n = 8; HFD, n = 8) and Grn / mice (SD, n = 8; HFD, n = 7).
(C-E) Gross appearance (C left), histology (H&E staining: C middle, mac-3 immunostaining: C right), adipocyte diameter (n = 100) (D), and number of mac-3
positive cells (n = 10) (E) in epididymal fat of WT and Grn / mice. Red arrows indicate mac-3 positive cells. Scale bars, 50 mm.
(F) Oral glucose tolerance test. Blood glucose (left) and serum insulin (right) levels in WT mice (SD, n = 8; HFD, n = 10) and Grn / mice (SD, n = 5; HFD, n = 9) were
determined at the indicated times after oral administration of glucose.
(G) Insulin tolerance test (ITT). Blood glucose levels in WT (SD, n = 8; HFD, n = 9) and Grn / (SD, n = 5; HFD, n = 9) mice were determined at the indicated times
after intraperitoneal injection of insulin (0.3 IU/kg) (left). Insulin sensitivity was assessed by inverse AUC of ITT (right).
All data are means SEM. *p < 0.05; **p < 0.01 in (A), (B), (D), (E), (F) (Students unpaired t-test), and in (G), compared with WT mice fed SD (Dunnets method);
N.S., not significant.
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
*
**
** **
C
D
N.S.
N.S.
**
(A) Effects of exogenous PGRN on IR and IRS-1 phosphorylation. 3T3-L1 adipocytes were treated with various
concentrations of recombinant mouse PGRN protein
(rmPGRN) for 20 hr and subsequently stimulated with
100 nM of insulin for 10 min. Activation of insulin signaling
was then assessed by phosphorylation of IR (Tyr1146)
and tyrosine phosphorylation of immunoprecipitated (IP)
IRS-1.
(B) Effect of exogenous PGRN on Akt phosphorylation
(n = 4). 3T3-L1 adipocytes were treated with various
concentrations of rmPGRN for 4 hr and subsequently
stimulated with 10 nM of insulin for 10 min. Activation of
insulin signaling was then assessed by phosphorylation of
Akt (Ser743).
(C) Effects of exogenous PGRN on glucose uptake.
3T3-L1 adipocytes were treated with various concentrations of rmPGRN for 20 hr and subsequently stimulated
with 10 nM of insulin for 10 min (n = 3).
(D) Effects of Grn knockdown (KD) on IR and IRS-1
phosphorylation. 3T3-L1 adipocytes were infected with
adenovirus carrying shRNA for Grn (GrnshRNA) or adenovirus carrying nontarget shRNA (control) at MOI of 100.
Activation of insulin signaling was then assessed by
phosphorylation of IR (Tyr1146) and tyrosine phosphorylation of immunoprecipitated (IP) IRS-1.
(E) Effect of Grn KD on insulin-stimulated Akt phosphorylation. Insulin-stimulated Akt phosphorylation in KD or
control cells was analyzed by immunoblot analysis (n = 4).
(F) Effects of Grn KD on glucose uptake (n = 3).
(G) Effect of Grn KD on the suppression by TNF-a of
insulin-stimulated Akt phosphorylation. KD or control cells
were treated with 10 ng/ml TNF-a for 16 hr. Insulinstimulated Akt phosphorylation was then analyzed by
immunoblot analysis (n = 4).
All data are means SEM. *p < 0.05; **p < 0.01; ***p <
0.0005 in (B), (C) (Dunnets method) and in (E), (F), (G)
(Students unpaired t-test); N.S., not significant.
1.6
1.2
0.8
0.4
0
-
EXPERIMENTAL PROCEDURES
Mice
We obtained male C57BL/6J from CLEA Japan (Tokyo, Japan), and ob/ob and
ob/+ mice from Charles River Japan (Yokohama, Japan). Grn+/ mice were
purchased from RIKEN BioResource Center (BRC) (Tsukuba, Japan). Genotyping of Grn / mice was performed as described previously (Kayasuga
et al., 2007). C57BL/6J mice were fed HFD from 7 through 33 weeks of age.
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
Il6
rn
G
2
C
cl
f
Tn
Il6
p
Le
oq
ip
Ad
lu
t4
4
bp
Fa
eb
Socs3
Pp
ar
pa
Socs3
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
Figure 6. Effects of Grn Deficiency on HFDInduced Elevation of IL-6 and SOCS3 In Vivo
rn
G
r1
Em
2
C
cl
Il6
Tn
ip
Ad
Le
oq
t4
lu
N.S.
WT
N.S.
Fa
bp
pa
eb
g
C
ar
Pp
Grn
Grn
Grn-/-
Grn-/-
WT
N.S.
N.S.
N.S.
Cell Metabolism
Role of PGRN in Insulin Resistance and Obesity
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and two tables and appears with this article online at doi:10.
1016/j.cmet.2011.12.002.
Emanuelli, B., Peraldi, P., Filloux, C., Chavey, C., Freidinger, K., Hilton, D.J.,
Hotamisligil, G.S., and Van Obberghen, E. (2001). SOCS-3 inhibits
insulin signaling and is up-regulated in response to tumor necrosis
factor-a in the adipose tissue of obese mice. J. Biol. Chem. 276, 47944
47949.
ACKNOWLEDGMENTS
Eriksen, J.L., and Mackenzie, I.R. (2008). Progranulin: normal function and role
in neurodegeneration. J. Neurochem. 104, 287297.
We thank for G.K. Honkawa for assistance with the manuscript. This study was
supported by a CREST grant from the Japan Science and Technology Agency
and Grant-in-Aid for Scientific Research and by grants for the Kobe Translational Research Cluster, the Knowledge Cluster Initiative, and the Global
Centers of Excellence Program Global Center of Excellence for Education
and Research on Signal Transduction Medicine in the Coming Generation
from the Ministry of Education, Culture, Sport, Science and Technology,
Japan.
Received: November 4, 2010
Revised: September 23, 2011
Accepted: December 2, 2011
Published: January 3, 2012
Fasshauer, M., Kralisch, S., Klier, M., Lossner, U., Bluher, M., Klein, J., and
Paschke, R. (2004). Insulin resistance-inducing cytokines differentially
regulate SOCS mRNA expression via growth factor- and Jak/Stat-signaling
pathways in 3T3-L1 adipocytes. J. Endocrinol. 181, 129138.
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Cell Metabolism
Article
p53-Induced Adipose Tissue Inflammation
Is Critically Involved in the Development
of Insulin Resistance in Heart Failure
Ippei Shimizu,1,5 Yohko Yoshida,1,5 Taro Katsuno,1 Kaoru Tateno,1 Sho Okada,1 Junji Moriya,1 Masataka Yokoyama,1
Aika Nojima,1 Takashi Ito,1 Rudolf Zechner,2 Issei Komuro,3 Yoshio Kobayashi,1 and Tohru Minamino1,4,*
1Department
of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
of Molecular Biosciences, University of Graz, A-8010 Graz, Austria
3Department of Cardiovascular Medicine, Osaka University School of Medicine, Osaka 565-0871, Japan
4PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan
5These authors contributed equally to this work
*Correspondence: t_minamino@yahoo.co.jp
DOI 10.1016/j.cmet.2011.12.006
2Institute
SUMMARY
Several clinical studies have shown that insulin resistance is prevalent among patients with heart failure,
but the underlying mechanisms have not been fully
elucidated. Here, we report a mechanism of insulin
resistance associated with heart failure that involves
upregulation of p53 in adipose tissue. We found
that pressure overload markedly upregulated p53
expression in adipose tissue along with an increase
of adipose tissue inflammation. Chronic pressure
overload accelerated lipolysis in adipose tissue. In
the presence of pressure overload, inhibition of lipolysis by sympathetic denervation significantly downregulated adipose p53 expression and inflammation,
thereby improving insulin resistance. Likewise, disruption of p53 activation in adipose tissue attenuated
inflammation and improved insulin resistance but
also ameliorated cardiac dysfunction induced by
chronic pressure overload. These results indicate
that chronic pressure overload upregulates adipose
tissue p53 by promoting lipolysis via the sympathetic
nervous system, leading to an inflammatory response of adipose tissue and insulin resistance.
INTRODUCTION
The p53 tumor suppressor pathway coordinates DNA repair,
cell-cycle arrest, apoptosis, and senescence to preserve
genomic stability and prevent oncogenesis. Activation of p53 is
driven by a wide variety of stress signals that have the potential
to promote tumor formation, such as DNA damage, telomere
shortening, oxidative stress, and oncogene activation (Harris
and Levine, 2005; Meek, 2009; Vousden and Prives, 2009).
Recently, the contribution of p53 to many undesirable aspects
of aging and age-associated diseases, such as cardiovascular
and metabolic disorders, has been recognized (Royds and Iacopetta, 2006; Vousden and Lane, 2007). It has been reported that
aging is associated with an increase of the p53-mediated transcriptional activity (Edwards et al., 2007) and that slight constitutive overactivation of p53 is associated with premature aging in
mice (Maier et al., 2004; Tyner et al., 2002). Activation of p53
has also been observed in aged vessels and failing hearts and
has been implicated in atherosclerosis and heart failure (Minamino and Komuro, 2007, 2008; Sano et al., 2007). Recent findings have indicated a role of p53 in determining the response
of cells to nutrient stress and in regulating metabolism (Vousden
and Ryan, 2009). It has also been demonstrated that excessive
calorie intake induces p53-induced inflammation in adipose
tissue, leading to insulin resistance and diabetes in mice (Minamino et al., 2009).
A close link between heart failure and diabetes has long been
recognized in the clinical setting (Ashrafian et al., 2007; Lopaschuk et al., 2007; Witteles and Fowler, 2008). Many mechanisms have been suggested to explain the increased incidence
of heart failure in diabetic patients, including the hypertrophic
influence of insulin, the adverse effects of hyperglycemia,
increased oxidative stress, and hyperactivity of neurohumoral
systems, such as the renin-angiotensin-aldosterone system
and the adrenergic system. Recently, increasing attention has
been paid to insulin resistance as a distinct cause of cardiac
dysfunction and heart failure in diabetic patients. A study of
Swedish patients without prior cardiac dysfunction found that
insulin resistance predicted the subsequent onset of heart failure
independently of established risk factors (Ingelsson et al., 2005).
In another clinical study, the plasma level of proinsulin (a marker
of insulin resistance) was found to be higher in patients who
subsequently developed heart failure than in control patients
20 years before the actual diagnosis of heart failure (Arnlov
et al., 2001). These findings indicate that insulin resistance
precedes heart failure rather than being a consequence of it.
Evidence has emerged that myocardial insulin resistance is
central to altered metabolism in the failing heart and may play
a crucial role in the development of heart failure (Ashrafian
et al., 2007; Lopaschuk et al., 2007; Witteles and Fowler,
2008). The adaptive response of the failing heart involves
a complex series of enzymatic shifts and changes in the regulation of transcriptional factors, which result in an increase of
glucose metabolism and a decrease of fatty acid metabolism
Cell Metabolism 15, 5164, January 4, 2012 2012 Elsevier Inc. 51
Cell Metabolism
Adipose Inflammation in Heart Failure
Cell Metabolism
Adipose Inflammation in Heart Failure
Figure 1. Pressure Overload Induces Systemic Insulin Resistance and Adipose Tissue Lipolysis and Inflammation
(A) Insulin tolerance test (ITT) and glucose tolerance test (GTT) in mice at 6 weeks after sham operation (Sham) or TAC (n = 30).
(B) Hematoxylin and eosin staining of adipose tissues of mice at 6 weeks after sham operation (Sham) or TAC (upper panel). In the lower panel, the infiltration of
macrophages was evaluated by immunofluorescent staining for Mac3 (green). Nuclei were stained with Hoechst dye (blue). Scale bar, 50 mm. The right graph
indicates the quantitative data on the infiltration of macrophages (n = 5).
(C) Real-time PCR assessing the expression of Emr1, Tnf (Tnfa), Ccl2 (MCP1), and Adipoq (Adiponectin) levels in adipose tissues of mice at 6 weeks after sham
operation (Sham) or TAC (n = 10).
(D) CT analysis of mice at 6 weeks after sham operation (Sham) or TAC. The graph shows the ratio of visceral fat tissue weight estimated by CT to whole body
weight (n = 7).
(E) Norepinephrine level in adipose tissue (left) and plasma (middle), and plasma free fatty acid (FFA) level (right) of mice at 6 weeks after sham operation (Sham) or
TAC (n = 10). Data are shown as the means S.E.M. *p < 0.05, **p < 0.01.
Cell Metabolism
Adipose Inflammation in Heart Failure
Cell Metabolism
Adipose Inflammation in Heart Failure
Figure 2. p53-Dependent Adipose Tissue Inflammation Provokes Systemic Insulin Resistance during Heart Failure
(A) Expression of p53 was examined in adipose tissues of mice by western blot analysis at indicated time points after sham operation (Sham) or TAC. Actin was
used as an equal loading control. The graph indicates the quantitative data on p53 expression (n = 3).
(B) Real-time PCR assessing the expression of Emr1, Tnf (Tnfa), Ccl2 (MCP1), and Cdkn1a (p21) levels in adipose tissue of adipocyte-specific p53-deficient mice
(adipo-p53 KO) and littermate controls (Cont) at 6 weeks after sham operation or TAC procedure (n = 12).
(C) Hematoxylin and eosin staining of adipose tissues of adipocyte-specific p53-deficient mice (adipo-p53 KO) and littermate controls (Cont) at 6 weeks after
sham operation (Sham) or TAC procedure. Scale bar, 50 mm. The right graph indicates the quantitative data on the infiltration of macrophages (n = 4).
(D) Insulin tolerance test (ITT) and glucose tolerance test (GTT) in adipocyte-specific p53-deficient mice (KO) and littermate controls (Cont) at 6 weeks after sham
operation (Sham) or TAC procedure (n = 16). Data are shown as the means S.E.M. *p < 0.05, **p < 0.01.
Cell Metabolism
Adipose Inflammation in Heart Failure
Figure 3. Surgical Transection of the Sympathetic Nerves Attenuates Adipose Tissue Inflammation and Systemic Insulin Resistance
(A) Real-time PCR assessing the expression of Emr1, Tnf (Tnfa), Ccl2 (MCP1), and Cdkn1a (p21) levels in adipose tissues of mice at 6 weeks after sham operation
(Sham) or TAC with or without surgical transection of the sympathetic nerves (Denervation) of epidydimal fat (n = 8).
(B) Insulin tolerance test (ITT) and glucose tolerance test (GTT) of mice at 6 weeks after sham operation (Sham) or TAC with or without surgical denervation (n = 20).
(C) Western blot analysis of p53 in adipose tissues of mice at 6 weeks after sham operation (Sham) or TAC with or without surgical denervation. The right graph
indicates the quantitative data on p53 expression (n = 3). Data are shown as the means S.E.M. *p < 0.05, **p < 0.01.
Cell Metabolism
Adipose Inflammation in Heart Failure
Figure 4. Treatment with a Lipolysis Inhibitor Ameliorates Adipose Tissue Inflammation and Systemic Insulin Resistance
(A) Real-time PCR assessing the expression of Emr1, Tnf (Tnfa), Ccl2 (MCP1), and Cdkn1a (p21) levels in adipose tissue of mice at 6 weeks after sham operation
(Sham) or TAC with or without acipimox treatment (n = 8).
(B) Insulin tolerance test (ITT) and glucose tolerance test (GTT) of mice at 6 weeks after sham operation (Sham) or TAC with or without acipimox treatment (n = 32).
(C) Western blot analysis of p53 in adipose tissues of mice at 6 weeks after sham operation (Sham) or TAC with or without acipimox treatment. Actin was used as
an equal loading control. The right graph indicates the quantitative data on p53 expression (n = 3). Data are shown as the means S.E.M. *p < 0.05, **p < 0.01.
Cell Metabolism
Adipose Inflammation in Heart Failure
Figure 5. Role of Lipolysis in the Regulation of Adipose p53 Expression and Inflammation
(A) Western blot analysis of p53 in adipose tissues of wild-type mice treated with PBS or isoproterenol (ISO). Actin was used as an equal loading control. The right
graph indicates the quantitative data on p53 expression (n = 3).
(B) Real-time PCR assessing the expression of Emr1, Tnf (Tnfa), and Ccl2 (MCP1) levels in adipose tissues of wild-type mice treated with PBS or isoproterenol
(ISO) (n = 8).
(C) Hematoxylin and eosin staining of adipose tissues of wild-type mice treated with PBS or isoproterenol (ISO). Scale bar, 50 mm. The right graph indicates the
quantitative data on macrophage infiltration (n = 4).
(D) The changes in weight of adipose tissues isolated from Atgl-deficient mice (KO) and littermate controls (Cont) after treatment with PBS or isoproterenol (ISO)
(n = 6).
(E) Expression of p53 was examined in adipose tissues of Atgl-deficient mice (KO) and littermate controls (Cont) treated with PBS or isoproterenol (ISO) by
western blot analysis. The right graph indicates the quantitative data on p53 expression (n = 3).
(F) Real-time PCR assessing the expression of Cdkn1a (p21) level in adipose tissues isolated from Atgl-deficient mice (KO) and littermate controls (Cont) after
treatment with PBS or isoproterenol (ISO) (n = 6). Data are shown as the means S.E.M. *p < 0.05, **p < 0.01.
enhances the activity of NF-kB, which regulates various cytokines including TNF-a and CCL2 (Benoit et al., 2006; Ryan
et al., 2000), we examined the relationship between p53 expression and NF-kB activation. We demonstrated that the
disruption of p53 expression significantly attenuated palmitic
acid-induced activation of NF-kB and upregulation of Ccl2
Cell Metabolism
Adipose Inflammation in Heart Failure
(Figures 7D and 7E), whereas knockdown of the NF-kB component p50 markedly inhibited palmitic acid-induced upregulation
of Ccl2 (Figure 7E). In addition, treatment with an antioxidant inhibited palmitic acid-induced DNA damage and upregulation of
p53 (Figures S7A and S7B). We also found that ROS and
Cell Metabolism
Adipose Inflammation in Heart Failure
Cell Metabolism
Adipose Inflammation in Heart Failure
tion rather than the increase of plasma free fatty acids per se is
involved in the impairment of insulin sensitivity and glucose tolerance associated with heart failure. We also noted that p53 was
modestly upregulated in the liver and skeletal muscle, presumably due to the increase of circulating free fatty acids. However,
we did not detect a strong inflammatory response in those
tissues under our experimental conditions (I. Shimizu and
T. Minamino, unpublished data), suggesting that upregulation
of adipose tissue p53 is more important for the development
of metabolic abnormalities during heart failure. This concept is
further supported by our finding that disruption of p53 activation
in adipose tissue nearly normalized insulin resistance and
glucose intolerance provoked by heart failure.
We observed that systolic cardiac function and survival with
chronic heart failure were significantly better for adipo-p53 KO
mice than their control littermates. Suppression of p53 activity
in adipose tissue by administration of a p53 inhibitor after the
onset of heart failure improved cardiac dysfunction and also
reduced adipose tissue inflammation and metabolic abnormalities in both the TAC and MI models. Inhibition of NF-kB activity
in adipose tissue also improved cardiac dysfunction, as well as
adipose tissue inflammation and insulin resistance. Improvement of cardiac dysfunction by disruption of p53 in adipose
tissue was not associated with a decrease of plasma free fatty
acid levels. Systemic inhibition of lipolysis (Atgl deficiency or acipimox treatment) and disturbance of lipolysis in adipose tissue
(denervation or guanethidine treatment) significantly reduced
plasma free fatty acid levels (Haemmerle et al., 2006). However,
the former intervention accelerated heart failure, whereas
cardiac dysfunction was improved by the latter. Thus, the beneficial effect of inhibiting p53-induced adipose tissue inflammation on cardiac function is independent of changes in circulating
free fatty acid levels, and lipolysis in cardiomyocytes appears to
have a crucial role in cardiac metabolism and energy production.
Although there is evidence suggesting that p53 has a protective
role against damage due to ROS and lipotoxicity (Bazuine et al.,
2009), our results indicate that chronic activation of p53 in
adipose tissue causes inflammation and that inhibition of p53induced adipose tissue inflammation is a potential target for
treating metabolic abnormalities and systolic dysfunction in
patients with heart failure.
Adipose tissue was traditionally considered to be a simple
energy storage organ, but it is now appreciated that it also has
endocrine functions and secretes a variety of factors referred
to as adipokines (Donath and Shoelson, 2011; Hotamisligil,
2006; Ouchi et al., 2011). With high calorie intake, the size and
number of adipocytes increase, and hypertrophic adipocytes
shift the balance toward production of proinflammatory adipokines. This shift in the adipokine profile causes the modification
of adipose tissue macrophages from the anti-inflammatory M2
type to the proinflammatory M1 type, and further increases
the production of proinflammatory molecules, which in turn
(G) The number of g-H2AX-positive nuclei (white arrows and inset) in adipose tissue of mice at 6 weeks after sham operation (Sham) or TAC procedure was
estimated by immunofluorescent staining for g-H2AX (red) (n = 5). Nuclei and plasma membranes were stained with Hoechst dye (blue) and Wheat Germ
agglutinin lectin (green). Scale bar indicates 50 mm.
(H) The number of p50-positive nuclei in adipose tissue of adipocyte-specific p53-deficient mice (adipo-p53 KO) and littermate controls (Cont) at 6 weeks
after sham operation (Sham) or TAC procedure was estimated by immunofluorescent staining for p50 (n = 6). Data are shown as the means S.E.M. *p < 0.05,
**p < 0.01.
Cell Metabolism
Adipose Inflammation in Heart Failure
accelerates the recruitment of activated macrophages into inflamed fatty tissue. Adipokines produced by inflamed adipose
tissue have been suggested to play a crucial role in the regulation
of glucose and lipid metabolism and to contribute to the development of diabetes (Donath and Shoelson, 2011; Hotamisligil,
2006; Ouchi et al., 2011). It has been reported that excessive
calorie intake leads to accumulation of ROS in adipose tissue
and subsequently causes DNA damage that activates p53 (Minamino et al., 2009). In contrast to obesity, heart failure decreases
body fat tissue mass by inducing lipolysis. Accelerated lipolysis
and a subsequent increase of free fatty acids are likely to cause
p53 activation because we found that the promotion of lipolysis
by treatment with isoproterenol upregulated adipose tissue
expression of p53, whereas inhibition of lipolysis by acipimox
or disruption of lipase activity attenuated p53 expression. These
results are consistent with a recent report describing that fasting-induced lipolysis promotes an immune response in murine
adipose tissue (Kosteli et al., 2010). Various molecular mechanisms of p53 activation by heart failure may be postulated,
including hypoxia, increased oxidative stress, and induction of
endoplasmic reticulum stress (Harris and Levine, 2005; Schenk
et al., 2008). Our in vitro and in vivo studies have indicated that
an increase of free fatty acids causes ROS-induced DNA
damage that upregulates p53 in adipose tissue. Activation of
p53 then upregulates the expression of proinflammatory adipokines via the NF-kB signaling pathway and promotes systemic
insulin resistance.
The b-blockers are competitive antagonists of b-adrenergic
receptors. At one time, b-blockers were contraindicated in
patients with heart failure due to their negative inotropic effect.
However, several large-scale clinical trials demonstrated the
efficacy of b-blockers for reducing morbidity and mortality in
heart failure patients with impaired systolic function, so
b-blockers are now recommended as first-line agent for these
patients (Hjalmarson et al., 2000; Leizorovicz et al., 2002; Packer
et al., 2001, 2002). A reduction of heart rate due to inhibition of
cardiac b1-adrenergic receptors is believed to be responsible
for most of the therapeutic benefits associated with b-blocker
treatment, although this is not the only mechanism of action
that may be important in heart failure. It is interesting that treatment with a nonselective b-blocker (carvedilol) achieved a more
marked improvement of survival in patients with chronic heart
failure than treatment with a b1-selective blocker (metoprolol)
(Poole-Wilson et al., 2003), whereas new-onset diabetes was
frequent in heart failure patients during treatment with the
b1-selective blocker (Torp-Pedersen et al., 2007). It has been
reported that carvedilol antagonizes the b3-adrenergic receptor
as well as the b1/2-adrenergic receptors (Schnabel et al., 2000).
Taking our results together with these reports, it seems that
inhibition of b3-adrenergic activity in adipose tissue partially
accounts for the better clinical outcome in patients treated with
this nonselective b-blocker. Recent evidence has suggested
that treatment with insulin sensitizers improves systolic function
of the failing heart in animal models (Asakawa et al., 2002; Nemoto et al., 2005) but such treatment increases the incidence of
heart failure in diabetic patients, presumably because of sodium
retention (Home et al., 2009). Inhibition of p53-induced adipose
tissue inflammation could be an alternative therapeutic target to
block the metabolic vicious cycle in patients with heart failure.
62 Cell Metabolism 15, 5164, January 4, 2012 2012 Elsevier Inc.
EXPERIMENTAL PROCEDURES
Animal Models
All animal study protocols were approved by the Chiba University review
board. C57BL/6 mice were purchased from the SLC Japan (Shizuoka, Japan).
TAC and MI were performed in 11-week-old male mice as described previously (Harada et al., 2005; Sano et al., 2007). Sham-operated mice underwent
the same procedure except for aortic constriction. Mice that expressed Cre
recombinase in adipocytes (Fabp4-Cre) were purchased from Jackson Laboratories. We then crossed Fabp4-Cre mice (with a C57BL/6 background) with
mice that carried floxed Trp53 alleles with a C57BL/6 background (Marino
et al., 2000) to generate adipocyte-specific p53 knockout mice. The genotype
of littermate controls was Fabp4-Cre- Trp53 flox/flox. The generation and genotyping of Atgl-deficient mice has been described previously (Haemmerle et al.,
2006). Surgical or chemical denervation was performed before TAC operation
as described previously (Demas and Bartness, 2001; Foster and Bartness,
2006), with slight modification. In brief, the epidydimal fat pad was gently
separated from the skin and the abdominal wall by using a dissecting microscope. For surgical denervation, a drop of 1% toluidine blue was applied to
the fat pad to facilitate visualization of the nerves. The nerves were then freed
from the surrounding tissue and vasculature and cut in two or more locations,
and the segments were removed to prevent possible reconnection. Chemical
denervation was performed by the local injection of guanethidine sulfate
(400 mg, Santa Cruz) into bilateral epidydimal fat. Sham-operated mice for
surgical denervation underwent the same procedure except for transection
of the nerve. For the control group for chemical denervation, saline was injected into adipose tissue rather than guanethidine. Acipimox (Sigma) were
provided in drinking water (at a concentration of 0.05%) for 6 weeks after
TAC operation as described previously (Guo et al., 2009). Isoproterenol
(30 mg/kg/day, Sigma) were delivered by infusion pump (DURECT Corporation) for 2 weeks as described previously (Iaccarino et al., 1999). The local
injection of pifithrin-a (2.2 mg/kg/week, Carbiochem) or BAY 11-7082
(20 mg/kg/week, Carbiochem) into bilateral epidydimal fat was performed to
inhibit adipose p53 or NF-kB activity, respectively, from 2 weeks to 4 weeks
after operation.
Physiological and Histological Analyses
Echocardiography was performed with a Vevo 770 High Resolution Imaging
System (Visual Sonics Inc, Toronto, Ontario, Canada). To minimize variation
of the data, the heart rate was always approximately 550650 beats per minute
when cardiac function was assessed. Epidydimal fat samples were harvested
and fixed in 10% formalin overnight. The samples were embedded in paraffin
and sectioned (Narabyouri research Co., Ltd). The sections were subjected to
immunohistochemistry or HE staining. The antibodies used are Mac3-specific
primary antibody (PharMingen) for macrophages, p50-specific primary antibody (Cell signaling), and phospho-H2AX-specific antibody (Cell signaling).
Laboratory Tests
For the intraperitoneal glucose tolerance test (IGTT), mice were starved for
6 hr and were given glucose intraperitoneally at a dose of 2 g / kg (body weight)
in the early afternoon. For the insulin tolerance test, mice were given human
insulin intraperitoneally (1 U / kg body weight) at 1:00 pm without starvation.
Blood glucose levels were measured with a glucose analyzer (Roche Diagnostics). We analyzed free fatty acid (Biovision, Inc) and norepinephrine levels
(LDN) by using ELISA-based immunoassay kits according to the manufacturers instruction.
Western Blot Analysis
The lysates were resolved by SDS-polyacrylamide gel electrophoresis.
Proteins were transferred to a polyvinylidene difluoride membrane (Millipore,
Bedford, MA), which was incubated with the primary antibody followed by
anti-rabbit or anti-mouse immunoglobulin-G conjugated with horseradish
peroxidase (Jackson, West Grove, PA).
Cell Culture
Human preadipocytes were purchased from Sanko (Tokyo, Japan) and were
cultured according to the manufacturers instructions. NIH 3T3-L1 cells were
cultured in high-glucose DMEM plus 10% fetal bovine serum.
Cell Metabolism
Adipose Inflammation in Heart Failure
Ex Vivo Culture
Epididymal fat was extracted from Atgl-deficient or littermate mice at 17 weeks
of age. Freshly isolated fat pads (100120 mg) were incubated in Dulbeccos
modified Eagles medium supplemented with 10% fetal bovine serum in the
presence of isoproterenol (10 mM) for 48 hr. Fat pads were treated with PBS
instead of isoproterenol in the control group.
Statistical Analysis
Data are shown as the mean SEM. Differences between groups were
examined by Students t-test or ANOVA followed by Bonferronis correction
for comparison of means. For survival analysis, the Kaplan-Meier method
and log-rank test were used. For all analyses, p < 0.05 was considered statistically significant.
SUPPLEMENTAL INFORMATION
Haemmerle, G., Lass, A., Zimmermann, R., Gorkiewicz, G., Meyer, C.,
Rozman, J., Heldmaier, G., Maier, R., Theussl, C., Eder, S., et al. (2006).
Defective lipolysis and altered energy metabolism in mice lacking adipose
triglyceride lipase. Science 312, 734737.
ACKNOWLEDGMENTS
Harada, M., Qin, Y., Takano, H., Minamino, T., Zou, Y., Toko, H., Ohtsuka, M.,
Matsuura, K., Sano, M., Nishi, J., et al. (2005). G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat. Med. 11, 305311.
We thank A. Berns (The Netherlands Cancer Institute) for floxed p53 mice,
T. Fujita (The Tokyo Metropolitan Institute of Medical Science) for reagents,
and E .Takahashi, M. Iijima, and I. Sakamoto for their excellent technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology of Japan
and grants from the Ono Medical Research Foundation; the Uehara Memorial
Foundation; the Daiichi-Sankyo Foundation of Life Science; the NOVARTIS
Foundation for the Promotion Science; the Japan Diabetes Foundation; the
Mitsui Life Social Welfare Foundation; the Naito Foundation; the Japanese
Society of Anti-Aging Medicine; and the Mitsubishi Pharma Research Foundation (to T.M.); a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture and Health and Labor Sciences Research
Grants (to I.K.); and a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science, Sports, and Culture, and Health and a grant from the
Uehara Memorial Foundation, Takeda Science Foundation, and Kowa Life
Science Foundation (to I.S.).
Received: June 10, 2011
Revised: October 27, 2011
Accepted: December 9, 2011
Published online: January 3, 2012
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Cell Metabolism
Article
Impaired Generation of 12-Hydroxylated Bile Acids
Links Hepatic Insulin Signaling with Dyslipidemia
Rebecca A. Haeusler,1 Matthew Pratt-Hyatt,2 Carrie L. Welch,1 Curtis D. Klaassen,2 and Domenico Accili1,*
1Department
SUMMARY
Cell Metabolism
Bile Acids and Insulin Action
Cell Metabolism
Bile Acids and Insulin Action
Cell Metabolism
Bile Acids and Insulin Action
Cell Metabolism
Bile Acids and Insulin Action
(C) Ratio of non-12a-OH BAs to 12a-OH BAs in bile, feces, and total pool.
(D) Composition of total BA pool in WTD-fed L-FoxO1 mice and controls (mean values of n = 56).
(E) Quantitation of total BAs, 12a-OH BAs, and non-12a-OH BAs in the total BA pool from WTD-fed mice (n = 56). *p < 0.05, **p < 0.01, by Welchs two-sample
t-test (A) or Students t-tests (B, C, and E). Data are presented as mean SEM. See also Figure S3.
Cell Metabolism
Bile Acids and Insulin Action
mechanism by which insulin regulates HGP has been well characterized (Lin and Accili, 2011), its control over TG synthesis,
secretion, and clearance is less defined (Choi and Ginsberg,
2011). In this work we show that insulin signaling through
FoxO1 controls production of 12a-OH BAs and we propose
that these molecules act as regulatory mediators, relaying a
signal from insulin to TG synthesis.
This discovery can be integrated into a new model for the role
of the insulin-Akt-FoxO1 pathway in regulating multiple aspects
of hepatic metabolism (Figure 5F). In normal physiology, FoxO1
maintains the production of 12a-OH BAs by regulating Cyp8b1
and, potentially, other BA synthetic genes. In turn, these BAs
maintain normal Fxr activity, curbing TG levels. In overt diabetes,
FoxO1 becomes trapped inside the nucleus, due to impaired
ability of insulin to cause its nuclear export, compounded by
hyperglycemia (Haeusler et al., 2010a; Kitamura et al., 2005).
In this condition, BAs would become skewed toward excess
12a-OH BAs. Indeed, diabetic models, such as alloxan-treated
rodents, NOD mice, and mice lacking hepatic insulin receptors,
show disproportionately high 12a-OH BAs (Akiyoshi et al.,
1986; Biddinger et al., 2008a; Uchida et al., 1985; Uchida
et al., 1996).
Compellingly, the pattern of increased 12a-OH BAs has also
been identified in human patients with diabetes (Brufau et al.,
2010). Contrary to rodents, however, CDCAthe most abundant
non-12a-OH BAis hydrophobic and also is the most potent
FXR ligand (Makishima et al., 1999; Parks et al., 1999); thus,
FOXO1 induction of CYP8B1 (which promotes CA formation) at
the expense of CDCA would cause a relative reduction in FXR
activation because CA and its conjugated form T-CA are less
potent FXR ligands (Makishima et al., 1999; Parks et al., 1999).
The constitutive activation of the FOXO1-CYP8B1-12a-OH BA
pathway during diabetes would be expected to result in reduced
FXR activity, raising TG.
Interestingly, L-FoxO1 mice express inadequate Cyp8b1 and
have a distinct functional defect in 12a-hydroxylation; however,
they do not phenocopy Cyp8b1 / mice (Li-Hawkins et al., 2002;
Murphy et al., 2005). The latter presumably have normal Fxr
activity because they express normal Shp and Bsep, and reportedly have no steatosis (Li-Hawkins et al., 2002; Murphy et al.,
2005). This is likely due to their compensatory upregulation of
Cyp7a1, which is associated with an expanded BA pool, including a substantial increase in CDCA (Li-Hawkins et al., 2002).
In contrast, L-FoxO1 mice show low Cyp7a1 and Cyp27a1 under
several different conditions and low Cyp7b1 after WTD feeding,
suggesting that FoxO1 is required for full expression of several
of these genes. Mice lacking Lrh-1 specifically in liver show
several similarities to L-FoxO1 mice, including reduced expression of Cyp8b1 and deficiency of 12-OH BAs (Lee et al., 2008;
Mataki et al., 2007). However, the two models are not identical:
Lrh-1 knockouts express normal levels of Cyp7a1 and they
have not been reported to develop steatosis. The latter may be
attributed to the fact that 1) they have not been challenged with
Cell Metabolism
Bile Acids and Insulin Action
Cell Metabolism
Bile Acids and Insulin Action
Figure 5. Defects in Bile Acid Composition, Lipid, and Glucose Metabolism in L-FoxO1 Mice
(A) Hepatic gene expression showing FXR activation due to GW4064 treatment (n = 67).
(B) Liver TGs from L-FoxO1 and control mice fed WTD supplemented with either GW4064 (0.035%) or CA (0.5%) for 3 weeks.
(C) Serum total and VLDL-TGs in L-FoxO1:Ldlr / and Ldlr / controls after GW4064 (4 daily treatments, orally) or supplementation of WTD with CA (1% for
1 week). For comparison in B and C, we also show lipid levels from mice fed chow and WTD (without treatment); these are the same mice whose mean values are
shown in Figure 1.
(D and E) Hepatic gene expression (D) and blood glucose levels (E) from P2 neonates, where nursing mothers ate either chow or chow supplemented with CA.
Expression of Cyp8b1 and Cyp7a1 from untreated mice are the same measurements presented in Figure 4B.
(F) Model of metabolic functions of FoxO1 in liver. Data are presented as mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001, by Students t-tests (B, C, and E) or
two-way analysis of variance (A and D). N.S., not significant; HGP, hepatic glucose production. See also Figure S4.
Scientific), cholesterol E, and free cholesterol (Wako). Lipoprotein fractions were
separated by density ultracentrifugation as described (Haeusler et al., 2010a).
Bile Acid Measurements and Metabolomics
A complete list of BA abbreviations is available in Table S4. The metabolomics
study was performed after 10 weeks on chow or WTD. Livers were collected
after a 5-hr fast and snap-frozen. Hepatic BAs (relative values) were measured
by LC-MS/MS (-ESI), with the exception of a-MCA, which was measured by
GC/MS (Metabolon). The relative increase of T-CDCA in L-FoxO1 mice is
underestimated in both WTD and chow-fed mice because it was detectable
in only 4 out of 16 control mice but 12 out of 16 L-FoxO1 mice; samples below
the limit of detection were considered to be at the lowest detectable level.
Cell Metabolism
Bile Acids and Insulin Action
Gallbladder bile was collected by syringe from mice fed the WTD for 10 weeks
and pooled (5 per genotype). Fecal samples were collected for two consecutive days following 8 weeks of WTD feeding. For total BA pool, gallbladder and
small intestine were collected together with whole liver, a small piece of which
was used to confirm genotype. For bile, feces, and total BA pool, BAs were
extracted and measured by LC-MS/MS as described (Alnouti et al., 2008).
Detailed methods are provided in Supplemental Information.
Gene Expression
RNA was isolated using TRIzol (Invitrogen). GeneChip Mouse Exon 1.0 ST
Arrays were from Affymetrix. Expression analysis was performed using Partek
Genomics Suite (Partek). For qPCR, cDNA was synthesized using qScript
(QantaBioSciences), and qPCR was performed using SyBr Green (New
England Biolabs). Genes were normalized to 18 S. Primer sequences are
available upon request.
Statistical Analyses
Data are presented as mean SEM. Data were analyzed by two-tailed
Students t-tests, with the following exceptions: metabolomics data were
analyzed by Welchs two-sample t-tests; microarray data was analyzed by
analysis of variance (ANOVA), using Partek Genomics Suite; and data comparing two variables (Figures 4D, 5A, 5D, S4E, and S4F) were analyzed by
two-way ANOVA; p < 0.05 was considered significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and four tables and can be found with this article online at
doi:10.1016/j.cmet.2011.11.010.
ACKNOWLEDGMENTS
This work was supported by NIH grants F32HL103103 to R.A.H, HL87123 and
DK58282 to D.A., and DK63608 (Columbia University Diabetes and Endocrinology Research Center). We thank A. Tall, I. Tabas, D. Mangelsdorf, J. Horton,
and U. Pajvani for valuable comments and discussions, Ana Flete for technical
assistance, and members of the Accili laboratory for discussion of the data.
R.A.H. and D.A. designed and analyzed the experiments and wrote the paper.
R.A.H. performed experiments. M.P.-H. and C.D.K. performed bile acid
measurements by LC-MS/MS. C.L.W. performed aortic root lesion analysis.
All authors edited the manuscript.
Received: July 18, 2011
Revised: October 6, 2011
Accepted: November 28, 2011
Published online: December 22, 2011
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Cell Metabolism
Article
Acetylation Negatively Regulates Glycogen
Phosphorylase by Recruiting Protein Phosphatase 1
Tengfei Zhang,1,2 Shiwen Wang,1,2 Yan Lin,2 Wei Xu,1,2 Dan Ye,2 Yue Xiong,1,2,4,* Shimin Zhao,1,2,*
and Kun-Liang Guan1,2,3,5,6,*
1State
SUMMARY
Glycogen phosphorylase (GP) catalyzes the ratelimiting step in glycogen catabolism and plays a
key role in maintaining cellular and organismal
glucose homeostasis. GP is the first protein whose
function was discovered to be regulated by reversible protein phosphorylation, which is controlled by
phosphorylase kinase (PhK) and protein phosphatase 1 (PP1). Here we report that lysine acetylation
negatively regulates GP activity by both inhibiting
enzyme activity directly and promoting dephosphorylation. Acetylation of GP Lys470 enhances its interaction with the PP1 substrate-targeting subunit, GL,
and PP1, thereby promoting GP dephosphorylation
and inactivation. We show that GP acetylation is
stimulated by glucose and insulin and inhibited by
glucagon. Our results provide molecular insights
into the intricate regulation of the classical GP and
a functional crosstalk between protein acetylation
and phosphorylation.
INTRODUCTION
Glycogen phosphorylase (GP) catalyzes phosphorolytic cleavage of glycogen to produce glucose-1-phosphate for glucosedependent tissues when body glucose is scarce. GP activity
plays an important role in glucose homeostasis and glycogen
metabolism. Defects in glycogen synthesis and breakdown
in liver, muscle, and other glucose-dependent tissues often
cause glycogen storage diseases (Stegelmeier et al., 1995).
For example, McArdle disease is caused by mutations in muscle
GP, and patients with this disorder are intolerant of exercise
and show early fatigue (Andreu et al., 2007; Tang et al., 2003).
Due to their critical roles in glucose homeostasis, regulations
of glycogen synthase (GS) and GP activities have been extensively investigated in hopes of finding therapeutic strategy for
type II diabetes.
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
mutant GP could dominantly inhibit wild-type GP. In the transfected cells, the ectopically expressed GP was much higher
(about 10-fold) than the endogenous protein GP (Figure S1D),
indicating that endogenous GP should have a negligible effect
on our assay results. Moreover, we purified WT homodimer,
WT-K2R heterodimer, and K2R homodimer and assayed their
catalytic activity. The WT-K2R heterodimer exhibited activity
that was the average of wild-type and K2R homodimers (Figure S1E), showing that the K2R mutant has no dominant inhibition on wild-type GP. Together, our results suggest that K470
and K796 are two major acetylation sites in GP responsible for
its inhibition by acetylation. To further test the effect of acetylation on GP activity and glycogen metabolism, we determined
cellular glycogen content in response to NAM and TSA treatment. Consistent with an inhibitory effect of acetylation on GP
activity, we found that inhibition of deacetylases by NAM and
TSA increased cellular glycogen levels (Figure S1F). Next, we
determined glycogen hydrolysis in cells expressing different
GP mutants. Expression of wild-type GP promoted a faster
glycogen hydrolysis than the GPK2Q mutant (Figure 1E), further
supporting a lower enzymatic activity of the acetylation mimetic
mutant GPK2Q.
GP Acetylation and Activity Are Regulated by Glucose,
Insulin, and Glucagon
Given that glucose concentration is a key factor in GP regulation,
we determined GP acetylation under different glucose concentrations. The acetylation level of ectopically expressed GP was
low when cells were maintained in glucose-free medium, and
increased significantly with elevated glucose concentrations
in a dose-dependent manner by as much as 4-fold (Figure 2A),
suggesting that acetylation of GP was upregulated by glucose.
Moreover, acetylation of endogenous GP in L02 human hepatocytes was increased by glucose (Figure 2B, Figure S2A). NAM
and TSA treatment of cells cultured in high-glucose medium
could further increase the acetylation level of endogenous GP
(Figure 2B), showing that deacetylation of GP occurs even under
high glucose.
Next, the effect of glucose on GP activity was determined. As
shown in Figure 2C, the activity of wild-type GP was significantly inhibited by increasing concentrations of glucose, while
GPK2Q was largely refractory to inhibition by glucose (Figure 2C). These results suggest that glucose inhibits GP activity
through K470 and K796 acetylation. To further test the function
of acetylation in mediating glucose-induced GP inhibition, we
measured the effect of glucose on GP activity after NAM and
TSA treatment. We hypothesized that since the inhibition of deacetylases would increase GP acetylation, high glucose may
not have a significant effect on GP activity in the presence of
deacetylase inhibitors. As expected, glucose increased GP
acetylation and decreased GP activity in the absence of deacetylase inhibitors (Figure 2D, lanes 14). However, in the presence of deacetylase inhibitors, glucose had little effect on GP
activity and did not further increase the elevated GP acetylation
(Figure 2D, lanes 58). These results further support the notion
that acetylation plays a major role in GP inhibition in response
to glucose.
Both insulin and glucagon are important signals that regulate
GP activity and glycogen metabolism in opposite manners
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
was increased with insulin treatment in time- and dose-dependent manners (Figure 2E, Figure S2B). Moreover, acetylation of
ectopically expressed GP increased within 30 min of insulin
Cell Metabolism 15, 7587, January 4, 2012 2012 Elsevier Inc. 77
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
Because of the inhibitory effect of acetylation on phosphorylation, the above data were unable to show whether acetylation
may directly affect GP activity. To address this question, we
prepared GP with or without acetylation and with or without
phosphorylation. Hyperacetylated GP was obtained by expressing Flag-GP in Changs liver cells maintained in high-glucose
(25 mM) medium supplemented with NAM and TSA, while hypoacetylated GP was obtained by glucose-free medium without
NAM and TSA. The immunopurified GP proteins were treated
with l-phosphatase or PhK in vitro. After verifying both the acetylation and the Ser-15 phosphorylation of GP by western blotting
(right panel, Figure 3F), GP activity was determined. We found
that phosphorylation by GP kinase (PhK) dramatically increased
GP activity regardless of whether GP was hyperacetylated (lane
1 versus lane 2, left panel, Figure 3F) or hypoacetylated (lane 3
versus lane 4). Acetylation did not affect GP activity when GP
was hypophosphorylated (lanes 1 versus lane 3). However,
when GP was hyperphosphorylated, acetylation exhibited an
inhibitory effect on GP in vitro (lane 2 versus lane 4). These results
indicate that acetylation inhibits GP only when it is hyperphosphorylated and active.
GP is also regulated by allosteric effect, including activation
of inactive b form by AMP. To explore the effect of acetylation
on allosteric regulation of GP, we determined the activity of
hypo- versus hyperacetylated GP in response to AMP. We found
that AMP induced a similar activation to both the hyperacetylated (lane 1 versus lane 1A, 20.5-fold, Figure 3F) and hypoacetylated GP (lane 3 versus lane 3A, 20.4-fold) when GP was
hypophosphorylated, indicating that acetylation does not
directly affect allosteric activation of GP by AMP. However,
when GP was fully activated (hypoacetylated/hyperphosphorylated), AMP could not activate GP further (lanes 4 versus 4A).
To provide more direct evidence to determine the effect of
acetylation on allosteric regulation, we treated immunopurified
GP with recombinant deacetylase CobB in vitro and measured
its allosteric activation by AMP after confirming the decrease
of acetylation (right panel, Figure 3G). We found that AMP activated GP equally regardless of the levels of GPs acetylation
(5.54-fold, 5.58-fold, and 5.50-fold, left panel, Figure 3G). We
therefore conclude that acetylation does not directly affect allosteric activation of GP by AMP.
Physiological Stimuli Regulate GP Acetylation-Induced
Dephosphorylation
The notion that acetylation negatively regulates GP-Ser-15
phosphorylation was further pursued by analyzing acetylation
and phosphorylation levels in response to physiological stimuli.
We found that glucose increased GP acetylation and decreased
phosphorylation in a dose-dependent manner (Figure 4A), indicating an inverse relationship between GP acetylation and
phosphorylation. Moreover, insulin treatment increased GP
acetylation and decreased GP phosphorylation (Figure 4B),
whereas glucagon treatment decreased GP acetylation and
increased GP phosphorylation (Figure 4C). A time-dependent
inverse correlation between acetylation and phosphorylation in
GP was observed in response to glucose, insulin, and glucagon
(Figure 4D, Figures S4A and S4B), supporting the notion that
acetylation may inhibit GP phosphorylation. It is worth noting
that glucagon can regulate GP activity through a direct signaling
Cell Metabolism 15, 7587, January 4, 2012 2012 Elsevier Inc. 79
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
(E) The effect of glucose on GP activity but not acetylation depends on Ser-15. GPS15D-transfected Changs liver cells were treated with different concentrations
of glucose. Acetylation, phosphorylation, and relative activity of GPS15D were determined. Activity of GP from glucose-free medium was set as 100% arbitrarily.
(F) Effect of acetylation and phosphorylation on GP activity and activation by AMP. Hyperacetylated GP was immunoprecipitated from Flag-GP-transfected
Changs liver cells maintained in high-glucose (25 mM) medium supplemented with NAM+TSA. Hypoacetylated GP was immunoprecipitated from FlagGP-transfected cells cultured in glucose-free medium without NAM+TSA. The immunopurified GP was incubated with l-phosphatase (l) or phosphorylase
kinase (PhK) in appropriate buffers for 2 hr. Both phosphatase and kinase were removed by washing with PBS, and the treated GP was measured for enzyme
activity with (hatched bars) or without AMP (5 mM, solid bars) as indicated. Relative enzyme activity was normalized against GP protein. Ser-15 phosphorylation
and lysine acetylation levels were determined by western blotting.
(G) GP allosteric activation by AMP is not affected by acetylation. Flag-GP was purified from Changs liver cells and then treated for 1 or 2 hr with buffer or CobB as
indicated. Relative activity of purified GP was measured in the absence (solid bars) or presence of 5 mM AMP (hatched bars) and normalized by protein quantity.
All error bars represent standard deviation (SD). n = 3 for each experimental group.
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
F
E
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
Figure 6. Acetylation
Interaction
Finally, to investigate whether the GP-GL interaction is regulated by physiological stimuli, we determined the interaction
between GL with either wild-type or K470Q mutant GP in
response to glucose concentration and insulin stimulation. We
found that the GP-GL interaction was increased by approximately 100% after switching from glucose-free medium to
25 mM glucose medium (Figure 6F). However, a similar glucose
switch did not affect the interaction between GPK470Q and GL.
Furthermore, insulin stimulated the interaction between the
wild-type GP, but not GPK470Q, and GL (Figure 6G). These results
support the notion that GP-GL interaction is regulated by K470
acetylation in response to nutrient and hormonal signals.
84 Cell Metabolism 15, 7587, January 4, 2012 2012 Elsevier Inc.
GP-GL
(A) Acetylation enhances GP-GL interaction. FlagGP was transfected into Changs liver cells either
alone or with HA-tagged GL or GLDC5. NAM+TSA
treatment was indicated. GP-GL and GP-GLDC5
interactions were determined by coimmunoprecipitation.
(B) Deacetylation of GP decreases GP-GL interaction in vitro. Flag-GP was expressed in HEK293
cells in the presence of deacetylase inhibitor,
and HA-GL was separately expressed. Both
proteins were affinity purified. Flag-GP was deacetylated with purified bacterial deacetylase
CobB in vitro as indicated. In vitro binding
between Flag-GP and HA-GL was performed.
Acetylation level of Flag-GP and the amount of
HA-GL coprecipitated by Flag-GP were determined by western blot.
(C) Glucose increases GP-GL interaction in vivo.
Human Myc-GP and HA-GL plasmids were intravenously injected into mice. The injected mice
were fasted overnight and then intraperitoneally
injected with glucose (1 g/kg). The mice were
sacrificed at 30 min postinjection. GP and GL
proteins expressed in the injected mouse liver
were immunoprecipitated by Myc beads for MycGP. The amount of GP and coprecipitated GL
proteins was measured, and the GL/GP ratio was
calculated.
(D) K470 is required for deacetylase inhibitors to
stimulate the GP-GL interaction. Flag-GP and
Flag-GPK470Q were transfected in Changs liver
cells, either alone or with HA-tagged GL. Cells
were treated with or without NAM+TSA. GP-GL
interactions were determined by coimmunoprecipitation.
(E) Quantification of GP K470 acetylation. Acetylation levels of K470 of GP expressed in Changs
liver cells treated with or without NAM+TSA were
quantified by iTRAQ.
(F and G) K470 in GP is required for regulation of
GP-GL interaction by glucose and insulin. Flag-GP
and Flag-GPK470Q were transfected in Changs
liver cells, either alone or with HA-tagged GL. Cells
were treated with or without glucose (F) or insulin
(G). GP-GL interactions were determined by
coimmunoprecipitation.
All error bars represent standard deviation (SD).
n = 34 for each experimental group.
Increases
DISCUSSION
Following the initial discovery of histone acetylation (Allfrey et al.,
1964; Phillips, 1963), extensive studies over the last four
decades have identified not only the enzymes that catalyze
reversible acetylation, the protein lysine acetyltransferases
(KATs, formerly termed histone acetyltransferases, HATs), and
deacetylases (commonly known as histone deacetylases, or
HDACs) but also many nonhistone substrates. Until relatively
recently, nearly all well-characterized acetylation substrates
were nuclear proteins, including transcription factors and coregulators (Yang and Seto, 2008). Recent studies, in particular
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
weakened, which in turn would increase GP Ser-15 phosphorylation and activity (Figure 7). Conversely, in cells with high
glucose concentration, GP is hyperacetylated and the acetylation enhances GPs binding with GL and thus PP1, leading to
decreased Ser-15 phopshorylation and activity of GP and eventually reduced glycogen degradation. Therefore, it appears that
high glucose can downregulate GP activity by two different
mechanisms: a conformational change by the direct binding of
glucose and acetylation-mediated dephosphorylation. It will be
important to determine whether these two regulations operate
separately or sequentially, and whether GP conformational
change brought by the initial glucose binding facilitates the
acetylation.
The role of acetylation in recruiting PP1 is clearly consistent
with the inverse correlation between acetylation and phosphorylation of GP. Glucagon can induce a direct signaling pathway to
phosphorylate and activate GP in a manner independent of
acetylation. However, we speculate that the decrease of GP
acetylation induced by glucagon may contribute to the magnitude and duration of GP activation in response to glucagon.
Glucagon stimulates GP by activating the GP kinase via the
classical phosphorylation cascade and also by dissociating GP
phosphatase via acetylation (Figure S7). It should be noted that
acetylation appears to have no direct role in GP allosteric activation by AMP, although AMP cannot further activate GP when GP
is fully active. Although the precise mechanism by which glucose
regulates GP acetylation remains to be elucidated, using
acetylation machinery to control GP adds another layer of
regulation and thus lends cells further versatility in integrating
multifaceted signaling pathways and nutrient conditions to this
enzyme that is not only central to the glycogen metabolism,
but is also interlocked with multiple energy metabolic pathways.
EXPERIMENTAL PROCEDURES
Mouse Liver Collection and Primary Hepatocytes
and Muscle Cell Isolation
Male BALB/c mice (46 weeks old, 2025 g) were divided into two groups, fed,
and overnight fasted. Fasting started from late afternoon and lasted for 16 hr
before experiments. The fasted mice were further subdivided into glucoseand insulin-treated groups, and the fed mice were treated with glucagon.
Mice blood glucose levels were measured by Accu-Chek Active Blood
Glucose Meter (Roche). At 30 min postinjection, mice were sacrificed and liver
samples were harvested. In addition, mouse primary hepatocytes and muscle
cells were isolated. Please refer to the Supplemental Experimental Procedures
for detailed information. Animal experiments were performed at Fudan Animal
Center in accordance with the animal welfare guidelines.
Glycogen Content Measurement
Cells were washed twice and then lysed with supersonic. The cell lysate in 1 ml
PBS (pH 4.8) was heated at a boiling point for 10 min to liberate stored
Cell Metabolism
Acetylation Inhibits Glycogen Phosphorylase
glycogen and to inactivate enzymes, which may produce extra glucose. After
centrifugation for 15 min at 13,000 rpm, 4U amyloglucosidase (Sigma) was
added into the supernatant. The resulting mixture was incubated for 1 hr at
50 C and followed by boiling for 10 min at 99 C. The cell lysate without
amyloglucosidase was included as a control. Subsequently, the glycogen
content was colorimetrically measured using a glucose assay kit (GAGO20,
Sigma). The mixtures were incubated for 30 min at 37 C, and absorbance at
540 nm was measured by using a UV/Visible spectrophotometer reader
(Ultrospec 3100 pro, Amersham Biosciences).
iTRAQ Quantification
Flag-GP proteins were expressed in Changs liver cells and quantified by
iTRAQ following the method modified according to Zhao (Zhao et al., 2010).
GP was immunopurified from cells untreated or treated with NAM and TSA,
resolved on 10% SDS-PAGE, and stained by Coomassie blue and sliced.
The dye of gel slice was removed by soaking with 50 mM NH4HCO3 and
50% acetonitrile, followed by water wash twice and removing water by
acetonitrile. The gel was dried and digested in 100 ml 50mM NH4HCO3 with
trypsin (trypsin:protein at 1:30) at 37 C overnight. The trypsin-treated peptides
were extracted by a volume containing 50% acetonitrile and 0.1% trifluoroacetic acid (TFA) and then followed by vacuum dry. Standard control peptides
and GP samples were separately labeled with different iTRAQ-labeling
reagents (ABI) as indicated in Table S1 and then subjected to LTQ-OrbiTrap
MS analysis. Quantification of peptides was calculated by comparing relative
intensity of the iTRAQ tags.
Cell Treatment
TSA (0.5 mM) and NAM (5 mM) were added to the culture medium 18 and 6 hr
before cell harvest, respectively. Glucose-free medium was prepared with
DMEM base (GIBCO, #11966) and supplemented with glucose (Sigma), insulin
(Sigma), and glucagon (Sigma) of different concentrations as indicated.
Glucose, insulin, and glucagon treatments were carried out by culturing cells
in DMEM medium for 24 hr before the desired medium was used to replace
DMEM medium.
Glycogen Phosphorylase Activity Assay and CobB Treatment
Flag-tagged proteins were expressed in Changs liver cells, eluted by Flag
peptides (Gilson Biochemical), and measured using the method of Jones
and Wright (Jones and Wright, 1970). The GP activity assay consists of
50 mM sodium glycerol-phosphate (pH 7.1), 10 mM potassium phosphate,
5 mM MgCl2, 0.5 mM NAD+, 1 mM DTT, 1.6 unit phosphoglucomutase, 1.6
unit glucose-6-phosphate dehydrogenase, and 0.2% glycogen in a total
volume of 0.3 ml. The reaction was started by adding GP into the volume
and assayed at 25 C. The reaction was monitored by measuring the increase
of fluorescence (excitation 350 nm, emission 470 nm, HITACHI F-4,600 fluorescence spectrophotometer) for NADH generation.
CobB deacetylation treatment was performed by using a method modified
elsewhere (Hallows et al., 2006). CobB was expressed in E. coli, purified
with nickel beads, and stored at 80 C in 10% glycerol. The CobB deacetylation
assay buffer consists of 40 mM HEPES (pH 7.0), 6 mM MgCl2, 1 mM NAD+, and
n1 mM DTT in a total volume of 0.1 ml. The reaction was started by adding
10 mg CobB and GP into the volume and was assayed at 37 C for 1 hr.
l-Phosphatase and Phosphorylase Kinase Treatment
Flag-GP was ectopically expressed in Changs liver cells and immunoprecipitated by Flag beads. Subsequently, the bead-linked GP was washed by
PBS (pH 7.4) three times before being treated with phosphatase and kinase
(PhK). The l-phosphatase assay consists of NEBuffer Pack for Protein
MetalloPhosphatases (50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij
35 [pH 7.5]) and 1 mM MnCl2 in a total volume of 0.4 ml. The reaction was
started by adding 50 units lambda protein phosphatase (#P0753S, NEB) and
GP into the volume and assayed at 30 C for 2 hr by shaking. The PhK reaction
assay consists of 41 mM glycerophosphate, 41 mM Tris, 0.2 mM CaCl2, 3 mM
ATP, MgCl2 (pH 8.2). Reaction was started by adding 2U PhK (#P2014, Sigma)
and GP into the volume and assayed at 30 C for 2 hr by shaking. The reaction
was terminated by washing the beads three times by PBS and eluting by Flag
peptide, and the activity was determined by fluorescence spectrophotometer
(Cohen, 1973; Shenolikar et al., 1979).
In Vitro Binding
Flag-GP purified by Flag beads was subject to in vitro deacetylation by CobB
before it was mixed with purified HA-GL. The binding was allowed in 4 C for
4 hr before the beads were washed three times by PBS. Proteins on beads
were denatured by SDS loading buffer and detected by western blot.
Statistical Analysis
Statistics were performed with a two-tailed unpaired Students t test. All data
shown represent the results obtained from triplicated independent experiments with standard deviations (mean SD). The values of p < 0.05 were
considered statistically significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures, one table, Supplemental
Experimental Procedures, and Supplemental References and can be found
with this article online at doi:10.1016/j.cmet.2011.12.005.
ACKNOWLEDGMENTS
We thank the members of the Fudan MCB laboratory for discussions
throughout this study. We appreciate Dr. P.T. Cohen for providing the GP
Ser-15 phospho-antibody. This work was supported by the 985 Program, 973
Program (grant numbers 2009CB918401, 2011CB910600, 2012CB910101,
and 2012CB910103), NSFC (grant numbers 30971485/C0706, 31030042,
31071192), Shanghai key project (grant numbers 09JC1402300 and
11JC1401100), the Shanghai Leading Academic Discipline Project (project
number B110), and National Institutes of Health (NIH) grants to Y.X. and K.-L.G.
Received: January 24, 2011
Revised: July 7, 2011
Accepted: December 9, 2011
Published online: January 3, 2012
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Cell Metabolism
Article
Lysosome-Related Organelles in Intestinal Cells
Are a Zinc Storage Site in C. elegans
Hyun Cheol Roh,1 Sara Collier,1 James Guthrie,2 J. David Robertson,2,3 and Kerry Kornfeld1,*
1Department
of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA
Reactor Center
3Department of Chemistry
University of Missouri, Columbia, MO 65211, USA
*Correspondence: kornfeld@wustl.edu
DOI 10.1016/j.cmet.2011.12.003
2Research
SUMMARY
INTRODUCTION
Zinc is a nutrient that is essential for all life. Zinc has roles in
many biological processes; protein-bound zinc contributes
to enzymatic activity and protein structure, and labile zinc
functions in signal transduction (Murakami and Hirano, 2008;
Vallee and Falchuk, 1993). Zinc is important for human health,
since zinc deficiency causes a broad range of defects in
multiple organ systems including skin, immune, skeletal, and
reproductive (Hambidge, 2000). Zinc deficiency is associated
with genetic diseases caused by mutations of zinc transporters, such as acrodermatitis enteropathica and inadequate
dietary intake, which is a major world-wide problem. Excess
zinc is also deleterious, since it may displace other trace metals
or bind low affinity sites, leading to protein dysfunction (Fosmire, 1990). Therefore, organisms require homeostatic mechanisms to control the levels and distribution of this essential
metal.
88 Cell Metabolism 15, 8899, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
FluoZin-3
A
WT
pgp-2
glo-1
glo-3
0
100
Supplemental Zn (M)
Zn content (ppm)
300
250
WT
pgp-2
glo-1
cdf-2
200
150
100
50
0
0
200
Supplemental Zn (M)
Figure 2. Gut Granules Are the Major Site of Zinc Storage
(A) Fluorescence images of live wild-type, pgp-2(kx48), glo-1(zu391), and
glo-3(zu446) animals cultured with FluoZin-3 and the indicated levels of
supplemental zinc. Images show the intestine with pharynx to the left and tail
to the right. Scale bar: 50 mm.
(B) Total zinc content of wild-type, pgp-2(kx48), glo-1(zu391), and cdf-2(tm788)
animals. Populations of animals consisting of a mixture of developmental
stages were cultured on NAMM dishes with the indicated levels of supplemental zinc. Total zinc content was determined by ICP-MS and calculated in
parts per million (ppm). Bars indicate mean values SEM of two independent
experiments (see also Figure S3).
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
Since LysoTracker labels acidic organelles, these results indicate that one lobe may lack the determinants that concentrate
the LysoTracker or that the LysoTracker dye may be present
but not fluorescent due to high pH. To examine a possible pH
difference, we used LysoSensor Green DND-153, which is highly
fluorescent in neutral compartments. Colocalization analysis
with CDF-2::mCherry demonstrated that LysoSensor Green
DND-153 was fluorescent only in one lobe of the bilobed gut
granules (Figure S5), similar to LysoTracker staining. These
results suggest that asymmetric staining of bilobed vesicles
may reflect an asymmetric distribution of the molecules that
bind these fluorescent probes.
To characterize additional differences between the two sides
of bilobed gut granules, we first investigated the distribution of
zinc using FluoZin-3. With 100 mM supplemental zinc, FluoZin-3
displayed asymmetric staining in bilobed gut granules; the
92 Cell Metabolism 15, 8899, January 4, 2012 2012 Elsevier Inc.
LysoTracker-positive lobe displayed weak FluoZin-3 fluorescence, whereas the LysoTrackernegative lobe displayed strong FluoZin-3
fluorescence (Figure 1C, right). To examine the
relationship between zinc levels and CDF-2
protein in bilobed gut granules, we examined
the colocalization of FluoZin-3 and CDF2::mCherry. While FluoZin-3 fluorescence was
asymmetric and strong in only one lobe of
bilobed granules, CDF-2::mCherry was localized to both lobes (Figure S4, right).
To define the molecular properties of bilobed
gut granules, we examined the localization of
several well-characterized endosomal or lysosomal marker proteins. The GTPase RAB-5
localizes to early endosomes (Chen et al.,
2006; Hermann et al., 2005). RAB-5 did not colocalize with LysoTracker or CDF-2 (data not
shown), suggesting that gut granules are
distinct from early endosomes. Lysosome associated membrane proteins (LAMPs) are localized predominantly in lysosomes in vertebrates,
and C. elegans LMP-1 localizes to late endosomal or lysosomal
vesicles of intestinal cells (Chen et al., 2006; Kostich et al., 2000).
A subset of LMP-1 colocalized with LysoTracker in gut granules
(Figure 4B). In addition, a subset of LMP-1 localized to other
membrane compartments that were LysoTracker negative,
including the plasma membrane, indicating this marker is not
specific for lysosomes. To determine whether LMP-1 was
present on both lobes of bilobed gut granules, we exposed
worms to high zinc and used CDF-2::mCherry to define bilobed
morphology. LMP-1 was present only in one lobe of bilobed gut
granules (Figure 4C). These results indicate that bilobed gut
granules displayed asymmetric molecular properties; one lobe
has late endosomal or lysosomal characteristics whereas the
other lobe lacks at least one lysosomal protein.
C. elegans PGP-2 is an ABC transporter that localizes specifically to the gut granule membrane (Schroeder et al., 2007). With
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
no supplemental zinc, CDF-2::mCherry, PGP-2::GFP, and autofluorescence completely colocalized in gut granules (Figure S6B,
left). With 100 mM supplemental zinc, CDF-2 and PGP-2 fully
colocalized on both sides of bilobed gut granules, while autofluorescence displayed an asymmetric pattern and was only
prominent on one lobe, similar to the LysoTracker staining
pattern (Figure S6B, right). These results indicate that bilobed
gut granules contain proteins that are distributed on both lobes,
such as PGP-2 and CDF-2, and at least one protein that is
asymmetrically localized to the LysoTracker positive lobe,
LMP-1 (Figure 5B).
Glo Genes Are Necessary for the Formation of Bilobed
Gut Granules
To elucidate the role of vesicular trafficking pathways in bilobed
gut granule formation, we reduced the activity of key genes
using the method of feeding RNAi. Animals were exposed to
RNAi starting at the first larval (L1) stage to allow normal embry-
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
B
WT
pgp-2
glo-1
glo-3
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1
0.8
0.6
0.4
0.2
0
50
100
150
200
Supplemental Zn (M)
C
1.4
WT
cdf-2
1.2
Length of worms (normalized)
50
100
150
200
Supplemental Zn (M)
WT
pgp-2
glo-1
l 1
glo-3
D
1.2
WT
cdf-2
cdf
2
d)
Length of worms (normalized
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
DISCUSSION
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
determine how zinc sequestration in lysosome-related organelles relates to other mechanisms of zinc tolerance such as
expression of zinc-binding proteins.
The second function of zinc storage in gut granules is to
provide a source of zinc that can be mobilized during dietary
deficiency. Animals shifted from high to low zinc conditions
displayed decreased zinc levels in gut granules after 48 hr, suggesting that stored zinc is released in response to deficiency.
Importantly, our data indicate that stored zinc is utilized for the
physiologic process of growth, since wild-type animals with
normal zinc storage grew more robustly than Glo and cdf-2
mutant animals with defective zinc storage. These results
demonstrate the existence of a specific site of zinc storage in
an animal that provides a physiologically important source of
zinc during dietary deficiency. Elegant studies reported by Eide
and colleagues show that zinc stored in the yeast vacuole is
mobilized by the ZIP protein Zrt3, and stored zinc can supply
the needs of as many as eight generations of progeny cells under
zinc starvation conditions (MacDiarmid et al., 2000; Simm et al.,
2007). In mammalian T cells, ZIP8 localizes to lysosomes and
mediates release of zinc that plays a regulatory role in T cell
activation (Aydemir et al., 2009; Begum et al., 2002). Zinc storage
and mobilization at the organismal level has been investigated
in vertebrates. Chickens fed a high zinc diet display accumulation of zinc in several tissues including the liver, bone, and small
intestine (Emmert and Baker, 1995). When these animals are
shifted to a zinc-deficient diet, the level of accumulated zinc
decreases, and the onset of zinc deficiency symptoms is
delayed compared to control chickens fed a low zinc diet, suggesting that accumulated zinc may be mobilized. In humans,
symptoms of zinc deficiency develop rapidly in response to dietary zinc deficiency, indicating there are only small pools of zinc
available for mobilization (King, 2011). Our studies indicate that
the capacity for zinc storage and mobilization of C. elegans is
intermediate between the large capacity of yeast and the small
capacity of mammals. The limited capacity of animals compared
to yeast may reflect the strategy of storing zinc in specialized cell
types, such as intestinal cells in C. elegans, compared to storage
in the vacuole of every yeast cell.
Lysosome-Related organelles Adopt a Bilobed
Morphology in Response to High Zinc
Gut granules displayed striking morphological changes in
response to high dietary zinc. In standard culture conditions,
gut granules were typically round in shape, autofluorescent,
positive for LysoTracker and FluoZin-3 staining, and positive
for membrane localization of CDF-2, LMP-1, and PGP-2. In
high dietary zinc, gut granules were frequently bilobed in shape,
and the two lobes displayed an asymmetric distribution of molecules. One lobe displayed molecular properties that were similar
to gut granules in standard culture conditions, whereas the other
lobe displayed strong FluoZin-3 staining, was positive for the gut
granule-specific proteins PGP-2 and CDF-2, and was negative
for the lysosomal markers Lysotracker and LMP-1. The high level
of zinc in one lobe raises the possibility that CDF-2 is more
abundant or active in the high zinc lobe. Using epifluorescence
microscopy, CDF-2 levels appeared to be similar on both lobes.
However, this analysis is complicated by autofluorescence from
the LysoTracker positive lobe. Using confocal microscopy that
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
minimizes signal due to autofluorescence, CDF-2 levels appeared to be higher on the lobe with high zinc, suggesting that
there may be a positive correlation between CDF-2 levels and
zinc accumulation. While these studies are suggestive, strong
conclusions about the relative levels of CDF-2 on the two lobes
will require a more quantitative analysis than described here. We
speculate that the bilobed structure may facilitate zinc storage
by generating a compartment that is specialized to accommodate a large amount of zinc. Alternatively, it may facilitate zinc
excretion through budding of a secretory vesicle containing
excess zinc. Secretory granules of mammalian paneth cells
also display nonhomogenous staining of the contents, but the
arrangement is distinct from the bilobed granules. Based on
EM analysis, paneth cell granules have a central core and a
distinct surround, called a biphasic structure, which differ in
carbohydrate composition (Leis et al., 1997).
We used genetic analysis to elucidate a pathway for the formation of bilobed gut granules (Figure 5B). Reducing the activity of
pgp-2 and glo-3 by the method of RNAi inhibited the formation
of bilobed morphology. Because glo genes were inactivated
after gut granule biogenesis during embryonic development,
the absence of bilobed morphology is not likely to be caused
by defects in gut granule biogenesis. Rather, glo genes are likely
to act in both gut granule biogenesis and the transition to bilobed
morphology in response to high levels of zinc. Interestingly,
animals exposed to RNAi of glo genes displayed vesicles that
contained CDF-2::GFP and were LysoTracker negative. These
vesicles were spatially separate from gut granules that contained
CDF-2::GFP and were LysoTracker positive, suggesting that glo
genes may be involved in a vesicle fusion event that generates
bilobed morphology. cdf-2 mutant animals displayed organelles
with bilobed morphology, based on membrane staining with
PGP-2, but these organelles did not display FluoZin-3 fluorescence. Thus, CDF-2 is not necessary for the formation of bilobed
morphology but is necessary to accumulate zinc in bilobed
organelles. Therefore, the formation of bilobed morphology is
separable from zinc transport and accumulation. Lysosome
biogenesis and function can be regulated by signaling pathways
in response to cellular conditions (Karageorgos et al., 1997), and
the transcription factor EB was recently demonstrated to play
a critical role in this process (Sardiello et al., 2009). These results
indicate lysosomes are not static organelles, but rather respond
dynamically to cellular conditions. Our results showed that environmental zinc induced transcription of cdf-2, a resident protein
of lysosome-related organelles, and caused lysosome-related
organelles to adopt a bilobed morphology. These findings are
consistent with the model that the transcriptional response
to environmental zinc causes dynamic changes in lysosomerelated organelle structure and function. These studies establish
a genetically tractable model system to dissect the contribution
of transcriptional programs to the biogenesis of specialized
lysosome-related organelles that play a critical role in coping
with the environmental challenge of high zinc.
EXPERIMENTAL PROCEDURES
General Methods and Strains
C. elegans strains were cultured at 20 C on nematode growth medium (NGM)
seeded with E. coli OP50 unless otherwise noted (Brenner, 1974). The wild-
type C. elegans and parent of all mutant strains was Bristol N2. The following
mutations and transgenes were used: pgp-2(kx48) I (Schroeder et al., 2007),
unc-119(ed3) III (Praitis et al., 2001), cdf-2(tm788) X (Davis et al., 2009),
glo-1(zu391) X (Hermann et al., 2005), glo-3(zu446) X (Rabbitts et al., 2008),
amIs4(cdf-2::GFP::unc-119(+)) (Davis et al., 2009), pwIs50 (lmp-1:GFP)
(Treusch et al., 2004), pwIs72 (Pvha-6::GFP::rab-5) (Hermann et al., 2005),
and kxEx98(pgp-2::GFP;rol-6D) (Schroeder et al., 2007). amEx132(cdf2::mCherry;rol-6D) and amEx142(cdf-2::mCherry::unc-119(+)) are described
here. Double mutant animals were generated by standard methods, and genotypes were confirmed by PCR or DNA sequencing.
Metal Sensitivity Assays
Gravid adult hermaphrodites were treated with NaOH and bleach, and eggs
were incubated in M9 solution overnight to allow hatching and synchronized
arrest at the L1 stage. L1 animals were transferred to NAMM dishes (Bruinsma
et al., 2008) supplemented with zinc sulfate (ZnSO4), cadmium chloride
(CdCl2), or copper sulfate (CuSO4) and seeded with concentrated OP50. After
3 days, animals were washed twice in M9 containing 0.01% Tween-20, paralyzed with 10 mM sodium azide (NaN3) in M9, and mounted on a 2% agarose
pad on a microscope slide. Images were captured with a Zeiss Axioplan 2
microscope equipped with a Zeiss AxioCam MRm digital camera. Length of
individual animals was measured as an indicator of growth using ImageJ
software (NIH) by drawing a line from the nose to the tail tip.
Quantitative Analysis of CDF-2::GFP Expression by Fluorescence
Microscopy
cdf-2(tm788);amIs4 animals were synchronized at L1 stage and cultured on
NGM dishes. L4 stage hermaphrodites were then cultured for 24 hr on
NAMM dishes supplemented with ZnSO4 and seeded with concentrated
OP50. Animals were paralyzed with 0.1% tricaine and 0.01% tetramisole in
M9, mounted on 2% agarose pads on microscope slides, and imaged with
a Zeiss Axioplan 2 microscope equipped with a Zeiss AxioCam MRm digital
camera using identical settings and exposure times. GFP fluorescence intensity was quantified using ImageJ software (NIH). Briefly, the Spot Enhancing
Filter 2D plugin was used to amplify signals from gut granules, and then
threshold settings were used to specifically select the fluorescent regions of
gut granules. The selected regions were overlaid on the original images and
analyzed for mean fluorescence intensity of the area.
Staining with FluoZin-3, LysoTracker, and LysoSensor
FluoZin-3 acetoxymethyl (AM) ester (Molecular Probes F24195) was reconstituted in dimethylsulfoxide (DMSO) to generate a 1 mM stock solution, diluted in
M9 and dispensed on NAMM dishes to yield a final concentration of 3 mM.
L4 stage hermaphrodites were cultured on these dishes for 1224 hr in the
dark, transferred to NGM dishes without FluoZin-3 for 30 min to reduce
FluoZin-3 in the intestinal lumen, and analyzed by fluorescence microscopy
as described above. The intestine on the anterior half of each animal was
analyzed because this structure was typically observed in the same focal
plane. Residual fluorescence from the intestinal lumen was manually removed
and excluded from the analysis.
LysoTracker RED DND-99 (1 mM, Invitrogen L7528), or LysoSensor Green
DND-153 (1 mM, Invitrogen L7534) were diluted in M9 and dispensed on
NAMM dishes to yield a final concentration of 2 mM. L4 stage hermaphrodites
were cultured on these dishes for 1224 hr in the dark, transferred to NGM
dishes without dye for 30 min, and imaged as described above. Confocal
microscopy was performed using an Olympus FV500 confocal microscope
system equipped with multiline argon (458/488/515 nm) and krypton
(568 nm) lasers.
Zinc Shift Assays
To monitor zinc levels in gut granules, we cultured L4 stage animals for
1216 hr on NAMM dishes containing FluoZin-3 and 200 mM ZnSO4 and
then analyzed them by fluorescence microscopy as described above. Next,
animals were transferred to NAMM dishes with FluoZin-3 containing 0 or
200 mM ZnSO4 or 100 mM TPEN and analyzed by fluorescence microscopy
after 24 hr and 48 hr. To analyze growth, we cultured synchronized L1 stage
animals on NAMM dishes supplemented with 0 or 50 mM ZnSO4 for 12 hr.
We chose 50 mM supplemental zinc because it caused relatively mild toxicity
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
Cell Metabolism
Lysosome-Related Organelles Store Zinc in Worms
Lichten, L.A., and Cousins, R.J. (2009). Mammalian zinc transporters: nutritional and physiologic regulation. Annu. Rev. Nutr. 29, 153176.
Sardiello, M., Palmieri, M., di Ronza, A., Medina, D.L., Valenza, M., Gennarino,
V.A., Di Malta, C., Donaudy, F., Embrione, V., Polishchuk, R.S., et al. (2009). A
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Chinwalla, A., Robertson, J.D., Mardis, E.R., and Kornfeld, K. (2011).
Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans.
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H. (2004). Caenorhabditis elegans functional orthologue of human protein hmucolipin-1 is required for lysosome biogenesis. Proc. Natl. Acad. Sci. USA
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protein that confers resistance to zinc by facilitating vesicular sequestration.
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Cell Metabolism
Article
Somatic Progenitor Cell Vulnerability
to Mitochondrial DNA Mutagenesis
Underlies Progeroid Phenotypes in Polg Mutator Mice
Kati J. Ahlqvist,1 Riikka H. Hamalainen,1 Shuichi Yatsuga,1 Marko Uutela,1 Mugen Terzioglu,6,7 Alexandra Gotz,1
Saara Forsstrom,1 Petri Salven,2 Alexandre Angers-Loustau,3 Outi H. Kopra,4,8 Henna Tyynismaa,1 Nils-Goran Larsson,6,7
Kirmo Wartiovaara,3,5 Tomas Prolla,9 Aleksandra Trifunovic,6,10 and Anu Suomalainen1,11,*
1Research
SUMMARY
100 Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
mtDNA Mutagenesis Affects Somatic Stem Cells
rearrangements, because of an inactivated exonuclease function of the mitochondrial replicative DNA polymerase gamma
(POLG) (Ameur et al., 2011; Kujoth et al., 2005; Trifunovic
et al., 2004; Williams et al., 2010). Their phenotype mimics
premature aging, starting from 68 months of age, with progressive hair graying, alopecia, osteoporosis, general wasting, and
reduced fertility (Kujoth et al., 2005; Trifunovic et al., 2004). Their
life span is limited to 1315 months because of severe anemia,
with age-dependent decline in erythro- and lymphopoiesis,
recently suggested to be due to HSC dysfunction (Chen et al.,
2009; Norddahl et al., 2011). POLG forms the minimal mtDNA
replisome together with the mitochondrial single-stranded DNA
binding protein and a replicative helicase, Twinkle (Korhonen
et al., 2004; Spelbrink et al., 2001). If dominant mutant Twinkle
is overexpressed in mice, large-scale mtDNA deletions accumulate in postmitotic tissues (hence the Deletor mice), leading to
progressive late-onset mitochondrial myopathy at 12 months of
age and respiratory chain (RC)-deficient neurons, but normal life
span without progeroid features (Tyynismaa et al., 2005).
We asked whether SSC dysfunction could contribute to the
mtDNA mutagenesis-linked premature aging, and utilized the
two mouse models with mtDNA maintenance defects, Mutator
and Deletor, of which only the former showed a premature aging
phenotype. We report here NSC and HSC dysfunction in murine
mitochondrial progeria.
RESULTS
Old Mutator Neurons and Skeletal Muscle Show Mild
Respiratory Chain Deficiency
To characterize the consequences of high mtDNA mutation
load in postmitotic cells, we analyzed the activity and presence
of RC enzymes of Mutator mice at the time when they already
manifest progeroid phenotype, with hair loss, kyphosis, and
severe anemia (1314 months, Figures 1A and 1B). Histochemical enzyme assay of cytochrome c oxidase (COX, partially encoded by mtDNA) and succinate dehydrogenase (SDH, nuclear
encoded) showed no COX-negative, SDH-positive neurons in
the hippocampus or dentate nucleus (Figures 1C and 1D), or
any area with large neurons, including the cortex and cerebellum. However, as previously described (Tyynismaa et al.,
2005), Deletor mice at the age of 18 months showed occasional COX-negative neurons in all areas harboring large neurons, including hippocampus and dentate nucleus (Figures 1C
and 1D). Immunohistochemistry for RC complexes I (Figure 1F
and see Figure S1C available online) and II and IV (Figure S1C)
showed high content of these complexes, with only occasional
CI-low Purkinje cells in cerebellum, and were similar to WT in
the cortex even at 46 weeks of age (Figures S1A and S1B). By
western blot, Mutator brains showed a tendency to reduced
amounts of complex I (80% of WT) and COX (72% of WT) (Figure 1H). No signs of gliosis were seen in the Mutator brain
even at the age of 46 weeks, in SVZ, hippocampus, cortex,
and striatum (Figures S1F), and the number of neurons was
similar in Mutators and WT littermates, when counted from
hippocampal regions CA1 and dentate gyrus (Figures S1G
and S1H). Skeletal muscle of Mutator showed less than 1% of
COX-negative fibers, whereas Deletors showed 5% of total
muscle fibers to be COX negative (Figure 1G), and the heart
Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc. 101
Cell Metabolism
mtDNA Mutagenesis Affects Somatic Stem Cells
nuclear-encoded mitochondrial outer membrane protein, porin (Figure 3C). Also the cytochrome c oxidase activity was normal (COX
activity/mitochondrial matrix enzyme, citrate
synthase [CS] activity, WT NSCs [n = 4 lines]
0.66 0.31; Mutator NSCs [n = 3] 0.56 0.06).
We next examined whether mtDNA mutations
affected the self-renewal capacity of NSCs
in vitro. The primary proliferation characteristics
of early passage (<6) NSCs, measured by incorporation of BrdU and by flow cytometry,
showed that mtDNA mutations did not affect
the proliferative activity of NSCs (Figure 3D).
To test the NSCs ability to self-renew, cultures
were diluted to clonal density, and the singlecell ability to produce new neurospheres was monitored. Mutator NSCs formed 3-fold less spheres when compared to WT
NSCs (Figure 3E).
We next studied whether the decline in self-renewal ability was
affected by increasing redox buffer capacity and antioxidant
102 Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
mtDNA Mutagenesis Affects Somatic Stem Cells
Figure 2. Adult Mutator Brains Show COX-Negative Cells and Decreased Levels of Nestin-Positive NSCs in the SVZ, but the Amount of
Proliferative Cells in the SVZ, as well as Olfactory Bulb Interneurons, is Wild-Type Like
(A) Midsagittal view of adult mouse brains showing the areas of neurogenesis in red. Newly formed neural progenitors migrate from SVZ through rostral migratory
stream (RMS) to olfactory bulb (OB), where they give rise to interneurons maintaining the OB function.
(B) The SVZ (arrows) had high numbers of COX-negative cells in Mutators, visualized by COX-SDH activity from cryosections (Scale bar, 10 mm), but not in the
Deletors or WT mice.
(C) Old Mutator brains at 14 months show decreased number of nestin-positive NSCs in SVZ region, whereas in Deletors at 18 months the NSC numbers did not
differ from WT mice.
(D) The relative optical density of the nestin signal was analyzed from SVZ region (6001,200 cells per sample) and normalized against WT sample. Shown as
mean SD, ***p < 0.0001; animals per genotype, n = 2; scale bar, 20 mm.
(E) Mutators show WT-like amounts of CDC47-positive, proliferating cells in the SVZ area. Five hundred to seven hundred cells per sample were counted, CDC47positive cells shown as percentage of total cells, mean SD. Animals per genotype: 14 weeks, n = 2; 25 weeks, n = 3; >40 weeks, n = 2. Shown is a representative
picture of CDC47 immunostaining, proliferating cells seen in brown, from 46-week-old Mutator and WT brains. Scale bar, 20 mm.
(F) The number of olfactory bulb periglomerular interneurons, of which a subset is calbindin positive, was similar to WT in old Mutator brains. Three hundred to four
hundred cells per genotype were counted, calbindin-positive cells are shown as percentage of total cells, mean SD. Analyzed animals: 12 weeks, n = 1; 25 weeks,
n = 2; >40 weeks, n = 2. Shown is a representative picture of calbindin immunostaining from 25-week-old WT and Mutator olfactory bulb (calbindin, red; nuclei, blue).
data show that Mutator NSCs have a WT-like capacity to differentiate to different cell types, but reduced self-renewal capacity,
which was attenuated by NAC supplementation. However, the
growth defect of long-term cultures was not affected by NAC.
These results suggest that the mechanism of NSC self-renewal
defect involves ROS/redox status.
Mutators Show Abnormal Fetal Erythropoiesis
and Disrupted Hematopoietic Progenitor Differentiation
in the Adult Bone Marrow
The Mutator NSC phenotype prompted us to ask whether other
stem/progenitor cell compartments were affected in the developing embryo. Adult Mutator mice have been shown to develop
severe progressive anemia after 6 months of age (Figure 1B)
(Chen et al., 2009; Trifunovic et al., 2004) with progressive
dysfunction of bone marrow HSCs (Chen et al., 2009; Norddahl
et al., 2011). We could replicate the previous findings from adult
bone marrow: despite their severe anemia, the adult Mutator
Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc. 103
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mtDNA Mutagenesis Affects Somatic Stem Cells
Figure 3. Cultured Mutator NSCs Accumulate mtDNA Point Mutations and Show Decreased Self-Renewal Capacity and Growth Defect in
Long-Term Culture, which Can Be Attenuated by NAC Supplementation
(A) Cultured Mutator (n = 3) NSCs from E15.5 embryos showed increase in point mutation load in the cytochrome B gene of mtDNA compared to WT (n = 4) NSCs
(Mutator 13.7 mutations/10 kb; WT 0.2 mutations/10 kb).
(B) Mutator NSCs (n = 3) showed slight reduction of RC complexes I and IV analyzed by western blot.
(C) Mutator NSCs (n = 3) RC protein levels were compared to WT NSCs (n = 4), and the amount of RC subunits was normalized to nuclear-encoded mitochondrial
porin.
(D) Mutator NSCs (n = 4) showed BrdU incorporation similar to that of WT (n = 8) NSCs, indicating unaffected proliferation capability in flow cytometric analysis
when 120,000240,000 cells per genotype were analyzed. Analysis was performed with low-passage NSCs (p < 6).
(E) Mutator NSCs (n = 13) produced significantly less neurospheres than did WT (n = 18; ***p = < 0.0001) or Deletor (n = 6; ***p = 0.0005) NSCs in clonal expansion
analysis, indicating reduced self-renewal capacity. Mutator NSCs with NAC (n = 11) showed improved self-renewal capacity (***p < 0.0001), while the treatment
had no significant effect on the WT NSCs (n = 7; p = 0.321). Altogether 10,50027,000 cells per genotype were analyzed. Self-renewing cells are shown as
a percentage of total cells (mean SD): Mutator, 6.5% 3.4%, WT, 19.9% 7.7%, and Deletor, 16.5% 2.8%; Mutator with NAC, 15.8% 5.3%, and WT with
NAC, 23.3% 6.5%.
(F) Mutator NSCs (n = 3) showed growth restriction in continuous long-term culture compared to WT NSCs (n = 5), and the difference between WT and Mutator
lines became statistically significant by the ninth week of culture.
(G) The growth restriction of Mutator NSCs was partially attenuated by NAC supplementation: Mutator NSCs with NAC (n = 6) grew slightly better compared to
Mutator NSCs but could not reach the same passage levels as WT or WT-NAC (n = 6) lines.
(H) Cultured Mutator NSCs were able to differentiate into both neuronal (Tuj-1, green) and glial (GFAP, red) cells when induced to differentiate with 2% FBS.
Hoechst (blue) was used to stain nuclei.
104 Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
mtDNA Mutagenesis Affects Somatic Stem Cells
increased in the fetal liver at E15.5 (Figure 4C). The proerythroblast population (CD71+) was similar to WT (results shown as
mean SD, E13-E13.5: WT [n = 7] 9.1% 2.6% of total cells;
Mutator [n = 3] 8.1% 0.5%; E15.5: WT [n = 15] 8.4% 2.8%;
WT NAC [n = 12] 7.5% 1.8%, Mutator [n = 7] 8.9% 1.5%;
Mutator NAC [n = 10] 8.0% 0.9%).
We next tested whether we could affect the fetal hematopoietic phenotype with NAC supplementation. Heterozygous
females were fed with NAC throughout the pregnancy, and
hematopoietic cells were extracted from the liver of E15.5
embryos and subjected to FACS analysis immediately after
extraction. The total amount of cells in the fetal liver of Mutators
was restored to WT levels by NAC supplementation (Figure 4A).
Also the proportions of different erythroid progenitor populations
were normalized to WT level in the Mutator embryos treated with
NAC (Figures 4B and 4C). We then analyzed the frequency of
CD11b-positive myeloid and B220-positive B-lymphoid progenitors in fetal liver and found a progressive increase in the amount
of B-lymphoid cells in the Mutators compared to WT littermates
(Figure 4D). However, NAC-treated Mutator E15.5 embryos
showed B220+ cell amounts similar to those of WT embryos (Figure 4D). These results show that, similar to the NCS defect, the
fetal hematopoietic phenotype can be ameliorated by NAC
treatment.
To analyze the function of fetal HPCs, we analyzed their ability
to produce hematopoietic colonies when plated on methylcellulose. Mutator fetal liver HPCs were able to produce mixed myeloerythroid colonies (CFU-GEMM), whereas erythroid (BFU-E)
and granulocyte-macrophage colonies (CFU-GM) were modestly increased in number (Figure S2E). Forty-week-old Mutator
bone marrow formed fewer mixed myeloerythroid, erythroid, and
granulocyte-macrophage colonies than did WT (Figure S2F).
These results show that different lineages of the hematopoietic
Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc. 105
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mtDNA Mutagenesis Affects Somatic Stem Cells
106 Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
mtDNA Mutagenesis Affects Somatic Stem Cells
Mouse Models
All animal experimentation was approved by the Ethical Review Board of
Finland. Mice with a knockin inactivating mutation (D257A) in the exonuclease
domain of DNA polymerase gamma, PolG (Mutator mice [Kujoth et al., 2005;
Trifunovic et al., 2005]), and mice ubiquitously overexpressing mouse Twinkle
EXPERIMENTAL PROCEDURES
Cell Metabolism 15, 100109, January 4, 2012 2012 Elsevier Inc. 107
Cell Metabolism
mtDNA Mutagenesis Affects Somatic Stem Cells
Brain Immunohistochemistry
Brains of Mutator and WT mice aged 12, 14, 25, 40, and 46 weeks and Deletors
of 72 weeks were collected and standard hematoxylin and eosin (TissueTek)
protocol was used to determine the morphology from paraffin sections. RC
complex subunits were visualized using rabbit anti-CI ND-1 (1:500), mouse
anti-CI NDUFS3 (1:100), mouse anti-CII 70 kDa subunit (1:200), and mouse
anti-CIV II (1:20). Mouse anti-CDC47 (1:200), rabbit anti-cleaved caspase 3
(1:100), mouse anti-nestin (1:200), and mouse anti-myelin basic protein
(1:200) were used to visualize proliferating cells, apoptotic cells, neural progenitors, and oligodendrocytes, respectively, together with the biotinylated
secondary antibodies from Vector ABC Elite kit (anti-mouse and anti-rabbit,
Vector), and developed using DAB Fast Kit (Sigma-Aldrich). Hematoxylin
(Tissue Tek) was used as a counterstain. COX-SDH immunohistological analysis was performed as previously described (Tyynismaa et al., 2005). Rabbit
anti-GFAP (1:400) and rabbit anti-calbindin (1:200) were used to visualize
astrocytes and subset of the periglomerular neurons of the olfactory bulb,
respectively, together with Alexa 594 chicken anti-rabbit secondary antibody.
Hoechst was used to stain nuclei. IgG from the primary antibody hosts was
used as a negative control for the staining. Analysis of optical density
(immunoperoxidase staining) or signal intensity (fluorescence staining) was
performed using ImageJ software.
(for K.J.A.). We thank Hanna Mikkola for advice and antibodies for HPC
FACS experiments and for very helpful discussions about hematopoietic
data. The authors are grateful for Anu Harju, Ilse Paetau, Tuula Manninen,
Markus Innila, Minna Teerijoki, Ivana Bratic, Jarmo Palm, Susanna Lauttia,
and Katja Piltti for advice and help in experimental procedures. Mika Hukkanen
is thanked for help in image analysis.
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Cell Metabolism
Article
Glucose-Independent Glutamine
Metabolism via TCA Cycling
for Proliferation and Survival in B Cells
Anne Le,1,* Andrew N. Lane,6,8,* Max Hamaker,2 Sminu Bose,1 Arvin Gouw,3 Joseph Barbi,4 Takashi Tsukamoto,5
Camilio J. Rojas,5 Barbara S. Slusher,5 Haixia Zhang,9 Lisa J. Zimmerman,9 Daniel C. Liebler,9 Robbert J.C. Slebos,9
Pawel K. Lorkiewicz,6 Richard M. Higashi,6,7 Teresa W.M. Fan,6,7,8,* and Chi V. Dang10,*
1Division
SUMMARY
110 Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
Glucose-Independent Glutamine-Dependent TCA Cycle
labeled glutamine, suggesting the existence of a glucose-independent TCA cycle. We also found under glucose-depleted
culture conditions that a glutamine-dependent and glucoseindependent TCA cycle may operate under both aerobic and
hypoxic conditions. Moreover, we observed an enhanced conversion of glutamine to glutathione under hypoxia; glutathione
is an important reducing agent for controlling the accumulation
of mitochondrial reduced oxygen species (ROS). Under moderate hypoxia, excess ROS is generated at complex II owing to
a mismatch between NADH production and terminal oxidase
activity (Wu et al., 2007). We therefore tested whether inhibition
of glutamine metabolism could induce oxidative stress under
hypoxia. We found that inhibition of glutaminase (GLS) by the
glutaminase-selective inhibitor BPTES (Robinson et al., 2007)
elevated ROS levels and diminished ATP levels in hypoxic cells.
In fact, we found that inhibition of glutaminase effectively kills
hypoxic cancer cells in vitro and delays tumor xenograft growth
in vivo.
RESULTS
Coexistence of Oxidative and Aerobic Glycolysis
Our genomic analysis of MYC target genes indicates that
the expression of genes involved in glycolysis and in mitochondrial respiration is coregulated by MYC (Dang, 2010; Kim
et al., 2007, 2008; Li et al., 2005). We thus determined the
metabolic consequences of MYC activation in a model cell line
(P493) of human Burkitt lymphoma grown in uniformly labeled
[U-13C]-Glc under aerobic (21% O2) or hypoxic (1% O2) conditions. Although the levels of metabolites at steady state are
the result of the balance between production and consumption,
the use of SIRM enabled us to determine not only steady levels of
metabolites but also the isotopomer and isotopologue distributions of metabolites derived from 13C-labeled glucose for reconstruction of metabolic pathways. Time course isotopomer data
on extracellular metabolites further provided flux measurement
for substrate import and product release (Figure 1A, see Figures S2C and S3B and Table S1 available online). P493 cells
contain a tetracycline-repressible MYC construct, such that
tetracycline withdrawal results in rapid induction of MYC and
tetracycline treatment results in MYC suppression (Figure S1A).
Induction of MYC resulted in an increase of 13C-glucose consumption and 13C-lactate production, which were further accentuated by hypoxia (Figure 1A). NMR analysis of 13C-labeled
metabolites derived from [U-13C]-Glc in cell extracts also corroborated the finding that overexpressed MYC resulted in lactic
fermentation even under aerobic conditions (Figure 1B). The
same cell extracts were further analyzed by GC-MS to quantify
glucose-derived 13C isotopologues of lactate (lactate with different number of 13C atoms) (Figure 1D). Levels of the m+3 isotopologue of lactate (triply 13C-labeled lactate or 13C3-lactate)
derived from glucose shown with MYC ON or OFF in aerobic
(A) or hypoxic (H) conditions (Figure 1D) also support the ability
of MYC to increase aerobic glycolysis (Figure 1C). Under hypoxic
conditions, glucose-derived lactate was increased but was
less dependent on MYC. The production of lactate (Table S1)
accounts for only part of the glucose consumed. Although
a complete carbon inventory has not been achieved, we estimate that a significant fraction of the glucose enters new
Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc. 111
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Glucose-Independent Glutamine-Dependent TCA Cycle
13
which reflects exchange of intracellular glutamine-derived glutamate for other amino acids such as cystine (see below).
As depicted in Figure 3A, labeled glutamine catabolism by
glutaminase led to the production of 13C5-aKG, which can enter
the TCA cycle for further oxidation. As shown in Figure 4, the synthesis of 13C4-succinate, -fumarate, and -malate (m+4) is consistent with the oxidation of 13C5-aKG via the forward reactions of
the TCA cycle (red circles, Figure S2D). These labeled TCA intermediates all responded to MYC status by increasing 60%
to >100% when MYC was ON, regardless of O2 availability (Figure 4). The levels of 13C4-citrate, which is synthesized in the
second turn from 13C4-oxaloacetate (OAA, derived from labeled
glutamine in the first turn) and acetyl-CoA (from unlabeled glucose or other unlabeled sources) by citrate synthase (CS), also
responded to MYC expression under both aerobic and hypoxic
conditions (Figure 4). These results show that MYC can drive
glutamine metabolism around the TCA cycle even under hypoxia.
112 Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc.
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Glucose-Independent Glutamine-Dependent TCA Cycle
Figure 2. Glucose Entry into the TCA Cycle Is Induced by MYC and Suppressed by Hypoxia
The cycle reactions are depicted without or with pyruvate carboxylation (green arrow), and the 13C isotopomer patterns are the result of one cycle turn. The
isotopologue distributions were determined by GC-MS. The incorporation of 13C atoms from 13C6-Glc into citrate, a-ketoglutarate (a-KG), succinate, fumarate,
and malate are denoted as m+n, where n is the number of 13C atoms. The m+5 (13C5)-citrate (green circle) is produced by condensation of m+2 acetyl-CoA with
m+3 OAA (from pyruvate carboxylation), while m+2 (13C2)-citrate is synthesized without input of pyruvate carboxylation. Red and green circles, 13C atoms derived
from 13C6-Glc without or with pyruvate carboxylation, respectively. GPT2, glutamate-pyruvate transaminase; PC, pyruvate carboxylase; CO2 indicates where
carbon dioxide is released. A, aerobic; H, hypoxic. The error bars represent SEM.
Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc. 113
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Glucose-Independent Glutamine-Dependent TCA Cycle
B
D
114 Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc.
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doubling every 34 2 hr in the presence of glucose (Figure S3A). The cells consumed glutamine (Figure S3B) to produce
13
C5-aKG (m+5) in a MYC-dependent fashion (circled red, Figure 5). The continued functioning of the TCA cycle under
glucose-deprived conditions was identified by the production
of various isotopologues of fumarate, malate, and aspartate,
particularly the 13C4-isotopologues (m+4, Figure 5). However,
these labeled isotopologues accumulated to much higher levels
(>100-fold for 13C4-Asp) than under glucose-replete conditions
(compare Figures 4 and 5 or Figure 1B and Figure S3C). This
could result from a lower supply of acetyl-CoA under glucosedeprived conditions such that excess glutamine-derived OAA
was transaminated to form aspartate. This is also consistent
with the lower levels of labeled citrate and aKG isotopologues
in glucose-deprived cells than those found in glucose-replete
cells (Figures 4 and 5). In the absence of glucose, it is also
notable that 13C incorporation from the glutamine tracer into all
TCA cycle intermediates decreased under hypoxia but increased
with MYC OFF (Figure 5). Furthermore, the 13C alanine isotopologues (e.g., 13C3-Ala or m+3 in Figure 5) showed the opposite
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116 Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc.
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Figure 7. Effects of a Glutaminase Inhibitor on Neoplastic Cells In Vitro and in a Tumor Xenograft Model In Vivo
(A) Effect of glutaminase inhibitor, BPTES, on aerobic and hypoxic P493 cell growth. Growth of control cells compared with cells treated with BPTES under both
aerobic and hypoxic conditions. All cells were grown at 1 3 105 cells/mL. Cell counts were performed in triplicate and shown as mean SD. Live cells were
counted daily in a hemocytometer using trypan blue dye exclusion.
(B) Effect of BPTES on steady-state ATP levels. P493 cells were treated with 2 mM BPTES for 20 hr and counted. ATP levels (mean SD, n = 3 experiments) were
determined by luciferin-luciferase-based assay on aliquots containing an equal number of live cells. *p = 0.0006; **p = 0.01 (t test).
(C) Effects of hypoxia and glucose deprivation on oxygen consumption rates. Oxygen consumption rates of aerobic (A) or hypoxic (H) P493 cells were determined
by a Clark-type oxygen electrode after culture in the presence or absence of glucose for 24 hr. Data are from duplicate experiments with SD and p values (t test)
shown.
(D) In vivo efficacy of BPTES. 2.0 3 107 P493 human lymphoma B cells were injected subcutaneously into tumor-bearing SCID mice. When the tumor volume
reached 100 mm3, 200 mg BPTES was injected every other day by intraperitoneal (i.p.) administration (12.5 mg/kg bodyweight) for 20 days. Control animals were
treated with daily i.p. injection of vehicle (2% [vol/vol] DMSO). BPTES inhibited lymphoma xenograft growth as compared to control. The tumor volumes were
measured using digital calipers every 4 days and calculated using the following formula: (length [mm] 3 width [mm] 3 width [mm] 3 0.52). The results represent
the average SEM (n = 7 each).
118 Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc.
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revealed BPTES-induced metabolic changes, such as diminished glutamate, a-ketoglutarate, succinate, fumarate, and
malate levels, consistent with an on-target effect on glutaminase
(Seltzer et al., 2010). Further, the uncompetitive nature of BPTES
inhibitory activity was recently underscored by the crystal structure of glutaminase (GAC form) cocrystallized with BPTES, which
sits at the oligomerization interface of the glutaminase tetramer
(Delabarre et al., 2011). Our finding of a glutamine-driven TCA
cycle and other recent discoveries of cancer-related metabolic
pathways (Dang et al., 2009b; Frezza et al., 2011; Possemato
et al., 2011) suggest that metabolic flexibility may be a common
feature of tumor cell metabolism. Hence, a broader and deeper
understanding of cancer cell metabolism and their ability to
reprogram canonical biochemical pathways under metabolic
stress can be a rich ground for uncovering strategies for therapeutic targeting of tumors.
(to T.W.M.F.) and 20061 (to A.N.L.); and National Institutes of Health National
Center for Research Resources (NIH NCRR) grant 5P20RR018733, Kentucky
Challenge for Excellence, the Brown Foundation, and Kentucky Lung Cancer
Research Program (postdoctoral fellowship to P.K.L.). The FT-ICR-MS instrumentation was supported by the National Science Foundations Office of
Experimental Program to Stimulate Competitive Research (NSF/EPSCoR)
grant number EPS-0447479. We thank Ramani Dinavahi, Julie Tan, and
Radhika Burra for excellent technical assistance and Stephen Rayport for the
heterozygous glutaminase mice. A.L., A.N.L., T.W.M.F., and C.V.D. designed
research and wrote the manuscript. A.L., A.N.L., M.H., S.B., J.B., C.J.R.,
H.Z., L.J.Z., R.J.C.S., P.K.L., R.M.H., and T.W.M.F. performed research; all
authors interpreted the data and commented on the manuscript.
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Cell Metabolism 15, 110121, January 4, 2012 2012 Elsevier Inc. 121
Cell Metabolism
Short Article
Coordination of Triacylglycerol
and Cholesterol Homeostasis by DHR96
and the Drosophila LipA Homolog magro
Matthew H. Sieber1,2 and Carl S. Thummel1,*
1Department
of Human Genetics, University of Utah School of Medicine, 15 N 2030 E, Room 2100, Salt Lake City, UT 84112-5330 USA
address: Department of Embryology, Carnegie Institution, 3520 San Martin Dr., Baltimore, MD 21218 USA
*Correspondence: carl.thummel@genetics.utah.edu
DOI 10.1016/j.cmet.2011.11.011
2Present
SUMMARY
INTRODUCTION
Coordinate regulation of lipid metabolism is central to human
health, and disruption of this process leads to a range of metabolic disorders, including obesity and cardiovascular disease.
Normal lipid homeostasis is maintained by balancing dietary lipid
uptake and synthesis with lipid catabolism and excretion. Under
normal feeding conditions, dietary lipids, such as triacylglycerol
(TAG) and cholesterol esters, are broken down into free fatty
acids, monoacylglycerols, and free sterols in the lumen of the
intestine. These digested lipids can then be absorbed by the
intestinal cells, where TAG is resynthesized and packaged
together with cholesterol, cholesterol esters, and carrier proteins
to form lipoprotein particles that are trafficked throughout the
body. These lipids can be either utilized by cells or deposited
in storage tissues, such as the adipose and liver. Under conditions of excess lipids, TAG and cholesterol esters are broken
down and free fatty acids can be utilized for energy, whereas
excess cholesterol is excreted from the body (Lusis and Pajukanta, 2008; van der Velde et al., 2010).
Nuclear receptors (NRs) are ligand-regulated transcription
factors that play essential roles in multiple aspects of lipid
homeostasis. Many NRs bind small lipophilic compounds,
such as fatty acids, sterols, and other metabolic intermediates,
and coordinate multiple aspects of metabolism by directing
specific changes in gene expression. One example of this is
LXRa (NR1H3), which binds oxysterols and promotes the modification and clearance of excess sterols (Kalaany and Mangelsdorf, 2006). In addition, LXRa is required to maintain proper
TAG levels, at least in part through the regulation of SREBPmediated fat synthesis (Schultz et al., 2000). Thus, LXRa
activity is central to both TAG and cholesterol homeostasis,
although much remains to be learned about the roles of specific LXR target genes in mediating these key metabolic
functions.
We have been studying a Drosophila homolog of LXR, DHR96,
as a simple system to understand the physiological and molecular roles for this family of NRs and their target genes. Biochemical and genetic studies of DHR96 have shown that it
shares the central metabolic functions of its mammalian counterpart. DHR96 binds cholesterol and is required for normal cholesterol homeostasis, with DHR96 null mutants exhibiting an 20%
increase in whole animal cholesterol levels due, at least in part, to
increased npc1b expression (Horner et al., 2009; Bujold et al.,
2010). In addition, DHR96 mutants display an 50% decrease
in whole animal TAG levels that can be attributed to an inability
to break down dietary TAG due to reduced expression of the
intestinal lipase Magro (CG5932) (Sieber and Thummel, 2009).
Interestingly, magro transcription responds to dietary cholesterol levels and this regulation is dependent on DHR96 function,
providing a potential link between cholesterol levels, DHR96,
and TAG homeostasis (Horner et al., 2009; Bujold et al., 2010).
Moreover, although Magro protein is most similar to mammalian
gastric lipase (38% identity, 56% similarity), the second most
similar protein is LipA (32% identity, 50% similarity), which has
both TAG lipase and cholesterol esterase activities (Ameis
et al., 1994). Genetic studies have demonstrated a central role
for LipA in maintaining proper cholesterol levels in mice (Du
et al., 2001). Similar phenotypes are seen in human LipA mutants
suffering from cholesterol ester storage disease (CESD) and
Wolmans disease (Burke and Schubert, 1972). These observations raise the possibility that magro, in addition to controlling
TAG homeostasis, may regulate cholesterol homeostasis and
122 Cell Metabolism 15, 122127, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
Regulation of Lipid Homeostasis
140
120
120
CE activity
100
80
80
60
40
40
magro RNAi
66
5
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00
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% cholesterol
A 160
GST
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g sterol
CE activity
88
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magro RNAi
6
6
4
4
2
2
22
00
00
55
10
15
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ng protein
20
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free
chol
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total
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esters
Cell Metabolism 15, 122127, January 4, 2012 2012 Elsevier Inc. 123
Cell Metabolism
Regulation of Lipid Homeostasis
130 m
Prov
24 m
Lysotracker
magro-GFP
magro-GFP
75 m
Overlay
Lysotracker
20 m
Overlay
124 Cell Metabolism 15, 122127, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
Regulation of Lipid Homeostasis
%total TAG
120
80
40
0
+
WT
120
80
40
0
Panc
DHR961
+
Act-GAL4/+
+
Act>mag
RNAi
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D
160
% cholesterol
120
160
% cholesterol
%total TAG
120
disease (Burke and Schubert, 1972). Patients with Wolmans disease also have digestive dysfunction, which
40
40
may be related to the defects in lipid uptake that we
0
0
observe in magro RNAi animals. In addition to these
+
+ Panc
+
+ Panc
shared phenotypes, however, mammalian LipA mutants
DHR961
Act-GAL4/+
Act>mag
WT
RNAi
display massive accumulations of lipid in the liver, spleen,
and intestinedefects that are not apparent in magro
RNAi animals (Du et al., 2001). Nonetheless, the parallels
then restoring magro function in these animals should rescue between Magro and LipA function in flies and humans establish
their defect in cholesterol homeostasis. Consistent with this Drosophila as a system to further our understanding of CESD
hypothesis, expressing wild-type magro specifically in the intes- and Wolmans disease and define the ancestral function for
tine of the DHR96 mutant is sufficient to reduce the cholesterol this class of acid lipases, demonstrating their central role in the
levels in these animals (Figure 4D). This rescue, however, is not intestine to coordinate TAG and cholesterol homeostasis.
Interestingly, the dual enzymatic functions of Magro appear to
complete, which is consistent with the multiple levels of cholesterol metabolism that are regulated by DHR96 (Horner et al., arise from distinct regions of the intestine. Disruption of magro
2009; Bujold et al., 2010). Moreover, specific expression of function specifically in the proventriculus blocks its TAG lipolytic
magro in the proventriculus of DHR96 mutants effectively activity but does not affect cholesterol levels in these animals
rescues their lean phenotype (Figure 4E) but has no significant (Figures 4B and 4C). In contrast, magro RNAi throughout the
effect on their elevated cholesterol levels (Figure 4F). Taken intestine affects both TAG and cholesterol homeostasis (Figtogether with our other data, these results indicate that the ure 1D). These region-specific activities are consistent with our
region-specific enzymatic activities of Magro correspond to the dietary supplementation studies with pancreatin and Orlistat
major lipid metabolic functions of DHR96. DHR96 regulation of (Figures 3 and S4). They are also consistent with the expression
magro expression in the proventriculus maintains an appropriate pattern of Magro-EGFP protein, providing insights into how the
level of TAG lipase activity in the intestinal lumen to facilitate the dual functions of this enzyme are manifested. Magro-EGFP is
breakdown of dietary lipid whereas DHR96 regulation of magro expressed abundantly in the large outer columnar cells in the
in the body of the intestine promotes the clearance of excess anterior half of the proventriculus (Figures 2A and S2). We also
see a stream of large acidic vesicles that originate from this
sterols.
region and move in a posterior direction toward the lumen of
the intestine (Figures 2F2H and S3). This apparent vesicular
DISCUSSION
trafficking of Magro is consistent with the cells at the anterior
end of the proventriculus having secretory functions, depositing
Magro and LipA Share Conserved Functions
peritrophic matrix components into the lumen that lies between
in Maintaining Lipid Homeostasis
Relatively little is known about the mechanisms that regulate the outer and inner cell layers of the proventriculus (King, 1988)
cholesterol metabolism in Drosophila. Most studies have (Figure S2). The peritrophic matrix is a meshwork of chitin and
focused on DHR96 and the Niemann-Pick (NPC) disease gene glycoproteins that provides a protective lining within the gut,
homologs, which play important roles in dietary cholesterol much like the mucosal layer of the mammalian intestine (Hegeabsorption and intracellular cholesterol trafficking (Niwa and dus et al., 2009). The observation that the Magro-EGFP vesicles
Niwa, 2011). In this paper, we identify the intestine as a key tissue reside in the same region of the proventriculus as the developing
for maintaining cholesterol homeostasis, and we define a central peritrophic matrix suggests that they are synthesized and exrole for the LipA homolog, Magro, in mediating this function. Like ported into the lumen of the gut in a similar manner. This also raiLipA, Magro has dual enzymatic activities, cleaving both TAG ses the possibility that the digestive enzymes that are embedded
and cholesterol esters, consistent with their common fatty acid in the peritrophic matrix may originate from vesicular trafficking
ester bonds (Ameis et al., 1994). Mouse LipA mutants display in the proventriculus. Like magro, many genes with predicted
a lack of stored fat in the form of white adipose tissue along digestive functions, including glucosidases, mannosidases, and
with excess cholesterol esters (Du et al., 2001), reflecting the endopeptidases, are regulated by DHR96 and expressed in the
major defects in magro RNAi animals. Similar phenotypes are intestine (Sieber and Thummel, 2009). Several genes that conseen in human LipA mutants suffering from CESD and Wolmans tribute to the peritrophic matrix are also regulated by DHR96.
80
80
Cell Metabolism 15, 122127, January 4, 2012 2012 Elsevier Inc. 125
Cell Metabolism
Regulation of Lipid Homeostasis
B120120
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100100
80 80
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126 Cell Metabolism 15, 122127, January 4, 2012 2012 Elsevier Inc.
Cell Metabolism
Regulation of Lipid Homeostasis
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Cell Metabolism
Erratum
Enteric Neurons and Systemic Signals
Couple Nutritional and Reproductive Status
with Intestinal Homeostasis
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1Department
128 Cell Metabolism 15, 128, January 4, 2012 2012 Elsevier Inc.