doi: 10.1152/ajpendo.00100.

2010
298:E1261-E1273, 2010. First published 30 March 2010; Am J Physiol Endocrinol Metab
Isabelle Leclerc and Guy A. Rutter
Gao Sun, Andrei I. Tarasov, James A. McGinty, Paul M. French, Angela McDonald,
secretion in vivo
-cell morphology and enhances insulin pancreatic
transgene modifies RIP2.Cre LKB1 deletion with the
You might find this additional info useful...
for this article can be found at: Supplementary material
00.2010.DC1.html
http://ajpendo.physiology.org/http://ajpendo.physiology.org/content/suppl/2010/05/10/ajpendo.001
47 articles, 17 of which you can access for free at: This article cites
http://ajpendo.physiology.org/content/298/6/E1261.full#ref-list-1
7 other HighWire-hosted articles: This article has been cited by
http://ajpendo.physiology.org/content/298/6/E1261#cited-by
including high resolution figures, can be found at: Updated information and services
http://ajpendo.physiology.org/content/298/6/E1261.full
can be found at: Metabolism
American Journal of Physiology - Endocrinology and about Additional material and information
http://www.the-aps.org/publications/ajpendo
This information is current as of August 16, 2012.
Physiological Society. ISSN: 0193-1849, ESSN: 1522-1555. Visit our website at http://www.the-aps.org/.
American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2010 the American
endocrine and metabolic systems on any level of organization. It is published 12 times a year (monthly) by the
publishes results of original studies about American Journal of Physiology - Endocrinology and Metabolism

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

LKB1 deletion with the RIP2.Cre transgene modies pancreatic -cell
morphology and enhances insulin secretion in vivo
Gao Sun,
1
Andrei I. Tarasov,
1
James A. McGinty,
2
Paul M. French,
2
Angela McDonald,
1
Isabelle Leclerc,
1
and Guy A. Rutter
1
1
Section of Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, and
2
Photonics
Group, Department of Physics, Imperial College London, London, United Kingdom
Submitted 11 February 2010; accepted in nal form 29 March 2010
Sun G, Tarasov AI, McGinty JA, French PM, McDonald A,
Leclerc I, Rutter GA. LKB1 deletion with the RIP2.Cre transgene
modies pancreatic -cell morphology and enhances insulin secretion
in vivo. Am J Physiol Endocrinol Metab 298: E1261E1273, 2010.
First published March 30, 2010; doi:10.1152/ajpendo.00100.2010.
The tumor suppressor liver kinase B1 (LKB1), also called STK11, is
a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphor-
ylates AMP-activated protein kinase (AMPK) and several related
protein kinases. Whereas deletion of both catalytic isoforms of AMPK
from the pancreatic -cell and hypothalamic neurons using the rat
insulin promoter (RIP2).Cre transgene (AMPKdKO) diminishes
insulin secretion in vivo, deletion of LKB1 in the -cell with an
inducible Pdx-1.CreER transgene enhances insulin secretion in mice.
To determine whether the differences between these models reect
genuinely distinct roles for the two kinases in the -cell or simply
differences in the timing and site(s) of deletion, we have therefore
created mice deleted for LKB1 with the RIP2.Cre transgene. In
marked contrast to AMPKdKO mice, LKB1KO mice showed
diminished food intake and weight gain, enhanced insulin secretion,
unchanged insulin sensitivity, and improved glucose tolerance. In line
with the phenotype of Pdx1-CreER mice, total -cell mass and the
size of individual islets and -cells were increased and islet architec-
ture was markedly altered in LKB1KO islets. Signaling by mam-
malian target of rapamycin (mTOR) to eIF4-binding protein-1 and
ribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-
mediated deletion, the expression of Glut2, glucose-induced changes
in membrane potential and intracellular Ca
2
were sharply reduced in
LKB1KO mouse islets and the stimulation of insulin secretion was
modestly inhibited. We conclude that LKB1 and AMPK play distinct
roles in the control of insulin secretion and that the timing of LKB1
deletion, and/or its loss from extrapancreatic sites, inuences the nal
impact on -cell function.
AMP-activated protein kinase; -cell; insulin secretion; food intake;
liver kinase B1; pancreas
LIVER KINASE B1 (LKB1, also called STK11) is a potent tumor
suppressor whose inactivation in Peutz-Jeghers syndrome
(PJS) (21) is characterised by melanotic macules, hamartoma-
tous polyps in the gastrointestinal tract, and increased risk of
all cancers (7). LKB1 is a partial mammalian homolog of the
Saccharomyces cerevisae kinases Elm1, Pak1, and Tos3,
which phosphorylate yeast snf1 (47), the yeast homolog of
mammalian AMP-activated protein kinase (AMPK) (47). Ac-
tivation of AMPK, a target of several glucose-lowering agents
used in diabetes treatment, including metformin and the thia-
zolidenediones (31), stimulates insulin action in peripheral
tissues, acting to phosphorylate and stimulate the TSC1:TSC2
complex, which subsequently inactivates mammalian target of
rapamycin/regulatory associated protein of mTOR (mTOR/
Raptor) (3). AMPK is also implicated in the control of -cell
survival (27, 39, 40) and insulin secretion (11, 12, 42). In the
mediobasal hypothalamus, changes in AMPK activity in pro-
opiomelanocortin (POMC)-, agouti-related peptide (AgRP)-,
and neuropeptide Y (NPY)-expressing neurons are also impli-
cated in the control of feeding and body weight (9, 33). Recent
data (4) have suggested that calmodulin-dependent protein
kinase kinase- (CaMKK), another upstream kinase for
AMPK (20), is involved in these cells.
Compelling evidence for a conserved role for an LKB1-
AMPK signaling cassette comes from studies in Caenorhab-
ditis. elegans, where inactivation of the corresponding ho-
mologs leads to reversal of the dauer phenotype (36), charac-
terized by metabolic inhibition and growth arrest. LKB1 and
AMPK are also implicated in the control of cell polarity. Thus,
deletion of the LKB1 homologues in Drosophila melanogaster
(dLKB1) and C. elegans (par4) disrupts epithelial cell polarity
(26, 32), and in Drosophila this change is rescued by trans-
genic overexpression of AMPK (48). Although forced over-
expression of LKB1 induces cell polarization in intestinal
epithelial cancer cell lines (5), the requirement for LKB1 in
maintaining the polarity of mammalian cells is less clear (44).
However, there is also growing evidence that the effects of
LKB1 in mammalian cells may be, at least in part, independent
of AMPK, since LKB1 phosphorylates 11 further kinases of
the AMPK subfamily in vitro (30, 41).
We (46) have recently demonstrated that deletion of both
catalytic isoforms of AMPK from the pancreatic -cell by use of
the rat insulin promoter (RIP2).Cre transgene (AMPKdKO
mouse) leads to impaired glucose tolerance and defective insulin
secretion in vivo but enhanced glucose-stimulated insulin secre-
tion from isolated islets. However, and in marked contrast, Fu et
al. (16) and Granot et al. (18) have demonstrated that deletion of
LKB1 from the pancreatic -cell (and probably intestinal incretin-
producing cells) from adult mice by means of an inducible
Pdx1-CreER transgene leads to increased insulin production and
enhanced -cell size. To determine whether these markedly di-
vergent phenotypes may reect true biological differences in the
roles of LKB1 and AMPK in the pancreatic -cell rather than
being the result of differences in the timing and sites of deletion,
using the RIP2.Cre transgene, we have therefore generated mice
in which LKB1 is deleted in the -cell.
Inactivation of LKB1 in -cells with this strategy led to
marked increases in -cell size and insulin production in vivo,
consistent with the ndings using Pdx1-CreER-mediated dele-
tion (16, 18). However, and in contrast to the latter model,
Address for reprint requests and other correspondence: G. A. Rutter, Section
of Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Dept.
of Medicine, Imperial College London, London, UK (e-mail: g.rutter@imperial.
ac.uk).
Am J Physiol Endocrinol Metab 298: E1261E1273, 2010.
First published March 30, 2010; doi:10.1152/ajpendo.00100.2010.
0193-1849/10 Copyright 2010 the American Physiological Society http://www.ajpendo.org E1261

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

-cell glucose signaling and insulin secretion were inhibited in
vitro. Importantly, the above alterations in LKB1 mice con-
trast sharply with the effects of deleting both AMPK subunits
using the same RIP2.Cre transgene (46), wherein unaltered
-cell mass and a decrease in mean -cell size are observed. In
addition, we observed a decrease in body weight and improved
glycemia but unaltered insulin sensitivity, suggestive of a role
for LKB1 in RIP2.Cre neurons to control satiety distinct from
that of AMPK. Overall, the present ndings indicate that LKB1
and AMPK control distinct signaling pathways in the -cell to
regulate insulin production. The results also support the view
that inhibition of LKB1, or its downstream targets, may be a
useful approach to increase -cell mass in some forms of
insulin-secretory insufciency, including type 2 diabetes, and
that these changes are likely to be mediated by member(s) of
the AMPK superfamily distinct from AMPK.
METHODS
Generation of Mutant Mice Selectively Lacking LKB1 in Pancreatic
-Cells and RIP2.Cre Neurons
Mice homozygous for oxd alleles of the lkb1/stk11 gene (Mouse
Models of Human Cancer Consortium, http://mouse.ncifcrf.gov/)
were rst crossed with heterozygous RIP2.Cre-expressing transgenic
mice (expressing Cre recombinase under the rat insulin 2 promoter;
Jackson Laboratory). The resulting double heterozygous LKB1
/
,Cre

were intercrossed with LKB1

/
mice to generate LKB1KO (LKB1
/
,
Cre

), LKB1het (LKB1
/
,Cre

), and LKB1Wt (LKB

/
,
Cre

). LKB1KO, LKB1het, and LKB1Wt mice were born at

the expected Mendelian ratios and kept on a mixed FVB/129S6 and
C57BL/6 background.
Mouse Maintenance and Diet
Mice were housed with 25 animals per cage in a pathogen-free
facility on a 12:12-h light-dark cycle. Mice were fed ad libitum with
a standard mouse chow diet or a high-fat diet [60% (wt/wt) fat
content; Research Diet, New Brunswick, NJ]. Where indicated, 4-wk-
old mice were transferred onto high-fat diet for a period of 6 wk. All
in vivo procedures stated were performed in the Imperial College
Central Biomedical Service (CBS) and approved by the UK Home
Ofce according to the Animals Scientic Procedures, Act of 1986.
Body Weight and Food Intake
Fed or 15-h overnight-fasted mice were weighed at the age of 68
wk. Food intake was measured daily at fed status either for 3
consecutive days or at 30 min and 1 h after refeeding the mice fasted
for 15 h using a metabolic cage.
In Vivo Physiological Studies
Intraperitoneal glucose tolerance test. Mice fasted for 15 h (water
allowed) were intraperitoneally injected with 1 g glucose/kg mouse
wt. Blood from the tail vein was obtained at 0, 15, 30, 60, 90, and 120
min after injection. Blood glucose levels were measured with an
automatic glucometer (Accuchek; Roche, Burgess Hill, UK).
Plasma insulin measurement. Mice fasted for 15 h were intraperi-
toneally injected with 3 g glucose/kg mouse wt. Blood from mices
tail veins was collected into a heparin-coated tube (Sarstedt, Beau-
mont Leys, UK) at 0, 15, and 30 min after injection. Plasma was
separated by centrifuging the blood at 2,000 g for 5 min. Plasma
insulin levels were measured using an ultrasensitive mouse insulin
ELISA kit (Mercodia, Uppsala, Sweden). Normal fed plasma insulin
levels were measured from blood collected from 6- to 8-wk-old
mices tail veins between 10:00 and 11:00 AM.
Insulin tolerance tests. Bovine insulin (Sigma, Dorset, UK; 0.75
U/kg) was intraperitoneally injected into fed mice. Blood glucose
levels were measured at 0, 15, 30, and 60 min after injection as above.
Islet Isolation and In Vitro Insulin Secretion Measurement
Islet isolation by in situ collagenase digestion, and in vitro insulin
secretion measurement, were performed as previously described (28).
RNA Extraction and RT-PCR
Total cellular RNA from mouse islets or other tissues was obtained
using TRIzol reagent (Invitrogen, Paisley, UK), and RNA was further
puried against DNA contamination with a DNA-free kit (Applied
biosystems, Warrington, UK). Total RNA (1.52 g) was then re-
verse transcribed into cDNA with a high-capacity reverse transcrip-
tion kit (Applied Biosystems) according to the manufacturers instruc-
tions. To detect deletion of LKB1 exons 3 to 6, two pairs of primers
within exon 1 (LKB fwd: AGGTGAAGGAGGTGCTGG) and 8
(LKB rev: TCTGGGCTTGGTGGGATA) were designed (Fig. 1A).
The PCR reaction was preheated at 95C for 2 min, and amplication
was performed for 30 cycles under the following conditions: 94C for
30 s, 57C for 30 s, and 72C for 1 min. At the end of the last cycle,
a prolonged extension step was carried out at 72C for 10 min.
qRT-PCR
The expression levels of lkb1/stk11, kir6.2, sur1, glut2, ki67,
insulin (ins1 and ins2), glucagon, gck, NPY, POMC, and AgRP
genes were quantitated by real-time PCR using the SYBR Green
method (11).
Immunoprecipitation, AMPK Activity Measurement, and Western
(Immuno-) Blot Analysis
To determine total AMPK activity in islet extracts, 10 g of total
protein from 150200 islets isolated from fed mice was incubated in
RPMI supplemented with 11 mMglucose for 35 days prior to AMPKassay
with synthetic SAMS peptide (HMRSAMSGLHLVKRR) as substrate
(28). To determine AMPK1 and -2 activities in the hypothalamus,
dissected tissue was placed in liquid nitrogen immediately after
extraction and lysed in 800 l of ice-cold lysis buffer [in mM: 50
TrisHCl (pH 7.4, 4C), 250 sucrose, 50 NaF, 1 Na pyrophosphate, 1
EDTA, 1 EGTA, 1 DTT, 0.1 benzamidine, and 0.1 PMSF, 5 g/ml
soybean trypsin inhibitor, and 1% (vol/vol) Triton X-100] [with 10%
(wt/vol) sucrose in lysis buffer for hypothalamus]. Total extracts (100
g of protein) were used for immunoprecipitating with anti-
AMPK1/2 antibodies (Upstate, Dundee) conjugated to protein
G-Sepharose and were subjected to AMPK activity measurement as
above. For Western blot analysis, 50 g of protein from 300400
islets was used.
Immunohistochemistry and Analysis of Islet Architecture
Isolated pancreata were xed in 10% (vol/vol) formalin and em-
bedded in parafn wax within 24 h of removal. Head-to-tail sections
(5 m lengthwise) were cut and incubated at 37C overnight on
superfrost slides. Slides were submerged sequentially in 100% (vol/
vol) xylene followed by decreasing concentrations of industrial meth-
ylated spirits for removal of parafn wax. Antigen epitopes were then
retrieved (de-cross-linked) in Tris-EDTA-0.05% (vol/vol) Tween
buffer (pH 9.0). Slides were subsequently blocked in 5% (vol/vol)
goat serum in Tris-bbuffered saline with 0.05% (vol/vol) Tween
(TBS-T) for 20 min at room temperature and then incubated in a
mixture of primary antibodies at the concentrations indicated at 4C
overnight, After being washed in TBS-T for three times of 5 min each
time, slices blotted with primary antibodies were visualized with
Alexa uor 568- or 488-conjugated IgG (1:500; Invitrogen, Paisley,
UK) under uorescent microscopy using a Zeiss Axiovert-200 con-
E1262 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

focal microscope with an Improvision/Nokigawa spinning disc and
running Volocity 5.0 (Improvision, Coventry, UK) software (10).
To quantify the number of rosette-like structures (i.e., 810 cells
arranged concentrically around an identiable central hub; see
RESULTS) in islets, we used E-cadherin and DAPI staining of pancre-
atic sections. Structures were included where the void at the center
was negative for DAPI. Ten islets from three pairs of mice per
genotype were assessed.
Antibodies
Antibodies used in Western (immuno-)blot analysis and immuno-
histochemistry were the following: rabbit anti-LKB1 (Millipore,
Watford, UK), rabbit anti-E-cadherin, anti-phospho-S6 ribosomal
protein (Ser
235/236
), anti-VEGFR2, rabbit anti-phospho-4E-BP1 (New
England Biolabs, Hitchin, UK), mouse anti-actin (C2) (Santa Cruz
Biotechnology, Heidelburg, Germany), rabbit anti-mouse GLUT2
(kind gift from Dr. Bernard Thorens, Lausanne), guinea pig anti-
insulin, rabbit anti-glucagon (Dako, Ely, UK), rabbit monoclonal
anti-Ki67 (Epitomics, Burlingame, USA), mouse anti--tubulin, anti-
-tubulin (Sigma Aldrich, Dorset, UK), and rat anti-ZO1 (kind gift
from Dr. Paolo Meda, Geneva).
Optical Projection Tomography (OPT) and Determination of
Relative -Cell Mass, Single -Cell Size, and Proliferation
Whole pancreatic optical projection tomography, to 19 m reso-
lution, was performed as described (2). Briey, whole pancreata xed
in 4% (wt/vol) paraformaldehyde for 23 h at 4C were dehydrated
and subjected to ve cycles of freezing and thawing (80C to room
temperature). Pancreata were then blocked overnight in Tris-buffered
saline, pH7.5, containing 0.05% (vol/vol) Tween 20, 0.01% (wt/vol)
sodium azide and 10% (vol/vol) goat serum, (Dako) before incubation
with guinea pig anti-swine insulin antibody (Dako, 1:1,000) dissolved
in the above buffer further supplemented with 5% (vol/vol) dimeth-
ylsulfoxide overnight at 4C. To visualize insulin-positive staining,
Alexa 594 goat anti-guinea pig antibody (Invitrogen, 1:1,000) was
applied. The sample was then embedded in 1% (wt/vol) low melting
temperature agarose, dehydrated in methanol, and cleared in benzyl
alcohol-benzyl benzoate (1:2) for optical tomographical scanning.
All specimens were scanned using two uorescent channels (exci-
tation: 580 15 nm and emission: 650 15 nm for insulin-positive
staining; excitation: 480 9 nm and emission: 650 25 nm for
autouorescence). The raw data for these two channels were recon-
structed in a pair of 3-D voxel data sets (voxel to m 1:19.5) using
Matlab software, and cell volumes (m
3
) from each channel were
Fig. 1. Generation of LKB1KO mice. A: schematic
representation of deletion of oxd lkb1 exons (exons
36) driven by RIP2.Cre expression. Location of primers
used for PCR as indicated. Black arrows, ox sites; gray
bar, LKB1 exons. B: RT-PCR analysis of effects on
LKB1 transcript levels of deleting exons 36 in pancre-
atic islets and hypothalamus (Hypo). Product sizes were
864 and 300 bp for oxd and null alleles, respectively.
NS, nonspecic. C: qRT-PCR (C) and Western blot
analysis (D) of LKB1 mRNA and protein levels in
pancreatic islets of LKB1KO mice and their heterozy-
gous (het) and wild-type (Wt) controls. Total AMPK
activity in islets (E) and hypothalamus (F) of LKB1KO
and LKB1het mice. Data are expressed as means SE;
n 45 mice per genotype. *P 0.05, **P 0.01,
***P 0.001.
E1263 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

measured using Volocity 5.0 (Improvision). Data from Volocity 5.0
were further analyzed in Microsoft Excel to provide size frequency
histograms and total volume graphs. Relative -cell mass was calcu-
lated by dividing the volume of insulin-positive area (total -cell
mass) by that of the autouorescence area (total pancreatic mass).
To calculate single -cell size, the area of 100 single -cells from
ve islets of each genotype from E-cadherin- and insulin-costained
pancreatic sections were measured using Image J (http://rsbweb.nih.
gov/ij/). To measure relative number of proliferating -cells, the
number of Ki67-positive -cells per islet was divided by total -cell
number; 1520 islets per genotype were analyzed.
Electrophysiological Measurements and Ca
2
Imaging
The plasma membrane potential of -cells was recorded in perfo-
rated-patch whole cell conguration using an EPC9 patch clamp
amplier controlled by Pulse acquisition software (HEKA Elektronik,
Lambrecht/Pfalz, Germany). The pipette tip was dipped into pipette
solution (see below),and then back-lled with the same solution
containing 0.24 mg/ml amphotericin B. Recordings were initiated
after 30-min exposure to substrate-free solutions at 37C, and the
duration of exposure to each concentration of effector(s) was 2 min.
Cells that were not responsive to tolbutamide were excluded from
analysis.
Series resistance and cell capacitance were compensated for auto-
matically by the acquisition software. Experiments were carried out
by periodically switching from current-clamp to voltage-clamp mode,
thus obtaining pseudo-simultaneous recordings of cell membrane
potential (V
m
) and K
ATP
conductance (G
KATP
). This controlled for the
leaks of the patch and veried that the depolarization (hyperpolariza-
tion) of the membrane was linked to K
ATP
channel closure (opening).
The current-clamp protocol involved continuous recording without
electrical stimulation. In the voltage clamp, the membrane potential
was held at 70 mV, and whole cell currents were evoked by
10-mV 0.5-Hz pulses. Data were ltered at 0.2 kHz and digitized at
0.5 kHz.
The pipette solution contained (in mmol/l): 76 K
2
SO
4
, 10 NaCl, 10
KCl, 1 MgCl
2
, and 5 HEPES (pH7.35 with KOH). The bath solution
contained (in mmol/l): 137 NaCl, 5.6 KCl, 10 HEPES (pH 7.4 with
NaOH), 2.6 CaCl
2
, and 1.1 MgCl
2
. All experiments were conducted
at 3337C, and the bath solution was perifused continuously.
For Ca
2
imaging, dispersed islets were incubated for 30 min in
Krebs-Ringer Buffer [KRB; in mmol/l: 125 NaCl, 3.5 KCl, 1.5 CaCl
2
,
0.5 NaH
2
PO
4
, 0.5 MgSO
4
, 3 glucose, 10 HEPES, and 2 NaHCO
3
, pH
7.4, and equilibrated with O
2
-CO
2
(95:5) and supplemented with 0.1%
(wt/vol) BSA] containing 3 mmol/l glucose and 200 nM FURA-RED
AM (Invitrogen UK). Cells were stimulated using the conditions
indicated and excited at 480/440 nm using an Olympus IX-81 micro-
scope coupled to an F-view camera and captured using Cell^R
software (Olympus UK) on a 40 oil objective. Data were expressed
at the ratio of the uorescence emission at 440/480 nm.
Statistical Analysis
Data are expressed as means SE. Signicance was tested by
Students two-samples unpaired or paired Students t-tests using
Excel, or ANOVA test using Graphpad 4.0. P 0.05 was considered
signicant.
RESULTS
LKB1KO Mice Are Lean and Hypophagic and Display
Improved Glucose Tolerance
To generate mice lacking LKB1 selectively in the pancreatic
-cell from midgestation (E911.5) and in a subset of hypo-
thalamic neurons (RIP2.Cre neurons) (17), we crossed ani-
mals bearing oxd Lkb1/Stk11 alleles with RIP2.Cre mice
(Fig. 1A). Demonstrating efcient deletion from -cells, levels
of endogenous LKB1 mRNA were markedly reduced in islets
from homo- (/) vs. heterozygous (/) mouse islets in the
presence of the Cre transgene, with the consequent increase in
the level mRNA encoded by the null allele (Fig. 1B). Similarly,
the null transcript was also present in hypothalamic extracts of
hetero- and homozygous mice (Fig. 1B). Levels of LKB1
Fig. 2. Food intake and glucose homeostasis in LKB1KO mice. AC: body weight (A), and food intake of LKB1KO mice fed (B) or refed after fasting for
15 h (C). DF: blood glucose (D and E) and plasma insulin (F) of LKB1KO mice fed or fasted for 15 h. Male mice from 68 wk old were used. Data are
expressed as means SE; *P 0.05, **P 0.01; n 710 mice per genotype.
E1264 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

mRNA (Fig. 1C) and immunoreactivity (Fig. 1D) in islets from
mice of each genotype were compared next. Each parameter was
decreased by 7080% in islets from LKB1KO (/,Cre

) vs.
wild-type (/,Cre

) mice, consistent with deletion from the

islet -cell compartment. Levels of LKB1 mRNA and immu-
noreactivity in LKB1het mouse islets were intermediate be-
tween those in LKB1Wt and LKB1KO mouse islets (Fig. 1,
C and D).
Since LKB1 is likely to be the major upstream kinase for
LKB1 in islets (I. Leclerc and G. A. Rutter, unpublished
results), AMPK activities were also measured as a further
readout of LKB1 activity. Total cellular AMPK activity in
isolated islets was reduced by 75% in LKB1KO vs.
LKB1het (Fig. 1E) (comparison with levels of AMPK activ-
ity in wild-type islets was not performed). AMPK1/2 activ-
ities were reduced by 25% in hypothalamic extracts from
LKB1KO vs. LKB1het mice (Fig. 1F), consistent with
deletion from a subset of RIP2.Cre neurons within this tissue
and/or regulation of AMPK in this tissue by distinct upstream
kinases (e.g., CaMKK) (20).
Examined at 68 wk, male (Fig. 2) and female (not shown)
LKB1KO mice displayed decreased fed body weight (Fig.
2A) and daily food intake (Fig. 2B) compared with LKB1Wt
or LKB1het mice. In addition, after 15 h of starvation,
LKB1KO mice tended to eat less in the rst 30 min after
refeeding (Fig. 2C). Reduced hypothalamic expression of the
orexigenic peptide NPY mRNA and the anorexigenic peptide
POMC were also observed (Suppl. Fig. 1A; supplementary
materials are found in the online version of this paper at the
Jounal website), although in each case the differences were
only apparent between wild-type and heterozygous mice, sug-
gesting they are unlikely to drive the marked differences in
feeding between knockout and heterozygous animals (Fig. 1).
Interestingly, we have observed in preliminary experiments
increases in the immunoreactivity of POMC in hypothalamic
extracts of LKB1KO mice (data not shown), suggesting that
LKB1-dependent posttranscriptional mechanisms may modify
the levels of this factor. Further studies will be required to
explore this possibility in detail.
Fasting and fed glycemia were signicantly decreased in
LKB1KO mice (Fig. 2, D and E), a change accompanied by
substantial increases in plasma insulin levels in the fed state
(Fig. 2F). Correspondingly, LKB1KO mice displayed dra-
matically improved glucose tolerance (Fig. 3A) and insulin
release (Fig. 3B) in vivo compared with LKB1Wt or
LKB1het mice but unchanged insulin sensitivity (Fig. 3C).
LKB1 Controls -Cell Size, Morphology, and Islet
Architecture
To examine in detail the possible mechanisms behind the
hyperinsulinism in LKB1KO mice, we used OPT (2). This
technique, which we have previously validated by comparison
with conventional measurements of -cell mass based on
histochemistry (46), allowed us to determine the mean and
total volume, size, distribution, and number of islets within the
entire pancreas (Fig. 4, AD, Suppl. Fig. 2, and Supplementary
movies: betaLKB1wt, betaLKB1Het, betaLKB1KO). Total
-cell mass was markedly increased with respect to LKB1wt
mice, and the mean volume of individual islets was signi-
cantly elevated by 30% in LKB1KO vs. LKB1het and
20% in LKB1KO vs. LKB1wt pancreata. The most
marked increase was apparent in the number of the largest
islets present (10
7
m
3
; 10,000 cells) and certain small
islets. Although there were no evident changes in the ratio of
- to -cells within islets (Fig. 4E), we observed a signicant
40% increase in the surface area of individual -cells, as
identied by staining with E-cadherin antibodies (see Methods;
Fig. 4F). Demonstrating an increase in -cell proliferation,
Ki67 staining was markedly increased in LKB1KO islets
(Fig. 4G).
Since mTOR signaling (3) is implicated in controlling cell
growth and protein synthesis, phosphorylation of ribosomal pro-
tein subunit S6 (rpS6) and eIF4-binding protein-1 (4E-BP1)
within islets was measured. As expected, increased phospho-rpS6
and decreased phospho-4E-BP1 were detected in LKB1KO
mice islets compared with LKB1Wt or LKB1het mouse islets
(Fig. 4H).
Fig. 3. Glucose and insulin tolerance of LKB1KO mice. Glucose tolerance
(A), and plasma insulin response (B) after intraperitoneal (ip) glucose injection,
and whole body insulin sensitivity (C) monitored after ip insulin injection of
LKB1KO or control mice. Male 6- to 8-wk-old mice old were used. Data are
expressed as means SE. For A and B, *P 0.05, **P 0.01 for LKB1KO
vs. LKB1Wt,

P 0.05 for LKB1KO vs. LKB1het; in C, *P 0.05 with

respect to time 0 for all genotypes; n 710 mice per genotype.
E1265 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

E1266 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

To determine whether LKB1 may be critical for the estab-
lishment of basolateral-apical polarity of -cells, we examined
the distribution of the adherens junction marker E-cadherin, as
well as actin, tubulin, and the tight junction protein zona
occludins 1 (ZO1). Unexpectedly, polarization was not com-
promised in LKB1-decient -cells (Fig. 5). Instead, mutant
islets displayed a signicant increase in the number of rosette-
like structures or islet acini (15) identied by E-cadherin and
ZO1 staining in which 810 cells were arranged around a
central void (6) (Fig. 5, A, B, E, and G). Strikingly, nuclei were
positioned in the cells comprising these rosettes away from the
apical pole, while actin and tubulin staining were concentrated
at the junctional zones (Fig. 5, C and D). Whereas the apical
voids were devoid of staining for VEGFR, this was enriched
at the basolateral surface, indicating the presence of capillaries
at the latter site (Fig. 5F).
LKB1 Deletion Diminishes -Cell Responses to Glucose
Ex Vivo
To determine whether the dramatic improvements in glucose
tolerance in vivo might also in part reect enhanced -cell
glucose sensitivity, we isolated islets and single cells from
LKB1KO and LKB1Wt or LKB1het mice. Glucose-stim-
ulated insulin secretion (GSIS) under static incubation was
diminished in LKB1KO mouse islets by 30% compared
with LKB1Wt mouse islets (Fig. 6A), although the absolute
insulin output, as assessed from six size-matched islets, was
not signicantly different between the genotypes (Fig. 6A). By
contrast, the insulin content of similarly sized islets from
LKB1KO mice was substantially increased (2.5-fold vs.
LKB1Wt and 1.5-fold vs. LKB1Het; Fig. 6A). Glucose-
stimulated changes in the conductance of ATP-sensitive K

channels (G
KATP
), membrane depolarization (V
m
), and in-
creases in intracellular free Ca
2
concentration were all mark-
edly attenuated in -cells from LKB1KO islets (Fig. 6, B and
C). In addition, membrane electrical activity was elevated at
basal and 3 mM glucose levels in LKB1KO islet cells,
suggesting blunted glucose sensing in these cells (Fig. 6B).
Providing a possible contribution to the impaired GSIS from
LKB1KO islets, we observed a substantial decrease in the
level of GLUT2 (slc2A2) immunoreactivity at the plasma
membrane of LKB1-deleted cells (Fig. 6D) and a correspond-
ingly large (90%) reduction in GLUT2 mRNA in isolated
islets (Suppl. Fig. 1B). Likewise, the levels of mRNAs encod-
ing the ATP-sensitive K
ATP
channel subunit kir6.2 (Suppl. Fig.
1B) were substantially (80%) decreased in LKB1KO vs.
LKB1wt islets. By contrast, we observed no signicant de-
creases in the expression of other genes involved in maintaining
the differentiated function of cells, including Pdx1, nor in SUR1
(ABCC8) or glucokinase (Gck; Suppl. Fig. 1), nor in neuroD1 or
MafA (LKB1
/
.RIP2.Cre

vs. LKB1
/
.RIP2.Cre

, not shown)
suggesting that a generalized decrease in -cell differentiation did
not occur.
Effect of Metabolic Stress on LKB1KO vs. Wild-Type Mice
Since LKB1 and AMPK are implicated in the cellular stress
responses, we determined how LKB1 deciency in -cells and
hypothalamus may impact on the diabetogenic effects of a
high-fat diet (1). After 6 wk on a high-fat diet, differences in
glucose tolerance (Fig. 7A) or insulin secretion (Fig. 7B) were
no longer apparent between genotypes, consistent with a
greater susceptibility of LKB1KO mice to the effects of this
metabolic stress.
DISCUSSION
LKB1 Regulates -Cell Size and Islet Architecture
The principal aim of the present study was to determine
whether, when compared using a near-identical strategy for
deletion from -cells with that used previously for AMPK
subunits (46), the loss of LKB1 may lead to similar changes in
insulin secretion and glucose homeostasis in vivo and in vitro.
This has seemed important given the dramatically different
phenotypes of mice deleted for LKB1 in adults using a Pdx-
1-CreER transgene (16, 18) and those deleted for AMPK using
the RIP2.Cre transgene (46), with the latter strategy resulting
in earlier (midgestational) deletion and some loss from hypo-
thalamic nuclei. Although the genetic background of the
LKB1-deleted mice generated here (FVB/129S6/C57BL6) and
in earlier studies (16, 18) differs slightly from that of those
deleted for AMPK (C57BL6) (46), it seems unlikely to us that
this difference could explain the markedly different phenotypes
of mice deleted for LKB1 vs. AMPK in the -cell.
Our ndings conrm the view that LKB1 is a critical
regulator of -cell development and function. First, we dem-
onstrated that the size of individual -cells was considerably
increased, as was that of individual islets, as assessed by OPT.
Interestingly, and as shown in Fig. 4D, the mean islet volume,
as determined by OPT, was increased 40% in LKB1 KO vs.
wild-type islets. This compares with an increase in average
(single) -cell area of 60% (Fig. 4F), equivalent to an
increase in volume of more than two fold. The difference
between these values seems likely to be due to the contribution
of an unchanged volume of other cell types within the islets,
and/or a lowered number of -cells per islet.
Fig. 4. Altered islet morphology in LKB1KO mice. A: representative optical projection tomographic (OPT) images of whole pancreas. OPT was performed as
described under METHODS. Red staining indicates insulin-positive structures (islets), while the outline of the whole pancreas was apparent as autouorescence and
is presented as white/gray shading. Images shown correspond to 3-D projections; (see also Suppl. Fig. 2 and Suppl. Movies betaLKB1wt, betaLKB1het, and
betaLKB1KO.avi.) Note presence of a particularly large islet in the LKB1KO pancreas (right image, arrow). B: distribution of islet volumes with marked (dotted
lines) section magnied. C: relative -cell mass; D: mean islet volume. Data are from 56 mice per genotype. Scale bar, 500 m. E: hematoxylin-eosin (H&E)
and immunouorescent staining of pancreatic sections using guinea pig anti-insulin (1:200; green) and rabbit anti-glucagon (1:100; red) antibodies. Nuclei are
shown with DAPI (blue) staining. Scale bar, 75 m. F: representative E-cadherin staining of pancreatic sections and quanication of single islet -cell sizes.
Average area of 100 single -cells from 5 islets of each genotype, costained in pancreatic sections for E-cadherin and insulin, were analyzed. Scale bar, 12 m.
G: staining for proliferation marker Ki67 and quantication, based on 1520 islets per pancreas; n 3 mice per genotype. H: Western blot analysis of mTOR
signaling pathway markers phospho-ribosomal protein (rp)S6 and phospho-4E-BP1 of pancreatic islet extracts from LKB1KO and control mice. Islets extracted
from fed mice were incubated in RPMI supplemented with 11 mM glucose for 16 h and lysed for analysis. Data are expressed as means SE. *P 0.05,
**P 0.01. In B, *P 0.05, for LKB1KO vs. LKB1Wt, $P 0.05 for LKB1KO vs. LKB1het.
E1267 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Fig. 5. -Cell polarity is enhanced in LKB1KO mice with
increased rosette-like (islet acini) structures and accumu-
lation of cell junction proteins. AE: immunouorescence
staining of pancreatic sections for the adherens junction
marker (A and B) E-cadherin (1:100, green), (C) actin
lament (1:50, red; note imaged areas were conrmed as
lying over an islet by inspection of corresponding bright
eld images), (D) -tubulin (1:100, green), (E) tight junc-
tion marker zona occludins 1 (ZO1, whole serum; green) or
capillary marker (F) VEGFR2 (1:100, green). White square,
voids at center of rosettes. G: quantication of no. of
rosette-like structures per islet in LKB1KO mice based on
E-cadherin and DAPI staining. Such structure in islet is
counted as 1 by E-cadherin staining where the voids at the
center of the rosette is absent of DAPI staining. Ten islets
from 3 pairs of mice per genotype were assessed. Data are
expressed as means SE. **P 0.01, ***P 0.001. Scale
bars, 50 m (A), 12 m (B), 10 m (C), and 25 m (DF).
E1268 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

As also recently reported (2), the distribution of islets in
wild-type mouse pancreata was highly nonuniform, with the
largest islets representing as much as 50% of the total -cell
mass. Using the same approach in LKB1KO islets, we dem-
onstrated that the abundance of the largest islets was signi-
cantly increased. This shift seems likely to be the result, at least
in large part, of an increase in the average volume of individual
-cells (60%), as well as increases in -cell proliferation.
TSC1/2 deletion in -cells, using the same RIP2.Cre mice,
leads to increased -cell mass (35), a phenotype similar to
what we describe here in LKB1KO mice. Increased phosphor-
ylation of rpS6 and decreased phosphorylation of 4E-BP1 in
LKB1KO mice islets suggested that the increased -cell size
and islet volume are likely to be due to elevated mTOR
signaling.
The present results also conrm the view (16, 18) that LKB1
is involved in the establishment of cell polarity in mammalian
epithelial cells, consistent with ndings from Shorning et al.
(44) in intestinal paneth and goblet cells, as well as the ndings
in -cells (16, 18). Thus, we found that rosette-like arrange-
ments of -cells (6), containing strikingly polarized -cells,
were twice as frequently observed in LKB1KO islets. We
considered the possibility that this may be due simply to an
increase in the size of individual cells. Arguing against this
possibility, Hamada et al. (19) demonstrated an increase in
-cell volume of 60% after transgenic overexpression of
mTORC1 in -cells, similar to the increase observed after lkb1
knockout. However, no obvious increase in the number of
rosettes was apparent in the case of mTORC1-deleted -cells
in contrast to the ndings described here, nor after induction of
-cell hypertrophy by dexamethasone (38). Similarly, such
structures did not appear to be more abundant in the islets of
transgenic mice expressing the cell cycle regulator CDK4/
R24C under the rat insulin promoter, in which a massive
increase in -cell mass (to almost 20% of pancreatic volume)
was observed, in this case due to enhanced proliferation (34).
We have considered the possibility that the effects of LKB1
deletion reect the requirement for this enzyme to activate
AMPK. However, deletion of the AMPK1 and AMPK2
subunits in -cells by use of the RIP2.Cre transgene leads to
defective insulin release in vivo, with none of the changes in
-cell mass or cellular architecture observed here in
LKB1KO islets (46). Instead, and as previously proposed (16,
18) the increased number of islet acini most likely involves
alternative kinases such as MARKS13. In this context, Par-1b
(MARK2) was recently shown to promote cell-cell adhesion
and association of E-cadherin with the actin cytoskeleton (13).
A loss of this interaction may be involved in the relocalization
of the latter to the apical pole as observed in LKB1KO
-cells. Par1b/MARK2 has also recently been shown to regu-
late glucose metabolism by adipose tissue in vivo (25), possi-
bly by interacting with syntaxin-4 (14) to control vesicular
trafcking of GLUT4 to the plasma membrane. As syntaxin-4
is implicated in the second phase of GSIS (45), it is possible
that abnormal phosphorylation of Par1b/MARK2 in the ab-
sence of LKB1 may also inuence the trafcking of -cell
secretory granules to the cell surface. Most importantly, -cells
from mice deleted globally for Par1b/MARK2
/
display a
similar redistribution of nuclei to that described here (18).
The results of the two very recent studies mentioned above
(16, 18), contrast with our own ndings in several important
respects. Notably, LKB1-decient -cells were found in the
present studies to display abnormalities in the expression of the
glucose transporter GLUT2, and the K
ATP
channel subunit
Kir6.2 (but not of several other genes important in maintaining
the glucose responsiveness of -cells; see RESULTS), with cor-
responding alterations in basal and glucose-induced changes in
membrane potential and intracellular free Ca
2
. Although
neither of the earlier studies examined the levels of expression
of -cell-specic genes in detail or involved studies of glucose
sensing at the single -cell level, the subcellular distribution of
GLUT2 immunoreactivity within individual -cells was re-
portedly altered by LKB1 deciency (18). We suspect that the
relatively well-preserved GSIS from isolated islets may in both
cases therefore represent alterations in -cell--cell (or -cell-
-cell contact) or between -cells and the capillary network.
Moreover, the well-preserved stimulation of insulin secretion
by glucose observed here in LKB1-deleted islets (Fig. 6A),
despite changes in K
ATP
channel conductance, resting mem-
brane potential, and a marked (50%) decrease in the rise in
intracellular free Ca
2
in response to the sugar (Fig. 6, BD),
may suggest a compensatory increase in K
ATP
-independent
(22) mechanisms of activation, serving to enhance the sensi-
tivity of the secretory machinery to Ca
2
changes. A more
detailed assessment of this possibility will require further
analysis, including a comparison of the transcriptome of islets
from LKBKO and wild-type islets. It should also be stressed,
however, that the substantial increase in -cell size and mass
(4-fold; Fig. 2, C and D) seems likely to predominate over
the relatively more minor changes in -cell glucose sensing in
LKB1-deleted -cells and to underlie the substantial increase
in vivo in insulin release under glucose challenge.
Does LKB1 Control Neuronal Function to Regulate Food
Intake and Body Weight?
A striking nding in the present study was that deletion of
LKB1 using the RIP2.Cre transgene led to a decrease in body
weight and feeding. We have considered the possibility that
these changes may simply reect the increased levels of insu-
lin, with the latter serving as a satiety factor (37). However, we
suspect that the metabolic and cellular changes resulting from
LKB1 deciency in the brain and in the pancreas are, at least
in large part, independent. Thus, mice inactivated for LKB1 by
Pdx1.CreER-mediated excision, which leads to deletion in all
pancreatic lineages but not in the hypothalamus, show similar
increases in circulating insulin levels and marked improve-
ments in glucose tolerance (23) without a change in body
weight.
Importantly, we show that changes in food intake and body
mass in LKB1KO mice were accompanied by signicant
decreases in the hypothalamic expression of the orexigenic
peptide NPY and of the anorexigenic peptide POMC at the
mRNA level, suggesting that RIP2.Cre neurons may control
the activity of neighboring cells in the melanocortin circuitry
(43). On the other hand, in preliminary experiments (not
shown) we have observed by Western blotting a small but
detectable increase in POMC immunoreactivity in hypotha-
lamic extracts from LKB1KO mice vs. wild-type controls,
suggesting possible posttranslational modications through
which LKB1 may control the levels of this peptide in feeding
centers. Further studies will be necessary to explore this
E1269 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

E1270 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

possibility and the mechanisms through which deletion of
LKB1 in RIP2.Cre neurons, not usually thought to express
either NPY or POMC (24), may inuence the levels of these
peptides in the ventromedial hypothalamus. Interestingly, de-
letion of insulin receptor substrate-2 (IRS-2), using an identical
strategy to that used here, has previously been shown to cause
hyperphagia, diminished -cell mass, and glucose intolerance
in mice (29). Conversely, potentation of insulin signaling by
deletion in RIP2.Cre cells of the inositol phospholipid 5=-
phosphatase PTEN (8) led to diminished growth and body
weight. LKB1 deletion in the same nuclei thus leads to a
strikingly similar phenotype to the latter model. This similarity
suggests that LKB1 may also oppose insulin, or possibly leptin,
signaling in RIP2.Cre neurons to enhance appetite and body
weight. Surprisingly, however, deletion of TSC1, also expected
to enhance mTOR signaling in the same neuronal population,
led to hyperphagia and obesity (35), suggesting that LKB1 may
engage other signaling pathways to inuence the activity of
these and associated neurons in the melanocortin network.
Interestingly, although LKB1-null mice displayed clear
decreases in hypothalamic and -cell AMPK activity (Fig. 1),
mice deleted for both AMPK catalytic subunits in the same
cells do not display a feeding or body weight phenotype (46),
indicating that the hypothalamic effects of LKB1 may be
mediated by alternative downstream kinases (30) (Table 1) or
other effectors. In any case, we stress that further studies will
be required in the future, perhaps involving the selective
deletion of LKB1 in the hypothalamus by the injection of Cre
recombinase into this brain region in LKB1 oxd mice, to
formally conrm or refute the possibility of distinct roles for
central and pancreatic islet LKB1 in the control of glucose
homeostasis and food intake.
Conclusion
Using an identical strategy for tissue-selective deletion in
mice, we have shown that LKB1 plays a distinct role(s) from
that of AMPK in controlling -cell mass and insulin secretion
in vivo and in vitro. Further dissection of the signaling path-
way(s) through which LKB1 acts at each site may provide
exciting new modalities for the treatment of metabolic disease
including type 2 diabetes.
ACKNOWLEDGMENTS
We thank Dr. Blerina Kola (Queen Mary, University of London) for useful
discussion and Lorraine Lawrence for the preparation of pancreatic slices.
GRANTS
This work was supported by grants to G. A. Rutter from the Wellcome Trust
(Programme Grant 081958/2/07/Z), The European Union (FP6 Save Beta),
Fig. 6. LKB1KO -cells display abnormal electrical, Ca
2
, and secretory responses and decreased GLUT2 immunoreactivity at the plasma membrane.
A: glucose-stimulated insulin secretion and total insulin content of 6 size-matched islets statically incubated with indicated glucose for 0.5 h from LKB1KO,
heterozygous, and WT mice; n 3 mice per genotype. B: representative traces of whole cell KATP channel conductance (GKATP) and plasma membrane potential
(Vm) from perforated patch clamp measurements. Three to six -cells from 3 pairs of mice of each genotype were recorded. C: representative traces and
quantication of free [Ca
2
] with fura 2-AM in dissociated -cells in LKB1KO mice. Cells (3860) from 3 pairs of mice of each genotype were analyzed.
D: immunouorescence staining and quantication of Glut2 expression at the plasma membrane of pancreatic islet -cells. Fifteen islets from 2 pairs of mice
per genotype were examined. Scale bar, 75 m. Data are expressed as means SE. *P 0.05, **P 0.01, ***P 0.001.
Fig. 7. In vivo metabolic advantages of LKB1 deletion from insulin-expressing
cells are lost on a high-fat diet. Glucose tolerance (A) and plasma insulin
changes (B) after ip glucose injection of LKB1KO mice on a high-fat diet for
6 wk (see METHODS).
Table 1. Relative abundance of LKB1 target kinases in
pancreatic islets
Protein Kinase
mRNA Level
(Relative Intensity)
LKB1/STK11 404
CaMKK2 152
AMPK1 (PRKAA1) 416
AMPK 2 (PRKAA2) 104
Snf-related kinase (SNRK) 396
Nuak1/Snf-1 line kinase-1/ARK5 314
Nuak2/SNARK 172
Salt-inducible kinase 1 (SIK1/MSK/SNF1LK1) 35
Salt-inducible kinase (SIK2/QIK/ SNF1LK2) 54
MARK1 214
MARK2 (PAR1B) 422
MARK3 (PAR1A/TAK1/MAP3K7) 568
MARK4 A
BRSK1 (SAD-A) A
BRSK2 (SAD-B) A
Pancreatic islets isolated from 12-wk-old wild-type male C57Bl/6 mice by
collagenase digestion and histopaque gradient purication as described (27)
were incubated in RPMI supplemented with 11 mM glucose for 16 h. Total
cellular RNA (400-600 ng) extracted from islets by use of RNAeasy kit
(Qiagen UK) was used for microarray analysis. Four independent hybridiza-
tions were performed on Mouse 430 2.0 chip (Affymetrix, Santa Clara, CA).
Data were exported to GeneSpringX to perform quality control and subsequent
downstream analysis. Robust multichip average (RMA) summary and quantile
normalization was employed to produce expression values for the probe sets.
Data are expressed as means of observations on islets from 4 separate mice,
which agreed within 10%. A, absent.
E1271 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

the Medical Research Council (G0401641) and National Institutes of Health
(RO1 DK-071962-01), and a JDRFI PostDoctoral Fellowship to A. I. Tarasov.
DISCLOSURES
No conicts of interest were reported by the authors.
REFERENCES
1. Ahren B, Simonsson E, Scheurink AJ, Mulder H, Myrsen U, Sundler
F. Dissociated insulinotropic sensitivity to glucose and carbachol in
high-fat diet-induced insulin resistance in C57BL/6J mice. Metabolism 46:
97106, 1997.
2. Alanentalo T, Asayesh A, Morrison H, Loren CE, Holmberg D,
Sharpe J, Ahlgren U. Tomographic molecular imaging and 3D quanti-
cation within adult mouse organs. Nat Methods 4: 3133, 2007.
3. Alessi DR, Sakamoto K, Bayascas JR. LKB1-dependent signaling path-
ways. Annu Rev Biochem 75: 137163, 2006.
4. Anderson KA, Ribar TJ, Lin F, Noeldner PK, Green MF, Muehlbauer
MJ, Witters LA, Kemp BE, Means AR. Hypothalamic CaMKK2 con-
tributes to the regulation of energy balance. Cell Metab 7: 377388, 2008.
5. Baas AF, Boudeau J, Sapkota GP, Smit L, Medema R, Morrice NA,
Alessi DR, Clevers HC. Activation of the tumour suppressor kinase
LKB1 by the STE20-like pseudokinase STRAD. EMBO J 22: 30623072,
2003.
6. Bonner-Weir S. Morphological evidence for pancreatic polarity of beta
cells within islets of Langerhans. Diabetes 37: 616621, 1988.
7. Boudeau J, Sapkota G, Alessi DR. LKB1, a protein kinase regulating cell
proliferation and polarity. FEBS Lett 546: 159165, 2003.
8. Choi D, Nguyen KT, Wang L, Schroer SA, Suzuki A, Mak TW, Woo
M. Partial deletion of Pten in the hypothalamus leads to growth defects
that cannot be rescued by exogenous growth hormone. Endocrinology
149: 43824386, 2008.
9. Claret M, Smith MA, Batterham RL, Selman C, Choudhury AI, Fryer
LG, Clements M, Al-Qassab H, Heffron H, Xu AW, Speakman JR,
Barsh GS, Viollet B, Vaulont S, Ashford ML, Carling D, Withers DJ.
AMPK is essential for energy homeostasis regulation and glucose sensing
by POMC and AgRP neurons. J Clin Invest 117: 23252336, 2007.
10. da Silva XG, Loder MK, McDonald A, Tarasov AI, Carzaniga R,
Kronenberger K, Barg S, Rutter GA. TCF7L2 regulates late events in
insulin secretion from pancreatic islet beta-cells. Diabetes 58: 894905,
2009.
11. da Silva XG, Leclerc I, Salt IP, Doiron B, Hardie DG, Kahn A, Rutter
GA. Role of AMP-activated protein kinase in the regulation by glucose of
islet beta-cell gene expression. Proc Natl Acad Sci USA 97: 40234028,
2000.
12. da Silva XG, Leclerc I, Varadi A, Tsuboi T, Moule SK, Rutter GA.
Role for AMP-activated protein kinase in glucose-stimulated insulin
secretion and preproinsulin gene expression. Biochem J 371: 761774,
2003.
13. Elbert M, Cohen D, Musch A. PAR1b promotes cell-cell adhesion and
inhibits dishevelled-mediated transformation of Madin-Darby canine kid-
ney cells. Mol Biol Cell 17: 33453355, 2006.
14. Elbert M, Rossi G, Brennwald P. The yeast par-1 homologs kin1 and
kin2 show genetic and physical interactions with components of the
exocytic machinery. Mol Biol Cell 16: 532549, 2005.
15. Esni F, Taljedal IB, Perl AK, Cremer H, Christofori G, Semb H.
Neural cell adhesion molecule (N-CAM) is required for cell type segre-
gation and normal ultrastructure in pancreatic islets. J Cell Biol 144:
325337, 1999.
16. Fu A, Ng AC, Depatie C, Wijesekara N, He Y, Wang GS, Bardeesy N,
Scott FW, Touyz RM, Wheeler MB, Screaton RA. Loss of Lkb1 in
adult beta cells increases beta cell mass and enhances glucose tolerance in
mice. Cell Metab 10: 285295, 2009.
17. Gannon M, Shiota C, Postic C, Wright CVE, Magnuson M. Analysis
of the Cre-mediated recombination driven by rat insulin promoter in
embryonic and adult mouse pancreas. Genesis 26: 139142, 2000.
18. Granot Z, Swisa A, Magenheim J, Stolovich-Rain M, Fujimoto W,
Manduchi E, Miki T, Lennerz JK, Stoeckert CJ Jr, Meyuhas O, Seino
S, Permutt MA, Piwnica-Worms H, Bardeesy N, Dor Y. LKB1 regu-
lates pancreatic beta cell size, polarity, and function. Cell Metab 10:
296308, 2009.
19. Hamada S, Hara K, Hamada T, Yasuda H, Moriyama H, Nakayama
R, Nagata M, Yokono K. Upregulation of the mammalian target of
rapamycin complex 1 pathway by Ras homolog enriched in brain in
pancreatic beta-cells leads to increased beta-cell mass and prevention of
hyperglycemia. Diabetes 58: 13211332, 2009.
20. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM,
Frenguelli BG, Hardie DG. Calmodulin-dependent protein kinase ki-
nase-beta is an alternative upstream kinase for AMP-activated protein
kinase. Cell Metab 2: 919, 2005.
21. Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola
A, Bignell G, Warren W, Aminoff M, Hoglund P, Jarvinen H, Kristo
P, Pelin K, Ridanpaa M, Salovaara R, Toro T, Bodmer W, Olschwang
S, Olsen AS, Stratton MR, de la Chapelle A, Aaltonen LA. A serine/
threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 391:
184187, 1998.
22. Henquin JC. Triggering and amplifying pathways of regulation of insulin
secretion by glucose. Diabetes 49: 17511760, 2000.
23. Hezel AF, Gurumurthy S, Granot Z, Swisa A, Chu GC, Bailey G, Dor
Y, Bardeesy N, DePinho RA. Pancreatic LKB1 deletion leads to acinar
polarity defects and cystic neoplasms. Mol Cell Biol 28: 24142425, 2008.
24. Hisadome K, Smith MA, Choudhury AI, Claret M, Withers DJ,
Ashford ML. 5-HT inhibition of rat insulin 2 promoter Cre recombinase
transgene and proopiomelanocortin neuron excitability in the mouse ar-
cuate nucleus. Neuroscience 159: 8393, 2009.
25. Hurov JB, Huang M, White LS, Lennerz J, Choi CS, Cho YR, Kim
HJ, Prior JL, Piwnica-Worms D, Cantley LC, Kim JK, Shulman GI,
Piwnica-Worms H. Loss of the Par-1b/MARK2 polarity kinase leads to
increased metabolic rate, decreased adiposity, and insulin hypersensitivity
in vivo. Proc Natl Acad Sci USA 104: 56805685, 2007.
26. Jansen M, Ten Klooster JP, Offerhaus GJ, Clevers H. LKB1 and
AMPK family signaling: the intimate link between cell polarity and energy
metabolism. Physiol Rev 89: 777798, 2009.
27. Kefas BA, Heimberg H, Vaulont S, Meisse D, Hue L, Pipeleers D, Van
de Casteele M. AICA-riboside induces apoptosis of pancreatic beta cells
through stimulation of AMP-activated protein kinase. Diabetologia 46:
250254, 2003.
28. Leclerc I, Woltersdorf WW, da Silva XG, Rowe RL, Cross SE,
Korbutt GS, Rajotte RV, Smith R, Rutter GA. Metformin, but not
leptin, regulates AMP-activated protein kinase in pancreatic islets: impact
on glucose-stimulated insulin secretion. Am J Physiol Endocrinol Metab
286: E1023E1031, 2004.
29. Lin X, Taguchi A, Park S, Kushner JA, Li F, Li Y, White MF.
Dysregulation of insulin receptor substrate 2 in beta cells and brain causes
obesity and diabetes. J Clin Invest 114: 908916, 2004.
30. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J,
Hawley SA, Udd L, Makela TP, Hardie DG, Alessi DR. LKB1 is a
master kinase that activates 13 kinases of the AMPK subfamily, including
MARK/PAR-1. EMBO J 23: 833843, 2004.
31. Long YC, Zierath JR. AMP-activated protein kinase signaling in meta-
bolic regulation. J Clin Invest 116: 17761783, 2006.
32. Martin SG, St Johnston D. A role for Drosophila LKB1 in anterior-
posterior axis formation and epithelial polarity. Nature 421: 379384,
2003.
33. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J,
Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, Kahn BB. AMP-kinase
regulates food intake by responding to hormonal and nutrient signals in the
hypothalamus. Nature 428: 569574, 2004.
34. Miyawaki K, Inoue H, Keshavarz P, Mizuta K, Sato A, Sakamoto Y,
Moritani M, Kunika K, Tanahashi T, Itakura M. Transgenic expres-
sion of a mutated cyclin-dependent kinase 4 (CDK4/R24C) in pancreatic
beta-cells prevents progression of diabetes in db/db mice. Diabetes Res
Clin Pract 82: 3341, 2008.
35. Mori H, Inoki K, Munzberg H, Opland D, Faouzi M, Villanueva EC,
Ikenoue T, Kwiatkowski D, MacDougald OA, Myers MG Jr, Guan
KL. Critical role for hypothalamic mTOR activity in energy balance. Cell
Metab 9: 362374, 2009.
36. Narbonne P, Roy R. Caenorhabditis elegans dauers need LKB1/AMPK
to ration lipid reserves and ensure long-term survival. Nature 457: 210
214, 2009.
37. Plum L, Belgardt BF, Bruning JC. Central insulin action in energy and
glucose homeostasis. J Clin Invest 116: 17611766, 2006.
38. Rafacho A, Cestari TM, Taboga SR, Boschero AC, Bosqueiro JR.
High doses of dexamethasone induce increased -cell proliferation in
pancreatic rat islets. Am J Physiol Endocrinol Metab 296: E681E689,
2009.
39. Riboulet-Chavey A, Diraison F, Siew LK, Wong FS, Rutter GA.
Inhibition of AMP-activated protein kinase protects pancreatic (beta)-cells
E1272 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

from cytokine-mediated apoptosis and CD8 T cell-induced cytotoxicity.
Diabetes 57: 415423, 2008.
40. Richards SK, Parton LE, Leclerc I, Rutter GA, Smith RM. Over-
expression of AMP-activated protein kinase impairs pancreatic beta-cell
function in vivo. J Endocrinol 187: 225235, 2006.
41. Rutter GA, Leclerc I. The AMP-regulated kinase family: Enigmatic
targets for diabetes therapy. Mol Cell Endocrinol 297: 4149, 2009.
42. Salt IP, Johnson G, Ashcroft SJ, Hardie DG. AMP-activated protein kinase
is activated by low glucose in cell lines derived from pancreatic beta cells,
and may regulate insulin release. Biochem J 335: 533539, 1998.
43. Schwartz MW, Porte D Jr. Diabetes, obesity, and the brain. Science 307:
375379, 2005.
44. Shorning BY, Zabkiewicz J, McCarthy A, Pearson HB, Winton DJ,
Sansom OJ, Ashworth A, Clarke AR. Lkb1 deciency alters goblet and
paneth cell differentiation in the small intestine. PLoS ONE 4: e4264,
2009.
45. Spurlin BA, Thurmond DC. Syntaxin 4 facilitates biphasic glucose-
stimulated insulin secretion from pancreatic beta-cells. Mol Endocrinol
20: 183193, 2006.
46. Sun G, Tarasov AI, McGinty J, McDonald A, DaSilva Xavier G,
Gorman T, Marley A, French PM, Parker H, Gribble F, Reimann F,
Prendiville O, Carzaniga R, Viollet B, Leclerc I, Rutter GA. Ablation
of AMP-activated protein kinase alpha1 and alpha2 from pancreatic
beta-cells and RIP.Cre neurons suppresses insulin release in vivo. Diabe-
tologia In press 2010.
47. Sutherland CM, Hawley SA, McCartney RR, Leech A, Stark MJ,
Schmidt MC, Hardie DG. Elm1p is one of three upstream kinases for the
Saccharomyces cerevisiae SNF1 complex. Curr Biol 13: 12991305,
2003.
48. Zhang L, Li J, Young LH, Caplan MJ. AMP-activated protein kinase
regulates the assembly of epithelial tight junctions. Proc Natl Acad Sci
USA 103: 1727217277, 2006.
E1273 LKB1 DELETION WITH RIP2.Cre TRANSGENE
AJP-Endocrinol Metab VOL 298 JUNE 2010 www.ajpendo.org

b
y

g
u
e
s
t

o
n

A
u
g
u
s
t

1
6
,

2
0
1
2
h
t
t
p
:
/
/
a
j
p
e
n
d
o
.
p
h
y
s
i
o
l
o
g
y
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m