RIP-Cre Mice Research Articles

2124-P: Raptor Levels Are Critical in the Adaptation of Beta Cells to High-Fat Diet

mTORC1 signaling is a central regulator of autophagy and altered autophagic activity has been implicated in the beta-cells of patients with type 2 diabetes, and in the beta-cells of obese mice. The importance of mTORC1 signaling and Raptor in beta-cells has been recently demonstrated using raptor knock-out models (βraKO mice). These studies have shown that complete deletion of Raptor impairs beta-cell proliferation and survival of the beta-cell, and both in function and insulin processing. However, nothing is known whether reduced mTORC1 levels impair beta-cell adaptation to diabetogenic conditions. Therefore, we decided to study mice with haploinsufficiency of Raptor in beta-cells (βraHET). Mice with heterozygous deletion of Raptor in beta-cells were generated by crossing raptorf/f with Rip-Cre mice (βraHET). βraHET and control mice were fed regular chow (RC) or high-fat diet (HFD). Mice fed with RC showed no difference at the metabolic level, islets morphology, or beta-cell function. βraHET mice fed HFD for eight weeks showed an impaired Intraperitoneal Glucose Tolerance Test, fed insulin secretion and a decreased beta-cell mass, compared to control mice. Isolated islets from βraHET mice fed HFD for eight weeks showed impaired insulin secretion either measured by perifusion or static incubation compared to control islets. Next, we tested whether isolated islets from βraHET mice are more susceptible to beta-cell stressors. Beta-cell death determined by TUNEL and cleavage Caspase 3 was increased (50 and 60%) in the βraHET islets after 24 h exposure to palmitate. In addition, autophagy markers such as LC3 and p62 were increased in islets from βraHET treated with palmitate. The increase in cell death and autophagy is specific to lipotoxicity since there βraHET islets exhibited no increase in cleavage Caspase 3 in TUNEL when they were treated with proinflammatory cytokines or ER Stress inducers. In conclusion, we demonstrate that adequate levels of Raptor are critical in the adaptation of the beta-cell to HFD. Disclosure M. Blandino-Rosano: None. E. Bernal-Mizrachi: None.

53-OR: Deletion of YTHDF2 in Beta Cells Results in Glucose Intolerance

N6-methyladenosine (m6A) is the most common and abundant mRNA modification. However, the role of m6A in regulating β-cell function is unclear. YTHDF2 is a ’reader’ protein that recognizes m6A-containing mRNAs and targets them for degradation. To address the functional role of YTHDF2 in β-cells, we generated an Ythdf2 β-cell knockout mouse using RIP-Cre transgenic mice. At the 3-month old age, the feeding and fasting glucose of βKO mice were similar to that in control mice (RIP-Cre mice). However, both male and female βKO mice are glucose intolerant after glucose challenge. The AUC was increased by 44.6% in male βKO mice and 71.1% in female mice, respectively (both P<0.01). The response to insulin was similar between βKO and control mice. Interestingly, although the fasting insulin levels were comparable between βKO and control mice, the feeding insulin levels in both male and female βKO mice were increased. The serum insulin levels from βKO mice were significantly lower at 15 min after glucose challenge. In addition, the insulin secretion from βKO islets was less due to decreased insulin protein. Surprisingly, β-cell mass in βKO mice was significantly higher than that in control mice (P<0.05). Both islets number and size were increased in βKO mice. To understand the molecular mechanism, we performed RNA-seq to detect the gene expression in βKO and control islets isolated from male mice. Among 626 altered genes in βKO islets, 421 genes were upregulated and 205 genes were downregulated. The genes response to ER stress were among the top up-regulated genes. The genes related to insulin secretion were downregulated. We applied m6A-seq in MIN6 cells and confirmed that Ire1α was indeed modified by m6A in its last exon and 3’ UTR region. We further confirmed that both mRNA and protein levels of Ire1α and spliced XBP-1 were significantly increased in βKO islets. In conclusion, YTHDF2 plays an important role in regulating insulin secretion, and YTHDF2 deficiency in β-cells induces glucose intolerance by regulating insulin secretion and ER stress. Disclosure D. Ren: None. J. Sun: None. Funding The University of Chicago (P30-DK020595)

363-OR: ChREBP-Beta Controls Both Proliferation and Cell Death of Pancreatic Beta Cells

Glucose is a key physiological driver of adaptive beta cell mass expansion, promoting beta cell proliferation both in vitro and in vivo. Carbohydrate response element binding protein β (ChREBPβ) is upregulated in response to glucose and is required for glucose-stimulated beta cell proliferation. To study the role of ChREBPβ in beta cells, we knocked out (KO) or overexpressed (OE) ChREBPβ in mouse pancreatic beta cells using Cre LoxP approaches with either MIP-CreERT (induced by tamoxifen at 3 months of age), or the RIP-Cre model which leads to recombination specifically in β cells, early during embryogenesis (E9). We performed intraperitoneal glucose tolerance tests (IPGTTs) and assessed pancreata for proliferation (Ki67), cell death (TUNEL) and beta cell mass in the 4 mouse models. ChREBPβ MIP-CreERT KO mice displayed impaired glucose tolerance, with an increase of 25.3 ± 7.9% (N=3-4, P<0.05) in the area under the curve (AUC) of IPGTT. Heterozygous or complete Chrebpβ KOs in the RIP-Cre model resulted in an increase in the AUC of IPGTT compared with control littermates (20.1 ± 13.7% and 56 ± 7.1%, respectively; n=2-4). Beta cells from isolated ChREBPβ KO islets had 60.4 ± 17.4% less proliferation compared to control islets in 6 mM glucose, and 22.9 ± 2.4% (P<0.05, N=5) less in 20 mM glucose. Inducible OE of ChREBPβ resulted in 97.7 ± 6% increase in AUC of IPGTT (N=3, p<0.01) and fasting blood glucose levels were increased by 89 ± 13.2% (P<0.01). Similarly, when ChREBPβ was OE early in the embryogenesis of β cells, we observed AUC for GTT was increased by 2.4 ± 0.5 fold compared to non-Cre expressing litter mates (N=5, p<0.05). In both mouse models, the OE of ChREBPβ resulted in β cell death as assessed by TUNEL along with increased in Ki67 staining. Remarkably, the RIP-Cre mice had almost no remaining β cells at 4 months of age and were overtly diabetic. Taken these data together, we propose a molecular regulatory mechanism where ChREBPβ controls both beta cell proliferation and cell death depending on its abundance. Disclosure L.S. Katz: None. G. Brill: None. M.A. Herman: None. D. Scott: None. Funding National Institutes of Health

324-LB: A Novel Link between Mig6 and PP2a in Beta-Cell Apoptosis

Hyperglycemia in both type 1 and 2 diabetes is toxic to pancreatic beta cells and exacerbates the decline in functional beta cell mass in both diseases. Thus, identifying the molecular mechanism for how this glucotoxicity negatively impacts beta cells is essential for identifying new therapeutic targets to treat both forms of diabetes. We identified that EGF receptor feedback inhibitor Mig6 is induced in islets cultured in glucotoxic conditions and that Mig6 promotes apoptosis in beta cells. To define how the adaptor protein Mig6 promotes apoptosis, we sought to identify its interacting partners. Following immunoprecipitation of flag-tagged Mig6 in Ins1-derived 832/13 cells cultured in normal and toxic conditions, mass spectrometric analysis revealed protein phosphatase 2A (PP2A). PP2A controls many cellular functions ranging from metabolism, cell cycle, DNA replication, growth and apoptosis, and PP2A can be exploited by tumor cells to promote cell survival. Thus, we hypothesized Mig6 interacts with PP2A to regulate beta cell apoptosis. We cultured 832/13 cells and mouse islets in low (5 mM) or high glucose (HG, 20 mM) for up to 48h and validated that glucotoxicity increased methylation and decreased phosphorylation of PP2A (indicating activation). HG also increased expression of Mig6, CHOP (ER stress), γH2AX (DNA damage response), and cleaved caspase 3 (CC3; apoptosis). Compared to controls, siRNA-mediated suppression of Mig6 in 832/13 cells increased methylated PP2A and decreased γH2AX and CC3, suggesting Mig6 antagonizes beta cell survival. We generated mice lacking Mig6 in beta cells (Mig6βKO) by crossing Mig6-floxed mice (Mig6fl/fl) with RIP-Cre mice. Beta cell mass and glucose homeostasis were similar between Mig6βKO and control Mig6fl/fl littermates. Islets from Mig6βKO mice had increased methylated PP2A during HG compared to controls. We speculate the Mig6-PP2A interaction limits PP2A activation; hence, disrupting Mig6-PP2A or inhibiting Mig6 could promote compensatory beta cell survival by activating PP2A. Disclosure A. Rezaeizadeh: None. B.M. Bauer: None. P.T. Fueger: None.

Prolactin receptors in Rip-cre cells, but not in AgRP neurones, are involved in energy homeostasis.

Among its many functions, prolactin has been implicated in energy homeostasis, particularly during pregnancy and lactation. The arcuate nucleus is a key site in the regulation of energy balance. The present study aimed to examine whether arcuate nucleus neuronal populations involved in energy homeostasis are prolactin responsive and whether they can mediate the effects of prolactin on energy homeostasis. To determine whether Agrp neurones or Rip-Cre neurones are prolactin responsive, transgenic mice expressing the reporter td-tomato in Agrp neurones (td-tomato/Agrp-Cre) or Rip-Cre neurones (td-tomato/Rip-Cre) were treated with prolactin and perfused 45minutes later. Brains were processed for double-labelled immunohistochemistry for pSTAT5, a marker of prolactin-induced intracellular signalling, and td-tomato. In addition, Agrp-Cre mice and Rip-Cre mice were crossed with mice in which the prolactin receptor gene (Prlr) was flanked with LoxP sites (Prlrlox/lox mice). The Prlrlox/lox construct was designed such that Cre-mediated recombination resulted in deletion of the Prlr and expression of green fluorescent protein (GFP) in its place. In td-tomato/Rip-Cre mice, prolactin-induced pSTAT5 was co-localised with td-tomato, indicating that there is a subpopulation of Rip-Cre neurones in the arcuate nucleus that respond to prolactin. Furthermore, mice with a specific deletion of Prlr in Rip-Cre neurones had lower body weights, increased oxygen consumption, increased running wheel activity and numerous cells in the arcuate nucleus had positive GFP staining indicating deletion of Prlr from Rip-Cre neurones. By contrast, no co-localisation of td-tomato and pSTAT5 was observed in td-tomato/Agrp-Cre mice after prolactin treatment. Moreover, Prlrlox/lox /Agrp-Cre mice had no positive GFP staining in the arcuate nucleus and did not differ in body weight compared to littermate controls. Overall, these results indicate that Rip-Cre neurones in the arcuate nucleus are responsive to prolactin and may play a role in the orexigenic effects of prolactin, whereas prolactin does not directly affect Agrp neurones.

Inhibition of Miro1 disturbs mitophagy and pancreatic β-cell function interfering insulin release via IRS-Akt-Foxo1 in diabetes.

Mitochondrial function is essential to meet metabolic demand of pancreatic beta cells respond to high nutrient stress. Mitophagy is an essential component to normal pancreatic β-cell function and has been associated with β-cell failure in Type 2 diabetes (T2D). Our previous studies have indicated that mitochondrial Rho (Miro) GTPase-mediated mitochondrial dysfunction under high nutrient stress leads to NOD-like receptor 3 (NLRP3)-dependent proinflammatory responses and subsequent insulin resistance. However, the in vivo mechanism by which Miro1 underlies mitophagy has not been identified. Here we show firstly that the expression of Miro is reduced in human T2D and mouse db/db islets and in INS-1 cell line exposed to high glucose and palmitate. β-cell specific ablation of Miro1 (Miro1f/f: Rip-cre mice, or (IKO) under high nutrient stress promotes the development of hyperglycemia. β-cells from IKO mice display an inhibition of mitophagy under oxidative stress and induces mitochondrial dysfunction. Dysfunctional mitophagy in IKO mice is represented by damaged islet beta cell mitochondrial and secretory capacity, unbalanced downstream MKK-JNK signalling without affecting the levels of MEK, ERK or p38 activation and subsequently, impaired insulin secretion signaling via inhibition IRS-AKT-Foxo1 pathway, leading to worsening glucose tolerance in these mice. Thus, these data suggest that Miro1 may be responsible for mitophagy deficiency and β-cell dysfunction in T2D and that strategies target Miro1 in vivo may provide a therapeutic target to enhance β-cell mitochondrial quality and insulin secretion to ameliorate complications associated with T2D.

Effects of Beta-Cell-Specific Insulin Resistance on Glucose Homeostasis and Body WeightImage 12

Type 2 diabetes is associated with obesity, tissue-specific insulin resistance, and eventually beta-cell failure and loss. It is unclear whether beta-cell-specific insulin resistance can play a causal role in diabetes pathogenesis. We assessed the progression of glucose and insulin tolerance, glucose-stimulated insulin secretion and body weight over a year in male and female Insrf/f:Ins1Cre/wt knockout mice and Insrwt/wt:Ins1Cre/wt controls. In contrast to published data on Insrf/f:Rip-Cre mice, we did not observe glucose intolerance in male Insrf/f:Ins1Cre/wt mice until after 9 months of high-fat feeding. In high-fat fed females, Insrf/f:Ins1Cre/wt mice had modestly improved glucose tolerance at 21 weeks. This was associated with a modest elevation of glucose-stimulated insulin release as well as increased insulin sensitivity at 5 months of age in Insrf/f:Ins1Cre/wt mice. High-fat fed Insrf/f:Ins1Cre/wt females were also heavier from 7 to 16 weeks of age. Increased insulin sensitivity was still present at 9 months of age in female high-fat fed Insrf/f:Ins1Cre/wt mice and was associated with a striking lack of oral glucose-stimulated insulin release. Our data suggest that beta-cell-specific modulation of insulin secretion via insulin receptor deletion can have peripheral consequences for body weight regulation and insulin sensitivity. Together, our studies provide new information about the function of beta cell insulin receptors and will provide more insight into the pathogenesis of type 2 diabetes.

Screening strategy to generate cell specific recombination: a case report with the RIP-Cre mice.

Conditional gene knockout technology is a powerful tool to study the function of a gene in a specific tissue, organ or cell lineage. The most commonly used procedure applies the Cre-LoxP strategy, where the choice of the Cre driver promoter is critical to determine the efficiency and specificity of the system. However, a considered choice of an appropriate promoter does not always protect against the risk of unwanted recombination and the consequent deletion of the gene in other tissues than the desired one(s), due to phenomena of non-specific activation of the Cre transgene. Furthermore, the causes of these phenomena are not completely understood and this can potentially affect every strain of Cre-mice. In our study on the deletion of a same gene in two different tissues, we show that the incidence rate of non-specific recombination in unwanted tissues depends on the Cre driver strain, ranging from 100%, rendering it useless (aP2-Cre strain), to ~5%, which is still compatible with their use (RIP-Cre strain). The use of a simple PCR strategy conceived to detect this occurrence is indispensable when producing a tissue-specific knockout mouse. Therefore, when choosing the Cre-driver promoter, researchers not only have to be careful about its tissue-specificity and timing of activation, but should also include a systematical screening in order to exclude mice in which atypical recombination has occurred and to limit the unnecessary use of laboratory animals in uninterpretable experiments.

Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity.

Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.

Abstract 62: Inhibition of Men1-deficient tumor growth by inactivation of the Rbp2 histone demethylase

Abstract Abstract: Mutation of the tumor suppressor gene MEN1 causes both human inherited and somatic tumors in multiple endocrine tumors. Dissection of mechanism on MEN1 tumor model will shed light on tumor biology and the targeting therapies on neuroendocrine tumors lacking Men1. Loss of tumor suppressor menin may be tumorigenic because it leads to decreased histone H3 lysine 4 trimethylation and therefore decreased expression of specific genes such as CDKN1B. Reversing the tumorigenesis caused by menin deficiency might therefore be achieved by inhibition of histone H3 lysine 4 demethylation. Rbp2 (also known as KDM5A) is an excellent candidate for such a demethylase because loss of the Rbp2 gene leads to increased expression of CDKN1B. Using a tissue-specific knockout approach, I have disrupted Men1 alone or both Men1 and Rbp2 in mouse pancreatic islets through RIP-Cre transgene (RIP = “rat insulin promoter”) and have observed that loss of Rbp2 dramatically inhibits tumor growth in Men1-deficient islets. Histological assays have found that 100 percent of Men1f/f RIP-Cre mice (13 out of 13) at 10 months old or older harbor tumors of the pancreatic islets, whereas only 7 out of 21 Men1f/f; Rbp2f/f RIP-Cre mice contain tumors or large hyperplastic islets. Furthermore, Inactivation of Rbp2 extends the life span of Men1-deficient mice. The median survival age of Men1f/f RIP-Cre mice in this cohort is 313 days, the median survival age of Men1f/f; Rbp2f/f RIP-Cre (n=72) is 486 days (p<0.0001). In addition, a number of targets shared by both Men1 and Rbp2 have been identified via expression profiling. Studies from this project may provide insight to the cause of endocrine tumors and may develop a molecular target for treatment of endocrine tumors and the MEN-1 syndrome through modulation of histone methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2011-62

Conditional ablation of Gsk-3β in islet beta cells results in expanded mass and resistance to fat feeding-induced diabetes in mice.

Glycogen synthase kinase 3β (GSK-3β) is an enzyme that is suppressed by insulin and when elevated results in insulin resistance in skeletal muscle and diabetes. Its role in beta cell development and function is little known. Because of the enzyme's anti-proliferative and pro-apoptotic properties, the hypothesis to be tested here was that beta cell specific deficiency of GSK-3β in mice would result in enhanced beta cell mass and function. Mice with beta cell deficiency of GSK-3β (β-Gsk-3β [also known as Gsk3b](-/-)) were generated by breeding Gsk-3β (flox/flox) mice with mice overexpressing the Cre recombinase gene under the control of the rat insulin 2 gene promoter (RIP-Cre mice), and glucose tolerance, insulin secretion, islet mass, proliferation and apoptosis were measured. Changes in islet proteins were investigated by western blotting. On a normal diet β-Gsk-3β ( -/- ) mice were found to have mild improvement of glucose tolerance and glucose-induced insulin secretion, and increased beta cell mass accompanied by increased proliferation and decreased apoptosis. On a high-fat diet β-Gsk-3β (-/-) mice exhibited improved glucose tolerance and expanded beta cell mass with increased proliferation relative to that in control mice, resisting fat-fed diabetes. Molecular mechanisms accounting for these phenotypic changes included increased levels of islet IRS1 and IRS2 proteins and phospho-Akt, suggesting enhanced signalling through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and increased islet levels of pancreas/duodenum homeobox protein 1 (PDX1). Inhibition of GSK3 in MIN6 cells in vitro led to increased IRS1 and IRS2 protein levels through inhibition of proteosomal degradation. These results are consistent with a mechanism whereby endogenous GSK-3β activity controls islet beta cell growth by feedback inhibition of the insulin receptor/PI3K/Akt signalling pathway.

Abstract 4166: Targeted expression of the Pleomorphic adenoma gene 1 in the pancreas of mice leads to islet hyperplasia

Abstract Pleomorphic adenoma gene 1 (PLAG1) encodes a transcription factor that is involved in several human neoplasias such as in pleomorphic adenomas of the salivary glands, lipoblastoma, hepatoblastoma and AML. The oncogenic capacity of PLAG1 has been further demonstrated in vivo in PLAG1 transgenic mice, in which the activation of the PLAG1 expression is Cre-mediated. Targeted expression of PLAG1 in the salivary gland or in the mammary gland leads to pleomorphic adenomas of the salivary glands and adenomyoepitheliomatous lesions of the mammary glands, respectively. Here, we investigated the impact of targeted PLAG1 expression in the pancreas. Pdx1-Cre-mediated targeted expression of PLAG1 in the whole pancreas, leads to islet hyperplasia. Although there was initially a balanced increase in the insulin, somatostatin and glucagon content of the pancreas, in older P1-Pdx1Cre mice the production of insulin and somatostatin far exceeded that of glucagon (82%, 275% and 9% respectively in 1 year old P1-Pdx1Cre mice as compared to littermate Pdx1-Cre mice). This was associated with hyperinsulinemia and an increased ability to dispose glucose in P1-Pdx1Cre mice, and progressive development of severe hypoglycaemia. Only 1/10 one year old mice developed a pancreatic tumor. We also investigated the consequence of overexpression of PLAG1 only in β-cells by using RIP-Cre mice. Similar to P1-Pdx1Cre mice, P1-RIPCre mice displayed islet hyperplasia, hyperinsulinemia and an increased ability to dispose glucose. Nevertheless, in contrast to P1-Pdx1Cre mice, P1-RIPCre mice stay normoglycemic and compensate for the hyperinsulinemia by the development of hepatic insulin resistance. In conclusion our data demonstrates that targeted expression of PLAG1 in the pancreas leads to islet hyperplasia rather than to pancreatic tumor formation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4166.

RIP-Cre Revisited, Evidence for Impairments of Pancreatic β-Cell Function

The Cre/loxP recombinase system for performing conditional gene targeting experiments has been very useful in exploring genetic pathways that control both the development and function of pancreatic beta-cells. One particular line of transgenic mice (B6.Cg-Tg(Ins2-cre)25Mgn/J), commonly called RIP-Cre, in which expression of Cre recombinase is controlled by a short fragment of the rat insulin II gene promoter has been used in at least 21 studies on at least 17 genes. In most of these studies inactivation of the gene of interest was associated with either glucose intolerance or frank diabetes. Experimental evidence has been gradually emerging to suggest that RIP-Cre mice alone display glucose intolerance. In this study experiments from three laboratories demonstrate that RIP-Cre mice, in the absence of genes targeted by loxP sites, are glucose intolerant, possibly due to impaired insulin secretion. In addition, we review the use of RIP-Cre mice and discuss possible molecular underpinnings and ramifications of our findings.

Essential role of STAT3 in body weight and glucose homeostasis.

STAT3 is a ubiquitous transcription factor that is indispensable during early embryogenesis. To study the functions of STAT3 postnatally, we generated conditional STAT3-deficient mice. To that end, STAT3(lox/lox) mice were crossed with mice expressing Cre under the control of rat insulin II gene promoter (RIP-Cre mice). Immunohistochemical and Western blot analyses showed that STAT3 is deleted from beta cells in the islets of Langerhans. Genomic DNA PCR revealed that STAT3 deletion also occurred in the hypothalamus. Hypothalamic Cre expression was further confirmed by crossing RIP-Cre/STAT3(lox/lox) mice with the ROSA26 Cre reporter strain and staining for lacZ activity. Double immunohistochemical staining confirmed that deletion of STAT3 occurred in leptin receptor (OB-Rb isoform)-positive neurons. RIP-Cre/STAT3(lox/lox) mice are mildly hyperglycemic and hyperinsulinemic at the time of weaning, become hyperphagic immediately after weaning, and exhibit impaired glucose tolerance. Body weight, body fat, and mRNA and protein levels of leptin are all significantly increased in RIP-Cre/STAT3(lox/lox) mice. Administration of recombinant leptin by intracerebroventricular infusion failed to cause complete loss of body fat in RIP-Cre/STAT3(lox/lox) mice. Transplantation of wild-type islets into RIP-Cre/STAT3(lox/lox) mice also failed to decrease adiposity or to correct other abnormalities in these mice. These data thus suggest that loss of STAT3 in the hypothalamus caused by RIP-Cre action likely interferes with normal body weight homeostasis and glucose metabolism.