Dual Role of Caspase 8 in Adipocyte Apoptosis and Metabolic Inflammation.

Caspases are cysteine-aspartic proteases that were initially discovered to play a role in apoptosis. However, caspase 8, in particular, also has additional nonapoptotic roles, such as in inflammation. Adipocyte cell death and inflammation are hypothesized to be initiating pathogenic factors in type 2 diabetes. Here, we examined the pleiotropic role of caspase 8 in adipocytes and obesity-associated insulin resistance. Caspase 8 expression was increased in adipocytes from mice and humans with obesity and insulin resistance. Treatment of 3T3-L1 adipocytes with caspase 8 inhibitor Z-IETD-FMK decreased both death receptor-mediated signaling and targets of nuclear factor κ-light-chain-enhancer of activated B (NF-κB) signaling. We generated novel adipose tissue and adipocyte-specific caspase 8 knockout mice (aP2Casp8-/- and adipoqCasp8-/-). Both males and females had improved glucose tolerance in the setting of high-fat diet (HFD) feeding. Knockout mice also gained less weight on HFD, with decreased adiposity, adipocyte size, and hepatic steatosis. These mice had decreased adipose tissue inflammation and decreased activation of canonical and noncanonical NF-κB signaling. Furthermore, they demonstrated increased energy expenditure, core body temperature, and UCP1 expression. Adipocyte-specific activation of Ikbkb or housing mice at thermoneutrality attenuated improvements in glucose tolerance. These data demonstrate an important role for caspase 8 in mediating adipocyte cell death and inflammation to regulate glucose and energy homeostasis. ARTICLE HIGHLIGHTS: Caspase 8 is increased in adipocytes from mice and humans with obesity and insulin resistance. Knockdown of caspase 8 in adipocytes protects mice from glucose intolerance and weight gain on a high-fat diet. Knockdown of caspase 8 decreases Fas signaling, as well as canonical and noncanonical nuclear factor κ-light-chain-enhancer of activated B (NF-κB) signaling in adipose tissue. Improved glucose tolerance occurs via reduced activation of NF-κB signaling and via induction of UCP1 in adipocytes.

Protective Renalase Deficiency in ß-Cells Shapes Immune Metabolism and Function in Autoimmune Diabetes.

Type 1 diabetes (T1D) is caused by the immune-mediated loss of pancreatic ß-cells that produce insulin. The latest advances in stem cell (SC) ß-cell differentiation methods have made a cell replacement therapy for T1D feasible. However, recurring autoimmunity would rapidly destroy transplanted SC ß-cells. A promising strategy to overcome immune rejection is to genetically engineer SC ß-cells. We previously identified Renalase (Rnls) as a novel target for ß-cell protection. Here we show that Rnls deletion endows ß-cells with the capacity to modulate the metabolism and function of immune cells within the local graft microenvironment. We used flow cytometry and single-cell RNA sequencing to characterize ß-cell graft-infiltrating immune cells in a mouse model for T1D. Loss of Rnls within transplanted ß-cells affected both the composition and the transcriptional profile of infiltrating immune cells in favor of an anti-inflammatory profile with decreased antigen-presenting capacity. We propose that changes in ß-cell metabolism mediate local immune regulation and that this feature could be exploited for therapeutic goals. ARTICLE HIGHLIGHTS: Protective Renalase (Rnls) deficiency impacts ß-cell metabolism. Rnls-deficient ß-cell grafts do not exclude immune infiltration. Rnls deficiency in transplanted ß-cells broadly modifies local immune function. Immune cell in Rnls mutant ß-cell grafts adopt a noninflammatory phenotype.

Protocol for genome-scale in vivo CRISPR screening to study protection of beta cells under autoimmunity in a type 1 diabetes mouse model.

Autoimmunity-induced pancreatic beta cell failure is the main characteristic of type 1 diabetes (T1D). Here, we describe a protocol for genome-scale in vivo CRISPR-Cas9 screening for use in a mouse model of T1D. Using a non-obese-diabetic-derived mouse beta cell line, NIT-1, and a genome-wide CRISPR-Cas9 knockout library (GeCKO-v2), we describe how to identify genes that confer resistance to autoimmune killing. This protocol can be applied in other mouse models of autoimmunity. For complete details on the use and execution of this protocol, please refer to Cai et al. (2020).1.

RIPK1 and RIPK3 regulate TNFα-induced ß-cell death in concert with caspase activity.

OBJECTIVE: Type 1 diabetes (T1D) is characterized by autoimmune-associated ß-cell loss, insulin insufficiency, and hyperglycemia. Although TNFα signaling is associated with ß-cell loss and hyperglycemia in non-obese diabetic mice and human T1D, the molecular mechanisms of ß-cell TNF receptor signaling have not been fully characterized. Based on work in other cell types, we hypothesized that receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) regulate TNFα-induced ß-cell death in concert with caspase activity. METHODS: We evaluated TNFα-induced cell death, caspase activity, and TNF receptor pathway molecule expression in immortalized NIT-1 and INS-1 ß-cell lines and primary mouse islet cells in vitro. Our studies utilized genetic and small molecule approaches to alter RIPK1 and RIPK3 expression and caspase activity to interrogate mechanisms of TNFα-induced ß-cell death. We used the ß-cell toxin streptozotocin (STZ) to determine the susceptibility of Ripk3+/+ and Ripk3-/- mice to hyperglycemia in vivo. RESULTS: Expression of TNF receptor signaling molecules including RIPK1 and RIPK3 was identified in NIT-1 and INS-1 ß cells and isolated mouse islets at the mRNA and protein levels. TNFα treatment increased NIT-1 and INS-1 cell death and caspase activity after 24-48 h, and BV6, a small molecule inhibitor of inhibitor of apoptosis proteins (IAPs) amplified this TNFα-induced cell death. RIPK1 deficient NIT-1 cells were protected from TNFα- and BV6-induced cell death and caspase activation. Interestingly, small molecule inhibition of caspases with zVAD-fmk (zVAD) did not prevent TNFα-induced cell death in either NIT-1 or INS-1 cells. This caspase-independent cell death was increased by BV6 treatment and decreased in RIPK1 deficient NIT-1 cells. RIPK3 deficient NIT-1 cells and RIPK3 kinase inhibitor treated INS-1 cells were protected from TNFα+zVAD-induced cell death, whereas RIPK3 overexpression increased INS-1 cell death and promoted RIPK3 and MLKL interaction under TNFα+zVAD treatment. In mouse islet cells, BV6 or zVAD treatment promoted TNFα-induced cell death, and TNFα+zVAD-induced cell death was blocked by RIPK3 inhibition and in Ripk3-/- islet cells in vitro. Ripk3-/- mice were also protected from STZ-induced hyperglycemia and glucose intolerance in vivo. CONCLUSIONS: RIPK1 and RIPK3 regulate TNFα-induced ß-cell death in concert with caspase activity in immortalized and primary islet ß cells. TNF receptor signaling molecules such as RIPK1 and RIPK3 may represent novel therapeutic targets to promote ß-cell survival and glucose homeostasis in T1D.

Genome-scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes.

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration-approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.

FAK signalling controls insulin sensitivity through regulation of adipocyte survival.

Focal adhesion kinase (FAK) plays a central role in integrin signalling, which regulates growth and survival of tumours. Here we show that FAK protein levels are increased in adipose tissue of insulin-resistant obese mice and humans. Disruption of adipocyte FAK in mice or in 3T3 L1 cells decreases adipocyte survival. Adipocyte-specific FAK knockout mice display impaired adipose tissue expansion and insulin resistance on prolonged metabolic stress from a high-fat diet or when crossed on an obese db/db or ob/ob genetic background. Treatment of these mice with a PPARγ agonist does not restore adiposity or improve insulin sensitivity. In contrast, inhibition of apoptosis, either genetically or pharmacologically, attenuates adipocyte death, restores normal adiposity and improves insulin sensitivity. Together, these results demonstrate that FAK is required for adipocyte survival and maintenance of insulin sensitivity, particularly in the context of adipose tissue expansion as a result of caloric excess.

Janus Kinase 2 (JAK2) Dissociates Hepatosteatosis from Hepatocellular Carcinoma in Mice.

Hepatocellular carcinoma is an end-stage complication of non-alcoholic fatty liver disease (NAFLD). Inflammation plays a critical role in the progression of non-alcoholic fatty liver disease and the development of hepatocellular carcinoma. However, whether steatosis per se promotes liver cancer, and the molecular mechanisms that control the progression in this disease spectrum remain largely elusive. The Janus kinase signal transducers and activators of transcription (JAK-STAT) pathway mediates signal transduction by numerous cytokines that regulate inflammation and may contribute to hepatocarcinogenesis. Mice with hepatocyte-specific deletion of JAK2 (L-JAK2 KO) develop extensive fatty liver spontaneously. We show here that this simple steatosis was insufficient to drive carcinogenesis. In fact, L-JAK2 KO mice were markedly protected from chemically induced tumor formation. Using the methionine choline-deficient dietary model to induce steatohepatitis, we found that steatohepatitis development was completely arrested in L-JAK2 KO mice despite the presence of steatosis, suggesting that JAK2 is the critical factor required for inflammatory progression in the liver. In line with this, L-JAK2 KO mice exhibited attenuated inflammation after chemical carcinogen challenge. This was associated with increased hepatocyte apoptosis without elevated compensatory proliferation, thus thwarting expansion of transformed hepatocytes. Taken together, our findings identify an indispensable role of JAK2 in hepatocarcinogenesis through regulating critical inflammatory pathways. Targeting the JAK-STAT pathway may provide a novel therapeutic option for the treatment of hepatocellular carcinoma.

Resolving Discrepant Findings on ANGPTL8 in ß-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy.

The ß-cell mitogenic effects of ANGPTL8 have been subjected to substantial debate. The original findings suggested that ANGPTL8 overexpression in mice induced a 17-fold increase in ß-cell proliferation. Subsequent studies in mice contested this claim, but a more recent report in rats supported the original observations. These conflicting results might be explained by variable ANGPTL8 expression and differing methods of ß-cell quantification. To resolve the controversy, three independent labs collaborated on a blinded study to test the effects of ANGPTL8 upon ß-cell proliferation. Recombinant human betatrophin (hBT) fused to maltose binding protein (MBP) was delivered to mice by intravenous injection. The results demonstrate that ANGPTL8 does not stimulate significant ß-cell proliferation. Each lab employed different methods for ß-cell identification, resulting in variable quantification of ß-cell proliferation and suggests a need for standardizing practices for ß-cell quantification. We also observed a new action of ANGPTL8 in stimulating CD45+ hematopoietic-derived cell proliferation which may explain, in part, published discrepancies. Overall, the hypothesis that ANGPTL8 induces dramatic and specific ß-cell proliferation can no longer be supported. However, while ANGPTL8 does not stimulate robust ß-cell proliferation, the original experimental model using drug-induced (S961) insulin resistance was validated in subsequent studies, and thus still represents a robust system for studying signals that are either necessary or sufficient for ß-cell expansion. As an added note, we would like to commend collaborative group efforts, with repetition of results and procedures in multiple laboratories, as an effective method to resolve discrepancies in the literature.

DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice.

Reactive oxygen species (ROS) have been linked to a wide variety of pathologies, including obesity and diabetes, but ROS also act as endogenous signalling molecules, regulating numerous biological processes. DJ-1 is one of the most evolutionarily conserved proteins across species, and mutations in DJ-1 have been linked to some cases of Parkinson's disease. Here we show that DJ-1 maintains cellular metabolic homeostasis via modulating ROS levels in murine skeletal muscles, revealing a role of DJ-1 in maintaining efficient fuel utilization. We demonstrate that, in the absence of DJ-1, ROS uncouple mitochondrial respiration and activate AMP-activated protein kinase, which triggers Warburg-like metabolic reprogramming in muscle cells. Accordingly, DJ-1 knockout mice exhibit higher energy expenditure and are protected from obesity, insulin resistance and diabetes in the setting of fuel surplus. Our data suggest that promoting mitochondrial uncoupling may be a potential strategy for the treatment of obesity-associated metabolic disorders.

PTEN deletion in pancreatic α-cells protects against high-fat diet-induced hyperglucagonemia and insulin resistance.

An aberrant increase in circulating catabolic hormone glucagon contributes to type 2 diabetes pathogenesis. However, mechanisms regulating glucagon secretion and α-cell mass are not well understood. In this study, we aimed to demonstrate that phosphatidylinositol 3-kinase (PI3K) signaling is an important regulator of α-cell function. Mice with deletion of PTEN, a negative regulator of this pathway, in α-cells show reduced circulating glucagon levels and attenuated l-arginine-stimulated glucagon secretion both in vivo and in vitro. This hypoglucagonemic state is maintained after high-fat-diet feeding, leading to reduced expression of hepatic glycogenolytic and gluconeogenic genes. These beneficial effects protected high-fat diet-fed mice against hyperglycemia and insulin resistance. The data demonstrate an inhibitory role of PI3K signaling on α-cell function and provide experimental evidence for enhancing α-cell PI3K signaling for diabetes treatment.

Rb and p107 are required for alpha cell survival, beta cell cycle control and glucagon-like peptide-1 action.

AIMS/HYPOTHESIS: Diabetes mellitus is characterised by beta cell loss and alpha cell expansion. Analogues of glucagon-like peptide-1 (GLP-1) are used therapeutically to antagonise these processes; thus, we hypothesised that the related cell cycle regulators retinoblastoma protein (Rb) and p107 were involved in GLP-1 action. METHODS: We used small interfering RNA and adenoviruses to manipulate Rb and p107 expression in insulinoma and alpha-TC cell lines. In vivo we examined pancreas-specific Rb knockout, whole-body p107 knockout and Rb/p107 double-knockout mice. RESULTS: Rb, but not p107, was downregulated in response to the GLP-1 analogue, exendin-4, in both alpha and beta cells. Intriguingly, this resulted in opposite outcomes of cell cycle arrest in alpha cells but proliferation in beta cells. Overexpression of Rb in alpha and beta cells abolished or attenuated the effects of exendin-4 supporting the important role of Rb in GLP-1 modulation of cell cycling. Similarly, in vivo, Rb, but not p107, deficiency was required for the beta cell proliferative response to exendin-4. Consistent with this finding, Rb, but not p107, was suppressed in islets from humans with diabetes, suggesting the importance of Rb regulation for the compensatory proliferation that occurs under insulin resistant conditions. Finally, while p107 alone did not have an essential role in islet homeostasis, when combined with Rb deletion, its absence potentiated apoptosis of both alpha and beta cells resulting in glucose intolerance and diminished islet mass with ageing. CONCLUSIONS/INTERPRETATION: We found a central role of Rb in the dual effects of GLP-1 in alpha and beta cells. Our findings highlight unique contributions of individual Rb family members to islet cell proliferation and survival.

Dichotomous role of pancreatic HUWE1/MULE/ARF-BP1 in modulating beta cell apoptosis in mice under physiological and genotoxic conditions.

AIMS/HYPOTHESIS: Diabetes mellitus represents a significant burden on the health of the global population. Both type 1 and type 2 diabetes share a common feature of a reduction in functional beta cell mass. A newly discovered ubiquitination molecule HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase (HUWE1 [also known as MULE or ARF-BP1]) is a critical regulator of p53-dependent apoptosis. However, its role in islet homeostasis is not entirely clear. METHODS: We generated mice with pancreas-specific deletion of Huwe1 using a Cre-loxP recombination system driven by the Pdx1 promoter (Pdx1cre (+) Huwe1 (fl/fl)) to assess the in vivo role of HUWE1 in the pancreas. RESULTS: Targeted deletion of Huwe1 in the pancreas preferentially activated p53-mediated beta cell apoptosis, leading to reduced beta cell mass and diminished insulin exocytosis. These defects were aggravated by ageing, with progressive further decline in insulin secretion and glucose homeostasis in older mice. Intriguingly, Huwe1 deletion provided protection against genotoxicity, such that Pdx1cre (+) Huwe1 (fl/fl) mice were resistant to multiple-low-dose-streptozotocin-induced beta cell apoptosis and diabetes. CONCLUSION/INTERPRETATION: HUWE1 expression in the pancreas is essential in determining beta cell mass. Furthermore, HUWE1 demonstrated divergent roles in regulating beta cell apoptosis depending on physiological or genotoxic conditions.

Overexpression of HIF-2α in pancreatic ß cells does not alter glucose homeostasis.

Both type 1 and type 2 diabetes are associated with insufficient functional ß-cell mass. Understanding intracellular signaling pathways associated with this decline is important in broadening our understanding of the disease and potential therapeutic strategies. The hypoxia inducible factor pathway (HIF) plays a critical role in cellular adaptation to hypoxic conditions. Activation of this pathway increases expression of numerous genes involved in multiple cellular processes and has been shown to impact the regulation of ß-cell function. Previously, deletion of HIF-1α or HIF-1ß in pancreatic ß-cells, as well as constitutive activation of the HIF pathway in ß-cells, was shown to result in glucose intolerance and impaired insulin secretion. The objective of this study was to delineate roles of HIF-2α overexpression in pancreatic ß-cells in vivo. We overexpressed HIF-2α in pancreatic ß-cells by employing the Cre-loxP system driven by the Pdx1 promoter to delete a stop codon. Our study revealed that pancreatic HIF-2α overexpression does not result in significant differences in glucose tolerance, insulin sensitivity or ß-cell area compared to wild-type littermates under basal conditions or after high fat diet. Together, our study shows excess HIF-2α in the pancreatic ß-cells does not play a significant role in ß-cell function and glucose homeostasis.

Retinoblastoma tumor suppressor protein in pancreatic progenitors controls α- and ß-cell fate.

Pancreatic endocrine cells expand rapidly during embryogenesis by neogenesis and proliferation, but during adulthood, islet cells have a very slow turnover. Disruption of murine retinoblastoma tumor suppressor protein (Rb) in mature pancreatic ß-cells has a limited effect on cell proliferation. Here we show that deletion of Rb during embryogenesis in islet progenitors leads to an increase in the neurogenin 3-expressing precursor cell population, which persists in the postnatal period and is associated with increased ß-cell mass in adults. In contrast, Rb-deficient islet precursors, through repression of the cell fate factor aristaless related homeobox, result in decreased α-cell mass. The opposing effect on survival of Rb-deficient α- and ß-cells was a result of opposing effects on p53 in these cell types. As a consequence, loss of Rb in islet precursors led to a reduced α- to ß-cell ratio, leading to improved glucose homeostasis and protection against 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.

Dual role of VAMP8 in regulating insulin exocytosis and islet ß cell growth.

Optimal insulin secretion required to maintain glucose homeostasis is the summation of total pancreatic islet ß cell mass and intrinsic secretory capacity of individual ß cells, which are regulated by distinct mechanisms that could be amplified by glucagon-like-peptide-1 (GLP-1). Because of these actions of GLP-1 on islet ß cells, GLP-1 has been deployed to treat diabetes. We employed SNARE protein VAMP8-null mice to demonstrate that VAMP8 mediates insulin granule recruitment to the plasma membrane, which partly accounts for GLP-1 potentiation of glucose-stimulated insulin secretion. VAMP8-null mice also exhibited increased islet ß cell mass from increased ß cell mitosis, with ß cell proliferative activity greatly amplified by GLP-1. Thus, despite the ß cell exocytotic defect, VAMP8-null mice have an increased total insulin secretory capacity, which improved glucose homeostasis. We conclude that these VAMP8-mediated events partly underlie the therapeutic actions of GLP-1 on insulin secretion and ß cell growth.

In vivo role of focal adhesion kinase in regulating pancreatic ß-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 ß-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 ß-cells. These RIPcre(+)fak(fl/fl) mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced ß-cell viability and proliferation resulting in decreased ß-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 ß-cells exhibited impaired insulin secretion with normal glucose sensing and preserved Ca(2+) 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 ß-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking.

Hepatocyte-specific deletion of Janus kinase 2 (JAK2) protects against diet-induced steatohepatitis and glucose intolerance.

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 ß-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.

The redundant role of JAK2 in regulating pancreatic ß-cell mass.

Janus kinase (JAK) 2 is a non-receptor tyrosine kinase that mediates the downstream effects of various growth factors, including growth hormone, prolactin, placental lactogen, and erythropoietin (EPO). EPO is a hematopoietic growth factor that is largely known for its role in promoting proliferation, differentiation and survival of cells in the erythroid lineage. Global loss of the EPO receptor (EPO-R) has been shown to be embryonically lethal in mice due to anemia attributed to defects in erythropoiesis. Interesting, mice with global deficiency of JAK2 share a similar developmental phenotype as the EPO-R knockout mice, demonstrating that JAK2 is essential in eliciting the biological effects of EPO, particularly in erythrocytosis. Recent studies from our group have shown that exogenous EPO protects mice against diabetes through direct effects on pancreatic ß-cells, and these protective effects are dependent on the presence of JAK2 in the ß-cells. Here, we briefly highlight the cytoprotective effects of exogenous EPO in the pancreatic ß-cells as well as our new findings on the redundant role of JAK2 in ß-cell expansion after high-fat feeding in mice.