Diana Choi

Erythropoietin protects against diabetes through direct effects on pancreatic β cells

In mouse models of type 1 and type 2 diabetes, administration of human erythropoietin protects against disease by acting directly on pancreatic β cells.

Hepatocyte-specific Deletion of Janus Kinase 2 (JAK2) Protects against Diet-induced Steatohepatitis and Glucose Intolerance

Background: JAK2 mediates signaling by a number of cytokines in the liver. Results: Hepatic JAK2 KO mice developed spontaneous steatosis but were protected from high fat diet-induced steatohepaitits and insulin resistance. Conclusion: Hepatic JAK2 is required for the development of diet-induced steatohepatitis and glucose intolerance. Significance: Understanding the role of JAK2 in metabolism will provide insights into the pathogenesis of the metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and is now considered to be the hepatic manifestation of the metabolic syndrome. However, the role of steatosis per se and the precise factors required in the progression to steatohepatitis or insulin resistance remain elusive. The JAK-STAT pathway is critical in mediating signaling of a wide variety of cytokines and growth factors. Mice with hepatocyte-specific deletion of Janus kinase 2 (L-JAK2 KO mice) develop spontaneous steatosis as early as 2 weeks of age. In this study, we investigated the metabolic consequences of jak2 deletion in response to diet-induced metabolic stress. To our surprise, despite the profound hepatosteatosis, deletion of hepatic jak2 did not sensitize the liver to accelerated inflammatory injury on a prolonged high fat diet (HFD). This was accompanied by complete protection against HFD-induced whole-body insulin resistance and glucose intolerance. Improved glucose-stimulated insulin secretion and an increase in β-cell mass were also present in these mice. Moreover, L-JAK2 KO mice had progressively reduced adiposity in association with blunted hepatic growth hormone signaling. These mice also exhibited increased resting energy expenditure on both chow and high fat diet. In conclusion, our findings indicate a key role of hepatic JAK2 in metabolism such that its absence completely arrests steatohepatitis development and confers protection against diet-induced systemic insulin resistance and glucose intolerance.

In Vivo Role of Focal Adhesion Kinase in Regulating Pancreatic β-Cell Mass and Function Through Insulin Signaling, Actin Dynamics, and Granule Trafficking

Focal adhesion kinase (FAK) acts as an adaptor at the focal contacts serving as a junction between the extracellular matrix and actin cytoskeleton. Actin dynamics is known as a determinant step in insulin secretion. Additionally, FAK has been shown to regulate insulin signaling. To investigate the essential physiological role of FAK in pancreatic β-cells in vivo, we generated a transgenic mouse model using rat insulin promoter (RIP)–driven Cre-loxP recombination system to specifically delete FAK in pancreatic β-cells. These RIPcre+fakfl/fl mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced β-cell viability and proliferation resulting in decreased β-cell mass was observed in these mice, which was associated with attenuated insulin/Akt (also known as protein kinase B) and extracellular signal–related kinase 1/2 signaling and increased caspase 3 activation. FAK-deficient β-cells exhibited impaired insulin secretion with normal glucose sensing and preserved Ca2+ influx in response to glucose, but a reduced number of docked insulin granules and insulin exocytosis were found, which was associated with a decrease in focal proteins, paxillin and talin, and an impairment in actin depolymerization. This study is the first to show in vivo that FAK is critical for pancreatic β-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking.

Vhl is required for normal pancreatic β cell function and the maintenance of β cell mass with age in mice

Type 2 diabetes is hallmarked by insulin resistance and insufficient β-cell function. Islets of type 2 diabetes patients have been shown to have decreased hypoxia-inducible factor (HIF)-1α/β expression. Target genes of the HIF pathway are involved in angiogenesis, survival, proliferation, and energy metabolism, and von Hippel-Lindau protein (VHL) is a negative regulator of this pathway. We hypothesized that increased HIF-mediated gene transcription by VHL deletion in the β-cells would increase β-cell mass and function. We generated β-cell-specific VHL-knockout mice using the Cre-loxP recombination system driven by the rat insulin promoter to assess the role of VHL in glucose homeostasis and β-cell function. VHL deletion in the pancreatic β-cells led to impaired glucose tolerance due to defects in glucose-stimulated insulin secretion and β-cell mass with age. VHL-knockout islets had decreased GLUT2, but increased glucose transporter 1 and vascular endothelial growth factor expression. Furthermore, there were significant aberrations in islet morphology in the VHL-knockout mice, likely due to increased islet vasculature. Given that erythropoietin (EPO) is a target gene of the HIF pathway, which is not expressed in islets, we tested whether activating EPO signaling by systemic administration with recombinant human EPO (rHuEPO) can overcome the β-cell defects that occurred with VHL loss. We observed improved glucose tolerance and restoration of GLUT2 expression in VHL-deficient β-cells in response to rHuEPO. Contrary to our hypothesis, loss of VHL and increased transcription of HIF-target genes resulted in impaired β-cell function and mass, which can be overcome with exogenous EPO. Our results indicate a critical role for VHL in β-cell function and mass, and that EPO administration improved β-cell function making it a potential strategy for diabetes treatment.

Perinatal survivin is essential for the establishment of pancreatic beta cell mass in mice

Aims/hypothesisPancreatic beta cells undergo dynamic remodelling during the perinatal period, with enhanced neogenesis, proliferation and apoptosis observed. The molecular mechanisms responsible for these processes have yet to be elucidated. Survivin is an inhibitor of apoptosis, first described as being exclusively expressed in tumour and embryonic tissues with regulatory functions in mitosis and apoptosis. The aim of the present study was to define the essential physiological role of survivin in the pancreas.MethodsThe expression profile of survivin was assessed in the mouse pancreas, and we generated a Pdx1 promoter-driven Survivin (also known as Birc5) knockout mouse using the Cre-loxP recombination system to determine the essential physiological function of survivin in the pancreas.ResultsSurvivin is transiently expressed in mouse pancreatic islets during the embryonic and neonatal periods. Targeted deletion of Survivin in the pancreas resulted in a significant decline in beta cell mass throughout the perinatal period, leading to glucose intolerance in the adult. Survivin-deficient islets showed decreased cell proliferation as a result of a delay in cell cycle progression with perturbations in cell cycle proteins. Survivin did not, however, play an essential role in beta cell apoptosis either during the physiological remodelling period or in response to streptozotocin. Islet development, islet architecture, microvasculature and apoptosis were not affected by the absence of survivin in the pancreas.Conclusions/interpretationSurvivin expression in the pancreatic islets during the perinatal remodelling period is essential for the establishment of beta cell mass through cell cycle regulation.

Deletion of Fas in the pancreatic β-cells leads to enhanced insulin secretion

Fas/Fas ligand belongs to the tumor necrosis factor superfamily of receptors/ligands and is best known for its role in apoptosis. However, recent evidence supports its role in other cellular responses, including proliferation and survival. Although Fas has been implicated as an essential mediator of beta-cell death in the pathogenesis of type 1 diabetes, the essential role of Fas specifically in pancreatic beta-cells has been found to be controversial. Moreover, the role of Fas on beta-cell homeostasis and function is not clear. The objective of this study is to determine the role of Fas specifically in beta-cells under both physiological and diabetes models. Mice with Fas deletion specifically in the beta-cells were generated using the Cre-loxP system. Cre-mediated Fas deletion was under the control of the rat insulin promoter. Absence of Fas in beta-cells leads to complete protection against FasL-induced cell death. However, Fas is not essential in determining beta-cell mass or susceptibility to streptozotocin- or HFD-induced diabetes. Importantly, Fas deletion in beta-cells leads to increased p65 expression, enhanced glucose tolerance, and glucose-stimulated insulin secretion, with increased exocytosis as manifested by increased changes in membrane capacitance and increased expression of Syntaxin1A, VAMP2, and munc18a. Together, our study shows that Fas in the beta-cells indeed plays an essential role in the canonical death receptor-mediated apoptosis but is not essential in regulating beta-cell mass or diabetes development. However, beta-cell Fas is critical in the regulation of glucose homeostasis through regulation of the exocytosis machinery.

Absence of Caspase-3 Protects Pancreatic β-Cells from c-Myc-induced Apoptosis without Leading to Tumor Formation

c-Myc is a powerful trigger of β-cell apoptosis, proliferation, and dedifferentiation in rodent islets in vivo. In a transgenic mouse model, c-Myc induction causes rapid β-cell apoptosis and overt diabetes. When suppression of apoptosis is achieved by overexpression of Bcl-xL in an inducible model of c-Myc activation, a full spectrum of tumor development, including distant metastasis, occurs. Caspase-3 is a key pro-apoptotic protein involved in the execution phase of multiple apoptotic pathways. To test whether caspase-3 is an essential mediator of apoptosis in this model of tumorigenesis, we generated caspase-3 knock-out mice containing the inducible c-myc transgene (c-Myc+Casp3-/-). In contrast to Bcl-xL-overexpressing c-Myc+ mice, c-Myc+Casp3-/- mice remained euglycemic for up to 30 days of c-Myc activation, and there was no evidence of tumor formation. Interestingly, caspase-3 deletion also led to the suppression of proliferation, perhaps through regulation of the cell cycle inhibitory protein p27, suggesting a possible mechanism for maintaining a balance between suppression of apoptosis and excessive proliferation in the context of c-Myc activation. Additionally, c-Myc-activated Casp3-/- mice were protected from streptozotocin-induced diabetes. Our studies demonstrate that caspase-3 deletion confers protection from c-Myc-induced apoptosis and diabetes development without unwanted tumorigenic effects. These results may lead to further elucidation of the mechanisms of c-Myc biology relevant to β-cells, which may result in novel therapeutic strategies for diabetes.

In Vivo Knockdown of Adipocyte Erythropoietin Receptor Does Not Alter Glucose or Energy Homeostasis

The growing prevalence of obesity and diabetes necessitate a better understanding of the role of adipocyte biology in metabolism. Increasingly, erythropoietin (EPO) has been shown to have extraerythropoietic and cytoprotective roles. Exogenous administration has recently been shown to have beneficial effects on obesity and diabetes in mouse models and EPO can modulate adipogenesis and insulin signaling in 3T3-L1 adipocytes. However, its physiological role in adipocytes has not been identified. Using male and female mice with adipose tissue-specific knockdown of the EPO receptor, we determine that adipocyte EPO signaling is not essential for the maintenance of energy homeostasis or glucose metabolism. Adipose tissue-specific disruption of EPO receptor did not alter adipose tissue expansion, adipocyte morphology, insulin resistance, inflammation, or angiogenesis in vivo. In contrast to the pharmacological effects of EPO, we demonstrate that EPO signaling at physiological levels is not essential for adipose tissue regulation of metabolism.

PTEN deletion and concomitant c-Myc activation do not lead to tumor formation in pancreatic beta cells.

Phosphatase and tensin homologue (PTEN) deleted on chromosome 10 is a dual-specific phosphatase and a potent antagonist of the phosphoinositide 3-kinase signaling pathway. Although first discovered as a tumor suppressor, emerging evidence supports PTEN as a potential therapeutic target for diabetes. PTEN deletion in β cells leads to increased β cell mass and protection from streptozotocin-induced diabetes. Importantly, PTEN deletion does not lead to tumor formation in β cells. To further assess the potential tumorigenic role of PTEN, we tested the biological role of PTEN in the context of activation of the proto-oncogene c-Myc. We generated and characterized β cell-specific PTEN knock-out mice expressing an inducible c-Myc transgene in β cells. Surprisingly, we found that PTEN loss did not confer protection from the overwhelming apoptosis and diabetes development seen with c-Myc activation. Importantly, despite the combined effect of the loss of a tumor suppressor and activation of an oncogene in β cells, there was no evidence of tumor development with sustained c-Myc activation.

Partial Deletion of Pten in the Hypothalamus Leads to Growth Defects that Cannot be Rescued by Exogenous Growth Hormone

The GH/IGF-I axis plays a critical role in mammalian body growth. GH is secreted by the anterior pituitary, and its actions are primarily mediated by IGF-I that is secreted by the liver and other tissues. Local and circulating IGF-I action is largely mediated by the phosphoinositide 3-kinase signaling pathway, and phosphatase with tensin homology (PTEN) is a potent negative regulator of this pathway. Here we show that RIPcre+Ptenfl/fl mice, which exhibit PTEN deletion in insulin-transcribing neurons of the hypothalamus in addition to pancreatic beta-cells, result in a small-body phenotype that is associated with an unexpected increase in serum IGF-I levels. We tested whether exogenous GH can override the growth defect in RIPcre+Ptenfl/fl mice. Our results showed no significant difference in their growth between the RIPcre+Ptenfl/fl mice injected with GH or vehicle. Together, PTEN in the hypothalamic insulin-transcribing neurons plays an essential role in body size determination, and systemic GH cannot overcome the growth defect in these mice.