Anti-Diabetic Effects of IGFBP2, a Leptin-regulated gene.

Kristina Hedbacker 1, Kivanc Birsoy 1, Robert W. Wysocki 1,2, Esra Asilmaz 1,

Rexford S. Ahima 3, I. Sadaf Farooqi 4, Jeffrey M. Friedman 1,2*

1 Laboratory of Molecular Genetics, Rockefeller University, New York, NY 10065, USA

2 Howard Hughes Medical Institute, New York, NY 10065, USA
3 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, and
Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania, USA.
4 University of Cambridge Metabolic Research Laboratories, Institute of Metabolic
Science, Addenbrooke's Hospital, Cambridge, CB2 0QQ, United Kingdom

*Corresponding author: Jeffrey Friedman friedj@mail.rockefeller.edu Phone number:

(212) 327-8000

RUNNING TITLE:

Leptin-regulated IGFBP2 corrects diabetes

SUMMARY

We tested whether leptin can ameliorate diabetes independent of weight loss by

defining the lowest dose at which leptin treatment of ob/ob mice reduces plasma
[glucose] and [insulin]. We found that a leptin dose of 12.5 ng/hour significantly lowers
blood glucose and that 25 ng/hour of leptin normalizes plasma glucose and insulin
without significantly reducing body weight, thus establishing that leptin exerts its most
potent effects on glucose metabolism. To find possible mediators of this effect, we
profiled liver mRNA using microarrays and identified IGF Binding Protein 2 as being
regulated by leptin with a similarly high potency. Over-expression of IGFBP2 by an
adenovirus reversed diabetes in insulin resistant ob/ob, Ay/a and diet-induced obese
mice, as well as insulin deficient streptozotocin-treated mice. Hyperinsulinemic clamp
studies showed a three-fold improvement in hepatic insulin sensitivity following IGFBP2
treatment in ob/ob mice. These results show that IGFBP2 can regulate glucose
metabolism, a finding with potential implications for the pathogenesis and treatment of
diabetes.

INTRODUCTION

Leptin treatment effectively corrects hyperglycemia and hyperinsulinemia in leptin-

deficient mice and humans and other forms of diabetes (Farooqi et al., 1999; Montague
et al., 1997; Muzzin et al., 1996). However, studies of the underlying mechanism are
complicated by the fact that leptin also causes marked weight loss, which by itself can
improve diabetes. To address this, we set out to define the lowest dose of leptin that
could correct insulin resistance and diabetes and identified two low doses at which an
infusion of leptin corrected hyperglycemia without normalizing food intake or reducing
body weight. Transcription profiles from the livers of ob/ob mice treated with these
doses identified several leptin regulated genes, including IGFBP2, which were
significantly induced even by the lowest dose of leptin (12.5 ng/hour subcutaneously), a
dose that does not significantly raise the plasma leptin level. IGFBP2 is a plasma
protein and one of 6 homologous proteins that can bind to IGFs. IGFBPs are generally
thought to inhibit the action of IGFs through high-affinity binding that prevents
interaction with IGF receptors (Dunger et al., 2004; Firth and Baxter, 2002; Kelley et al.,
1996; Kelley et al., 2002; Rosenzweig, 2004). A loss of IGF1 is known to cause
diabetic-like symptoms, as is over-expression of IGFBP1 (Crossey et al., 2000;
Rajkumar et al., 1999).
It has been suggested that IGFBP2 also inhibits IGF-1 (Firth and Baxter, 2002;
Kelley et al., 1996; Kovacs et al., 1999; Sadri and Lautt, 2000). IGF-1 is known to have
antidiabetic effects that are independent of the insulin receptor (Di Cola et al., 1997;
Froesch et al., 1996; Guler et al., 1987; Kovacs et al., 1999; Sadri and Lautt, 2000;
Zenobi et al., 1994). If true, this predicts that over-expression of IGFBP2 should worsen
diabetes in ob/ob mice; however, this was inconsistent with the finding that IGFBP2 is
induced by a low dose of leptin that improves diabetes and raised the possibility that it
could have the opposite effect. We thus considered that acute over-expression of
IGFBP2 could improve glucose metabolism. We tested this by replicating its expression
in liver using a recombinant adenovirus, which, under the conditions of these studies,
was only expressed in liver.
Here we show that acute IGFBP2 over-expression corrected hyperinsulinemia
and hyperglycemia not only in leptin-deficient ob/ob mice but also in DIO and Ay Type 2
diabetic mice which are leptin-resistant and insulin-resistant. IGFBP2 also normalized
glucose levels of insulin-deficient mice that were treated with streptozotocin. Finally,
hyperinsulinemic euglycemic clamp studies were used to establish the physiologic
mechanism by which IGFBP2 corrects diabetes in ob/ob mice.

RESULTS

Low-dose leptin treatment of ob/ob mice corrects hyperglycemia and

hyperinsulinemia independently of body weight.
We performed a dose-response study of leptin treatment with the goal of finding a
specific low dose of leptin that does not reduce weight and food intake but that has
positive effects on diabetes. Osmotic pumps were implanted subcutaneously in ob/ob
mice with increasing doses of leptin: 0, 12.5, 25, 50, and 100 ng/hour leptin for 12 days.
Daily weight and food-intake were measured (FIG 1A, 1B). At 0, 4, 8 and 12 days, mice
were fasted for 6 hours, anesthetized and blood was drawn. (The phlebotomy
procedure resulted in a modest weight loss in all of the groups).
As expected, mice receiving the highest doses of leptin, 50 and 100 ng/hour,
showed a significant decrease in food intake and body weight with normalization of
plasma glucose and insulin. A dose of 25 ng/hour of leptin also normalized plasma
glucose and insulin despite the fact that these animals failed to lose weight during the
course of the experiment. This dose resulted in a steady state plasma [leptin] of 0.7
ng/ml which is barely above background. Finally, a leptin dose of 12.5 ng/hour, which
fails to increase plasma leptin above background levels, significantly reduced plasma
glucose levels from 400 ng/mL to 208 ng/mL, p<0.05. This dose did not significantly
reduce either daily food intake or body weight. As a further control for this and
subsequent studies, we food restricted ob/ob mice to less than 4 grams of food/day
which is less than the food consumed by animals receiving the 12.5, 25, and 50 ng/hour
doses of leptin. These food-restricted animals failed to show any significant
improvement in plasma glucose or insulin (FIG 1A, 1B, 3B, 3C, and 3D).
These results identify two very low doses of leptin that either correct (25 ng/hour)
or improve (12.5 ng/hour) diabetes without causing the animals to lose weight during
the treatment. These data indicate that, for those parameters studied, the potency of
leptin is greatest for plasma glucose. In an effort to establish the mechanism
responsible for this effect of leptin, we next set out to identify genes that were
significantly regulated by leptin at the 12.5 and 25 ng/hour doses.

Leptin regulation of IGFBP2

We hybridized liver RNA prepared from animals receiving 12 days of 0, 12.5 or 25
ng/hour leptin to generate transcriptional profiles using Illumina microarrays. The
aggregate results for gene expression for each of these experiments are provided
(Supplemental FIG 1). Data were analyzed by generating an ordered list of induced
genes, comparing the levels of gene expression in liver RNA prepared from the animals
treated with 25 and 12.5 ng/hour of leptin to the PBS control mice. For the 25 ng/hour
group, the two most significantly induced genes were Serpina1e, a protease inhibitor,
and MUP, a testosterone sensitive urinary protein. The third most prominently induced
gene on this list was IGFBP2, a plasma protein with IGF binding activity. IGFBP2 has
been shown to inhibit IGF activity in some assays though its precise biologic function is
unknown (Firth and Baxter, 2002; Kelley et al., 1996). IGFBP2 was upregulated 5.28-
fold at the 25 ng/hour leptin dose, (p<0.01) and 1.6 fold in the 12.5 ng/hour mice
compared to PBS controls, (p<0.01) (Suppl. FIG 1).
We also used a Taqman assay to confirm that IGFBP2 mRNA was regulated by
leptin. IGFBP2 mRNA levels were upregulated 2-fold in the 12.5 ng/hour mice
compared to controls (0.6 vs. 0.2, p=0.11), 3-fold in 25 ng/hour mice (0.9 vs. 0.2,
p<0.05), 10-fold in 50 ng/hour mice (2.7 vs. 0.2, p<0.05), and 13-fold in100 ng/hour
mice (3.2 vs. 0.2, p<0.05) (FIG 2A).
The effect of leptin treatment on plasma levels of IGFBP2 was measured using
an Elisa (EIA) assay. IGFBP2 circulates at a level of 396 ng/mL in wild type mice and its
baseline levels are significantly reduced in ob/ob to 31 ng/ml, p<0.01 (FIG 2B). Leptin
significantly increased plasma IGFPB2 levels in ob/ob mice by the 4th day of treatment,
with the 100 ng/hour leptin dose increasing it 8-fold by the 12 th day (246 vs. 31 ng/mL in
ob/ob mice, p<0.01). IGFBP2 levels increased 4.3-fold in the 25 ng/hour group (132 vs.
31 ng/mL in ob/ob mice, p<0.01) by the 12th day. There was also a similar trend toward
increased levels of IGFBP2 in the 12.5 ng/hour group (FIG 2B).
Because low doses of leptin markedly improve insulin sensitivity, we considered
the possibility that the leptin mediated induction of IGFBP2 was insulin dependent. We
tested this possibility in two ways. First, ob/ob mice were severely food restricted (0.5
grams/day for 12 days – this is the voluntary food intake of ob/ob mice receiving 200
ng/hr leptin) which resulted in insulin levels falling by 90%. Despite this reduction in
plasma insulin, plasma IGFBP2 remained constant between food-restricted and controls
(35 ng/mL in food restricted vs. 34 ng/mL eating ad libitum, p>0.7) (FIG 2C). We further
examined the relationship between insulin levels and IGFBP2 expression by injecting
wildtype animals with a bolus of insulin and found that an acute injection of insulin failed
to increase plasma IGFBP2 levels at 1 and 3 hours post-injection (Supplementary figure
4). Consistent with these results, the baseline levels of IGFBP2 were similar among
several different animals with markedly different baseline levels of insulin, including wild
type mice, Type I diabetic mice resulting from streptozotocin treatment (STZ), and Diet
Induced Obese (DIO) mice (FIG 2C and 5B).

Ad-IGFBP2 treatment corrects hyperglycemia, hyperinsulinemia, and hepatic

steatosis in ob/ob diabetic mice.
We next tested whether over-expression of IGFBP2 can correct hyperglycemia and
hyperinsulinemia in diabetic mice. A mouse IGFBP2 cDNA was cloned into an empty
shuttle adenoviral vector under the control of the CMV promoter. This is a gutless
adenoviral vector that fails to express adenoviral genes, thus mitigating secondary
effects of the viral infection. As a control, we used additional adenovirus strains with no
insertion or with an insertion of a luciferase reporter. The luciferase virus allowed us to
assess the sites of gene expression from the viral vector. Five days after intravenous
injections of the Ad-luciferase adenovirus, mice received an intraperitoneal injection of
luciferin and were imaged using a CCD camera (IVUS, Caliper Technology). These data
showed that viral gene expression was limited to the liver, the site of endogenous
IGFBP2 expression, and also at the site of injection in the tail (Supplemental Figure 2A).
Animals injected with the IGFBP2 adenovirus showed a highly significant
increase in plasma IGFBP2 levels to at least 4000 ng/mL (data not shown). The
IGFBP2 and empty (control) adenoviruses were injected into ob/ob mice followed by
measures of daily body weight and food intake (FIG 3A and 3B) and plasma glucose
and insulin five days after viral injection (FIG 3C and 3D). Mice treated with the IGFBP2
adenovirus showed a modest decrease in food intake with a stabilization of body weight
while the control ob/ob mice continued to gain weight (FIG 3A and 3B). At 5 days post-
injection, the ob/ob mice that had received the IGFBP2 treatment had completely
normalized plasma glucose and insulin. While control mice had blood glucose levels of
over 300 mg/dL, IGFBP2 treated animals had blood glucose levels under 100 mg/dL
(320 vs. 94 mg/dL for the controls, p<0.01) (FIG 3C). Plasma insulin levels were also
normalized in the IGFBP2 treated mice (85 vs. 5 ng/mL, p<0.01) (FIG 3D). ob/ob mice
pair-fed to the IGFBP2-treated ob/ob animals failed to show a reduction of blood
glucose or plasma insulin (FIG 3A, 3B, 3C and 3D).
We performed glucose tolerance tests in the IGFBP2-treated mice. While plasma
glucose levels of the control mice peaked above 550 mg/dL at 30 minutes post glucose-
injection, the blood glucose of IGFBP2-treated mice remained below 300 mg/dL (559
vs. 288 mg/dL, p< 0.01) (FIG 4A). GTT of IGFBP2 treated ob/ob showed a complete
correction of glucose clearance in response to a glucose challenge (compare FIG 4A
and FIG 5E).
To assess the mechanism of these effects, we utilized hyperinsulinemic-
euglycemic clamps to study insulin sensitivity, hepatic glucose output and glucose
uptake in treated and control mice. The glucose infusion rates (GIR) required to
maintain blood glucose levels at ~140 mg/dL were 3.6-fold higher in IGFBP2-treated
animals compared to controls (36 mg/kg/min in IGFBP2-treated ob/ob vs. 10 mg/kg/min
in controls, p<0.01) (FIG 4B), thus showing a marked effect of IGFBP2 to improve
insulin sensitivity. Hepatic glucose production (HGP) during the insulin clamp of
IGFBP2-treated mice was 11 mg/kg/min compared to 32 mg/kg/min in controls (p<0.01)
(FIG 4D). In response to the super-physiological 30mU/kg/min insulin infusion, the HGP
was suppressed by 77% of the basal glucose production in IGFBP2 treated ob/ob
versus 34% suppression in controls, p<0.01) (FIG 4E). In contrast, the glucose
disappearance rate (Rd) during the clamp was not significantly different between
IGBP2-treated and control ob/ob mice (FIG 4C). We also measured 2-deoxyglucose
uptake in WAT and gastrocnemius/soleus muscle at the end of the clamp. WAT glucose
uptake was not affected by IGFBP2 treatment (Supplemental figure 3A). Muscle
glucose uptake was slightly increased in IGFBP2-treated ob/ob relative to wildtype but
the difference was not statistically significant (25 nmol/g/min in IGFBP2 treated vs. 17 in
controls, p=0.1) (Supplemental figure 3B). Together, these results demonstrate that
IGFBP2 treatment markedly increases insulin sensitivity primarily by suppressing
hepatic glucose output.
The improvement in hepatic insulin sensitivity by IGFBP2 treatment was
associated with a significant reduction of hepatic steatosis. Liver histology showed a
drastic reduction in lipid droplets in the livers of IGFBP2-treated ob/ob mice (FIG 3F).
Liver triglycerides were significantly reduced by IGFBP2 and low-dose leptin treatment
(Figure 3E).
We next tested the effect of IGFBP2 over-expression in wild type mice and other
leptin-resistant diabetic mouse strains. In each case, plasma [glucose] was significantly
decreased in the animals receiving the IGFBP2 adenovirus. By the 6th day after IGFBP2
infection, blood glucose in wild type mice fell 37% (90 vs. 142 mg/dL, p<0.01); in DIO
animals, blood glucose fell 32% (117 vs. 171 mg/dL, p<0.01), and finally blood glucose
fell 34% in Ay mice (134 vs. 202 mg/dL, p<0.05) (FIG. 5A). There was a trend for
reduction in insulin levels in all these mice although it only reached significance in Ay
mice (FIG 5B). The levels of blood glucose during glucose tolerance tests were also
significantly reduced at all time points including the peak level at 30 minutes in wild
type, Ay, and DIO mice treated with the IGFBP2 adenovirus vs. controls (FIG 5E, 5F,
5G and 5H). Changes in food intake and body weight were minimal and in most cases
not statistically significant (FIG 5C and 5D).

Ad-IGFBP2 treatment corrects hyperglycemia in insulin deficient mice.

Previous studies have shown that leptin treatment corrects hyperglycemia in Type 1
diabetic mice (Yu et al., 2008). We next tested whether IGFBP2 can improve diabetes in
this setting of insulin deficiency. IGFBP2 was injected into streptozotocin-induced Type
1 diabetic/insulin deficient mice. Plasma insulin was not detectable 6 weeks post low-
dose STZ-treatment even when using an ultra-sensitive mouse insulin EIA kit (FIG 5B
and data not shown). At day 5 after IGFBP2-injections, control mice had fasting (4 hour)
glucose levels of 509 as compared to 136 mg/dL of the IGFBP2 treated group, p<0.01
(FIG 5A). Glucose tolerance tests were also markedly improved at 45 minutes (654 vs.
342 mg/dL, p<0.05) (FIG 5G). Food intake and body weight were not changed in either
groups (FIG 5C and 5D).

IGFBP2 levels in leptin-deficient patients.

Finally we assessed whether IGFBP2 regulation by leptin is evident in human subjects.
To this end, we obtained sera from three leptin deficient patients and fourteen age and
weight-matched controls, before and after 6 months of daily subcutaneous low-dose
leptin treatment. These patients and their treatment have been previously described
(Farooqi et al., 2002). There was a two-fold decrease in baseline serum IGFBP2 levels
in the leptin-deficient (LEP/LEP) versus controls (leptin deficient patients had 71 ng/mL
serum IGFBP2 vs. 132 ng/mL of controls, p=0.04) (FIG 6A). After low-dose leptin
treatment, IGFBP2 levels increased in 2 out of 3 patients; from 50 to 88 ng/mL in
Patient 1, 90 to 132 ng/mL in Patient 2, while one patient showed no change (74 and 74
ng/mL). The average levels of IGFBP2 before and after leptin treatment were 71 ng/mL
and 98 ng/mL respectively, p=0.2 (FIG 6B).

DISCUSSION
Leptin has been shown to improve hyperglycemia and insulin resistance in a number of
clinical settings in animals and humans. These conditions include leptin
mutations/deficiency, lipodystropy, Rabson-Mendenhall syndrome, resulting from insulin
receptor mutations, and more recently Type 1 diabetes in NOD mice, which have a
complete insulin deficiency (Asilmaz et al., 2004; Farooqi and O'Rahilly, 2004;
Montague et al., 1997; Oral et al., 2002; Petersen et al., 2002; Yu et al., 2008). The
mechanism by which leptin exerts these salutary effects is poorly understood. In this
report, we sought to explore the mechanism responsible for leptin’s anti-diabetic effects
and first showed that leptin can correct diabetes of ob/ob mice at low doses that do not
significantly reduce body weight. IGFBP2 gene expression is induced by these low
doses of leptin treatment and IGFBP2 plasma levels increase in response to leptin
treatment of ob/ob and wild type mice. Over-expression of IGFBP2 using a recombinant
adenovirus resulted in a striking reduction of plasma glucose and insulin in all animals
tested including ob/ob, wild type mice, as well as Ay, DIO, and streptozotocin treated
animals. Overall, these data confirm that leptin can improve glucose homeostasis
independent of its ability to reduce weight and suggest that IGFBP2 may account for a
portion of leptin’s anti-diabetic effects.
IGFBP2 is a 34 kD plasma protein produced by liver. It was originally isolated
based on its ability to bind to IGF1 and IGF2 and is one of six IGF binding proteins that
circulate in plasma (Baxter and Martin, 1989; Kelley et al., 2002; Martin and Baxter,
1986). Further studies in vitro suggested that IGFBP2 and the other IGFBPs function as
IGF inhibitors by chelating these ligands (Hoflich et al., 1998; Jones and Clemmons,
1995). However, IGFBP2 circulates at equimolar, or lower molar, concentrations
compared to IGF1 which is not a typical characteristic of hormone inhibitors that
generally circulate in molar excess of the ligand. This raises the possibility that IGFBP2
may not inhibit IGF-1 in vivo or might have IGF-1 independent effects. In vivo, IGFBP2
has been invoked as playing a role to modulate IGF signaling in growth and
development, cancer and diabetes. The precise function of IGFBP2 is poorly
understood and it is not well established whether it inhibits or activates IGF1 signaling
in vivo or if it has actions independent of IGF (Wolf et al., 2000). (These possibilities are
not mutually exclusive).
 An IGFBP2 knockout does not have a major metabolic phenotype and it as been
suggested that other IGFBPs can compensate for its loss (Pintar et al., 1995; Wood et
al., 1993). However, a possible role for IGFBP2 in the regulation of metabolism has
been suggested by its association to diabetes in several human whole genome studies
(Cauchi et al., 2008a; Cauchi et al., 2008b; Grarup et al., 2007; Hertel et al., 2008;
Horikawa et al., 2008; Sanghera et al., 2008; Saxena et al., 2007; Scott et al., 2007;
Zeggini et al., 2007). In addition, a CMV-IGFBP2 transgene has been shown to
modestly prevent weight gain and hyperglycemia in DIO mice. However, in that report, it
was not shown whether the effect of IGFBP2 on diabetes was independent of its effect
on body weight nor was the potential therapeutic benefit of acute IGFBP2 over-
expression established (Wheatcroft et al., 2007).
The results reported here show an ability of IGFBP2 to profoundly reduce plasma
glucose and insulin when acutely over-expressed from an adenoviral vector in wild type
and ob/ob mice as well as markedly hyperglycemic STZ-induced Type 1 diabetic mice
and even in leptin resistant DIO and Ay mice, both of which have Type 2 diabetes (Lin
et al., 2000; Prpic et al., 2003; Van Heek et al., 1997). Thus IGFBP2 can improve
glucose metabolism in leptin sensitive and leptin resistant animals, an observation that
is consistent with the finding that IGFBP2 is regulated by, and thus downstream of,
leptin signaling. IGFBP2 levels are not higher in DIO and Ay animals than in wild type
mice, which is consistent with the fact that they are leptin resistant. The observation that
these leptin resistant animals still respond to exogenous IGFBP2 similarly to leptin
sensitive animals is also consistent with IGFBP2 being downstream of leptin signaling in
brain.
In these studies, we used an adenovirus to over-express IGFBP2 because this
protein has 7 disulphides and is difficult to produce as a recombinant protein in sufficient
amounts to perform the studies reported here. Adenoviral expression results in IGFBP2
protein levels that are significantly higher than the level at which it normally circulates in
the blood stream and further studies will be required to determine whether the observed
effects are physiologic. Efforts to produce bioactive recombinant IGFBP2 are underway.
The mechanism by which IGFBP2 reduces blood glucose is also under
investigation. Hyperinsulinemic euglycemic clamp show that IGFBP2 markedly
improves hepatic insulin-sensitivity in ob/ob mice with a three-fold increase in the
glucose infusion rate. However, it is important to point out that despite the infusion of
more than 10 times the insulin dose typically given to wild type mice, the glucose
infusion rate was still lower, and the HGP in IGFBP2-treated ob/ob mice was still higher,
than the levels seen in wild type mice (Ahima et al., 2006; Qi et al., 2006). We propose
that the improvement in hepatic insulin sensitivity in IGFBP2-treated ob/ob mice is due,
at least in part, to a reduction in hepatic steatosis. A similar negative correlation
between hepatic steatosis and insulin sensitivity has been described in ob/ob mice
treated with an insulin-sensitizing aminosterol (Takahashi et al., 2004) or antisense
oligonucleotides against the lipid droplet protein ADRP (Imai et al., 2007). Further
studies will be required to determine to what extent the amelioration of steatosis can
account for the beneficial effects of IGFBP2 on hepatic glucose production. Further
studies will also be necessary to determine whether IGFPB2 improves steatosis and
hepatic insulin sensitivity by an autocrine mechanism or indirectly via modulating the
activity of other organs or the levels of other hormones.
Since IGFBP2 does not fully correct HGP but nonetheless normalizes plasma
glucose, insulin and a GTT in ob/ob mice, it is possible that it could also have additional
insulin independent effects to reduce blood glucose. This possibility is consistent with
the observation that the IGFBP2 adenovirus can also normalize glucose levels in insulin
deficient STZ mice. While it is conceivable that IGFBP2 can sensitize these mice to a
low level of residual insulin, it is also possible that IGFBP2 acts through an insulin or
even IGF independent mechanism. Studies to further explore the hepatocellular
mechanism by which IGFBP2 reduces blood glucose are currently underway.
The data in this report also establish that the potency of leptin for reducing
hyperglycemia is greater than that for correcting food intake and body weight. While an
early study of leptin showed direct effects on hyperglycemia independent of weight loss
in ob/ob mice, the study used daily IP bolus injections of leptin as opposed to the
continuous doses used in this study (Pelleymounter et al., 1995). In other studies, leptin
improved diabetes to a greater extent than pair-feeding, suggesting that leptin had
independent effects on glucose metabolism (Levin et al., 1996; Schwartz et al., 1996).
However, these studies did not identify the mechanisms by which leptin improved
diabetes without reducing weight. Finally a study of low dose leptin infusions to treat
diabetic lipodystrophic animals was limited by the fact that the underlying condition of
these animals made it difficult to assess whether there was significant weight loss
(Asilmaz et al., 2004). Thus our data confirm that leptin regulates glucose metabolism
independent of its effects on energy balance and identify conditions under which this
effect can be studied without a confounding effect on body weight.
The low doses of leptin used in these studies also reveal a potent effect of leptin
to regulate several liver genes, some of which could also play a role in mediating
leptin’s effects. Previous studies of a brain and liver specific knockout of the leptin
receptor, and a brain specific leptin receptor transgenic mouse, have indicated that the
brain is the primary site of leptin’s actions and that its effects on liver are indirect (Cohen
et al., 2001). The nature of the signal responsible for the induction of IGFBP2 by leptin
is unknown but does not appear to be insulin, as acute increases or subacute
decreases of plasma [insulin] do not alter circulating IGFBP2 levels (Supplementary
figure 4 and Figure 2C). Further studies are necessary to establish whether leptin
regulation of IGFBP2 is mediated by efferent neural outputs from the CNS and/or a
result of modulation of its gene expression by other hormones.
Finally, consistent with the mouse data, we found lower IGFBP2 levels in leptin-
deficient human subjects compared to control subjects, and that IGFBP2 levels
increased after leptin treatment in two out of three patients. This raises the possiblity
that IGFBP2 could have therapeutic effects in human diabetes, which would require the
use of recombinant protein rather than a viral vector. A variety of eukaryotic expression
systems will be tested for their ability to yield bioactive IGFBP2.
In summary, we have developed a protocol in which leptin treatment potently
improves diabetes independent of its ability to correct weight and food intake. This
protocol was used to identify IGFBP2 as a leptin regulated gene whose expression is
correlated with leptin’s anti-diabetic effect. IGFBP2 over-expression reduces blood
glucose in wild type and diabetic mice and potently suppresses heptic glucose
production suggesting that it may play a role in mediating some portion of leptin’s anti-
diabetic effects. Further studies will reveal whether IGFBP2 shows similar anti-diabetic
effects in clinical settings.
EXPERIMENTAL PROCEDURES

Mice and Diet

Leptin dose experiment: 8 week old male C57Bl6 Lep-ob/ob mice were obtained
from the Jackson laboratory (Bar Harbor, ME), acclimated to our mouse facility under
controlled light conditions (12 hr light/12 hr dark), temperature (22°C), single-caged and
fed ad libitum a standard mouse chow for at least a week. Alzet 2002 mini-osmotic
pumps (Alzet, Palo Alto, CA) were filled with indicated concentrations of leptin (Amgen,
Thousand Oaks, CA) and incubated at 37°C in sterile 0.9% NaCl overnight and
implanted subcutaneously in 9-11 weeks old mice. Mice were single-caged and their
weight and food-intake were recorded daily. At 0, 4, 8, and 12 days of treatment, mice
were anesthetized and blood was collected intraorbitally. At day 12, mice were
anesthetized, livers were collected fresh frozen in liquid N2 or fixed in Accustain
Formalin Solution Neutral Buffered 10% Formalin (Sigma-Aldrich, St Louis, MO)
overnight for histology.
Ad-IGFBP2 experiments: 8 week old male C57Bl6 Lep-ob/ob or wild type mice
were acclimated as above. Ay mice were ordered from Jackson laboratory and kept
until at least 12 weeks of age. Wildtype male mice were ordered from Jackson
laboratories and kept on a standard high-fat-diet chow until at least 15 weeks of age.
Mice received intrajugular vein injections under anesthesia (isoflurane) of 1.2x10^11
particles of Ad-CMV-empty, Ad-CMV-Luciferase or Ad-CMV-IGFBP2 (ViraQuest Inc.). 1
week after injections, plasma levels of IGFBP2 were confirmed with an IGFBP2 EIA kit
(Alpco).
Streptozotocin-treated mice: 5 week old male C57Bl6 wild type mice were
obtained from the Jackson laboratory (Bar Harbor, ME) and received a daily
intraperitoneal dose of 50 mg/kg Streptozotocin (Sigma) for 5 days. 4 weeks later,
hyperglycemic mice were injected with Ad-IGFBP2 as described above.

Metabolic studies
Glucose tolerance test: Mice were injected intraperitoneally with 1 unit
glucose/gram body weight. Blood glucose was recorded at 0, 30, 60, and 120 minutes
post-injection. In STZ mice, blood glucose was recorded at 0 and 45 minutes post-
injection.
Hyperinsulinemic euglycemic clamp: This has been previously described in (Qi et
al., 2006). 10 week old male ob/ob mice were treated with Ad-CMV-IGFBP2 or Ad-
CMV-control via a tail vein injection, and insulin clamp was performed 10 days later. An
indwelling catheter was inserted in the right internal jugular vein under sodium
pentobarbital anesthesia and extended to the right atrium. After regaining their
presurgery weight (4 days), the mice were fasted for 6 hours, a bolus injection of 5 µCi
of [3-3H] glucose was administered, followed by continuous intravenous infusion at 0.05
µCi/min. Baseline glucose kinetics was measured for 60 min. A priming dose of regular
insulin (40 mU/kg, Humulin; Eli Lilly, Indianapolis, IN) was given intravenously, followed
by continuous infusion at 30 mU•kg-1•min-1. Blood glucose was maintained at 120-140
mg/dL via a variable infusion rate of 30% glucose. At the end of the 120-minute clamp,
10 µCi 2-deoxy-D-[1-14C]glucose was injected to estimate glucose uptake. The mice
were euthanized, and liver, perigonadal fat (WAT), and soleus/gastrocnemius muscle
were excised, frozen immediately in liquid nitrogen, and stored at –80°C for subsequent
analysis of glucose uptake. The rates of basal glucose turnover and whole body glucose
uptake are measured as the ratio of [3H] glucose infusion rate (dpm) to the specific
activity of plasma glucose. Hepatic glucose production (HGP) during clamp is measured
by subtracting the glucose infusion rate (GIR) from the whole body glucose uptake (Rd).
Liver triglycerides: Liver triglycerides were determined using Sigma TR0100
Triglyceride Determination Kit.

Human subjects
Fasting plasma samples were obtained from three children with leptin deficiency before,
and one and six months after treatment with recombinant human leptin as reported
previously (Farooqi et al., 2002). All samples were stored at minus 80C and thawed
once prior to analysis. Results were compared to age and BMI matched controls on
whom fasting plasma samples had been obtained.

RT-PCR and microarray

Total RNA was isolated by homogenizing liver tissue in TRIzol reagent (Invitrogen) and
purifying the RNA using Qiagen RNA prep kit (Qiagen). Real-time PCR was performed
using the TaqMan system (Applied Biosystems) according to the manufacturer’s
protocol as previously described (Birsoy et al., 2008). Microarrays were done using
MouseRef-8 v2 BeadChip (part# 11288185) after labeling the RNA with Ambion’s
Illumina TotalPrep RNA Amplification Kit.

Serum Assays
Blood glucose was determined using an Ascensia Elite XL glucometer (Bayer) or
QuantiChrom Glucose Assay Kit (BioAssay Systems, Hayward, CA). For all other
assays, mice were bled intraorbitally while anesthetized with isoflourane. Blood was
spun for 10 minutes and plasma collected. Plasma insulin was determined using an
Insulin (mouse) EIA kit (Alpco Diagnostics, Windham, NH). Plasma leptin and IGFBP2
levels were determined using a Leptin (mouse/rat) EIA kit and an IGFBP-2 (mouse/rat)
EIA kit, respectively (Alpco Diagnostics, Windham, NH). Plasma IGF 1 was determined
using a Mouse IGF-I Quantikine ELISA Kit (R&D Systems).

Histology
Paraffin-embedded, 10% formalin-perfused livers were sectioned and stained with
Hematoxylin and Eosin.

Statistical Analysis
All data was analyzed for statistical significance using the student’s t-test. P-values as
indicated.

ACKNOWLEDGMENTS

We would like to thank Roger Unger, Domenico Accili, Allyn Mark, Stephen O’Rahilly
and Paul Cohen, for constructive criticism on experiments and this manuscript. We
would also like to thank Shaheen Kabir, and Susan Korres for technical assistance.
Lastly, we would like to thank Ravindra Dhir at the University of Pennsylvania Diabetes
Endocrinology Research Center (DERC) Mouse Metabolic Phenotyping Core for
performing the clamp and radioisotopic tracer studies. The DERC is supported by NIH
grant P30-DK-19525.
FIGURE LEGENDS

Figure 1
Low-dose leptin treatment of ob/ob mice corrects blood glucose and
hyperinsulinemia independently of body weight. Mice receiving 12 day leptin
treatment. Dose of leptin as indicated in 1A. Mice fasted for 6 hours prior to receiving
anesthesia and blood collection at day 0, 4, 8, and 12. For each group, n≥4. A) Percent
change in body weight during 12 day leptin treatment. Arrows show day 4 and 8 of
treatment. Dotted line indicates food restricted animals (see figure 3A, 3B. 3C and 3D).
B) Food intake in grams each day during treatment. Dotted line indicates food restricted
animals (see figure 3A, 3B. 3C and 3D). C-E) ng/mL plasma leptin, mg/dL blood
glucose, and ng/mL plasma insulin at day 12 of leptin treatment. Blood glucose and
plasma insulin for food-restricted animals can be found in Figure 3C and 3D. Error bars
show standard error. * p<0.05, ** p<0.01.

Figure 2
Leptin regulation of IGFBP2. A) Relative IGFBP2 mRNA expression in liver samples
from ob/ob mice leptin-treated for 12 days as indicated. B) Plasma IGFBP2 levels in
ob/ob mice during treatment with leptin at the indicated concentrations. C) Plasma leptin
(left y-axis) and plasma IGFBP2 (right y-axis) in mice as follows: wt=wild type,
wt+leptin=wildtype after 8 days of 1 ug/hr leptin, DIO=diet-induced obese (high-fat diet
for 15 weeks), STZ=streptozotocin diabetic, ob/ob mice, ob/ob+leptin=ob/ob after 12
days of 100 ng/hour leptin treatment, ob/ob food restr. = ob/ob after 12 days of food
restriction to 0.5g/day (insulin levels 1/10th of free-feeding – data not shown), Ay=agouti,
Srebp-1c. Grey bars indicate IGFBP2. Black bars indicate leptin levels in same animals.
Error bars show standard error. * p<0.05, ** p<0.01.

Figure 3
IGFBP2 treatment corrects hyperglycemia, hyperinsulinemia, and hepatic
steatosis in ob/ob diabetic mice. ob/ob mice treated with Ad-control (blue) or Ad-
IGFBP2 (red) or untreated and pair-fed to Ad-IGFBP2. N≥4. A) and B) Body weight and
food intake in grams. Food intake is average for 24 hours. Mice injected with adenovirus
on day ‘0’. Arrow indicates 18-hour fast (for GTT). X-axis indicates day of experiment.
Dotted line shows body weight and food intake of mice pair-fed to the IGFBP2 treated
mice. C) and D) plasma glucose and plasma insulin in treated, control, and pair-fed
mice. E) Milligrams triglycerides per gram liver tissue in ob/ob control, ob/ob+IGFBP2,
ob/ob+12 days 100 ng.hr leptin and ob/ob+ 12 days of 25 ng/hr leptin. F) H&E stained
liver paraffin sections of treated and control mice. 10x and 40x as indicated. * p<0.05, **
p<0.01.

Figure 4
IGFBP2 treatment corrects hyperglycemia in wild type and diabetic mice. Red=
IGFBP2 treated ob/ob mice, blue= control treated ob/ob mice. * p<0.05, ** p<0.01. n=7.
A) Glucose tolerance test (GTT) in ob/ob mice. B) Glucose infusion rate (GIR) in
mg/kg/min necessary to keep hyperinsulinemic mice euglycemic during clamp studies.
C) Rd (whole body glucose disappearance in mg/kg/min. D) Hepatic glucose production
(HGP) in mg/kg/min. E) Percent suppression of hepatic glucose production in response
to hyperinsulinemic clamp.

Figure 5
Comparison of effect of IGFBP2 treatment on blood glucose, insulin, daily food
intake, body weight and glucose tolerance in wild types, ob/ob, Ay, STZ and DIO
mice.
Red= IGFBP2 treated, blue= control treated. * p<0.05, ** p<0.01. wt=wildtype,
Ay=Agouti, DIO=diet-induced obese (15 weeks on high-fat diet), STZ=streptozytocin-
induced Type 1 diabetic mice. Blood glucose and insulin taken on day 6 post-infection.
Daily food intake is average of day 4-14 post-infection. Change in weight is total change
in weight from day 4-14 post-infection.

Figure 6
IGFBP2 is regulated by Leptin in humans.
A) Serum IGFBP2 in leptin deficient and age and weight-matched controls. B) Serum
IGFBP2 in 3 leptin deficient patients before (light grey), and 6 months after (dark grey)
low-dose leptin treatment.
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