L-leucine alters pancreatic β-cell differentiation and function via the mTor signaling pathway

Leucine (Leu) is an essential branched-chain amino acid, which activates the mammalian target of rapamycin (mTOR) signaling pathway. The effect of Leu on cell differentiation during embryonic development is unknown. Here, we show that Leu supplementation during pregnancy significantly increased fetal body weight, caused fetal hyperglycemia and hypoinsulinemia, and decreased the relative islet area. We also used rat embryonic pancreatic explant culture for elucidating the mechanism of Leu action on β-cell development. We found that in the presence of Leu, differentiation of pancreatic duodenal homeobox-1-positive progenitor cells into neurogenin3-positive endocrine progenitor cells was inefficient and resulted in decreased β-cell formation. Mechanistically, Leu increases the intracellular levels of hypoxia-inducible factor 1-α, a repressor of endocrine fate in the pancreas, by activating the mTOR complex 1 signaling pathway. Collectively, our findings indicate that Leu supplementation during pregnancy could potentially increase the risk of type 2 diabetes mellitus by inhibiting the differentiation of pancreatic endocrine progenitor cells during a susceptible period of fetal life.     

Nutritional programming of pancreatic β-cell plasticity

Nutritional insufficiency during pregnancy has been shown to alter the metabolism of the offspring and can increase the risk of type 2 diabetes. The phenotype in the offspring involves changes to the morphology and functional capacity of the endocrine pancreas, and in the supporting islet microvasculature. Pancreatic β-cells possess a plastic potential and can partially recover from catastrophic loss. This is partly due to the existence of progenitors within the islets and the ability to generate new islets by neogenesis from the pancreatic ducts. This regenerative capacity is induced by bone marrow-derived stem cells, including endothelial cell progenitors and is associated with increased angiogenesis within the islets. Nutritional insults in early life, such as feeding a low protein diet to the mother, impair the regenerative capacity of the β-cells. The mechanisms underlying this include a reduced ability of β-cells to differentiate from the progenitor population, changes in the inductive signals from the microvasculature and an altered presence of endothelial progenitors. Statin treatment within animal models was associated with angiogenesis in the islet microvasculature, improved vascular function and an increase in β-cell mass. This demonstrates that reversal of the impaired β-cell phenotype observed following nutritional insult in early life is potentially possible.     

Activation of protein kinase C-ζ in pancreatic β-cells in vivo improves glucose tolerance and induces β-cell expansion via mTOR activation

OBJECTIVE

PKC-ζ activation is a key signaling event for growth factor–induced β-cell replication in vitro. However, the effect of direct PKC-ζ activation in the β-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in β-cell expansion and function in vivo in mice and the mechanisms associated with these effects.

RESEARCH DESIGN AND METHODS

We characterized glucose homeostasis and β-cell phenotype of transgenic (TG) mice with constitutive activation of PKC-ζ in the β-cell. We also analyzed the expression and regulation of signaling pathways, G1/S cell cycle molecules, and β-cell functional markers in TG and wild-type mouse islets.

RESULTS

TG mice displayed increased plasma insulin, improved glucose tolerance, and enhanced insulin secretion with concomitant upregulation of islet insulin and glucokinase expression. In addition, TG mice displayed increased β-cell proliferation, size, and mass compared with wild-type littermates. The increase in β-cell proliferation was associated with upregulation of cyclins D1, D2, D3, and A and downregulation of p21. Phosphorylation of D-cyclins, known to initiate their rapid degradation, was reduced in TG mouse islets. Phosphorylation/inactivation of GSK-3β and phosphorylation/activation of mTOR, critical regulators of D-cyclin expression and β-cell proliferation, were enhanced in TG mouse islets, without changes in Akt phosphorylation status. Rapamycin treatment in vivo eliminated the increases in β-cell proliferation, size, and mass; the upregulation of cyclins Ds and A in TG mice; and the improvement in glucose tolerance—identifying mTOR as a novel downstream mediator of PKC-ζ–induced β-cell replication and expansion in vivo.

CONCLUSIONS

PKC-ζ, through mTOR activation, modifies the expression pattern of β-cell cycle molecules leading to increased β-cell replication and mass with a concomitant enhancement in β-cell function. Approaches to enhance PKC-ζ activity may be of value as a therapeutic strategy for the treatment of diabetes.Diabetes appears when β-cell mass is insufficient to maintain normal glucose homeostasis. Therefore, deciphering the molecular mechanisms that induce β-cell expansion can be of great value for therapeutic approaches aimed at increasing β-cell mass in diabetes. Atypical protein kinase C (PKC)-ζ, a relatively novel downstream target of phosphatidylinositol (PI) 3-kinase–phosphoinositide-dependent kinase-1 (PDK-1) in β-cells, is critical for mitogenic signal transduction in a variety of cell types, including fibroblasts, glial cells, and oocytes (1,2). PKC-ζ is expressed in insulinoma cells, as well as in rodent and human islets, and it is phosphorylated/activated by growth factors and nutrients such as glucose and free fatty acids (3–8). Importantly, activation of PKC-ζ is required for growth factor–stimulated β-cell proliferation in vitro (3,4). Furthermore, PKC-ζ overexpression enhances insulin-like growth factor-1 and insulin- and serum-induced proliferation in insulinoma cells in vitro (9). Taken together, these results highlight PKC-ζ as a critical signaling target for growth factor–mediated β-cell proliferation in vitro. Indeed, constitutively active PKC-ζ (CA-PKC-ζ) increases β-cell proliferation in insulinoma and primary mouse and human islet cells in vitro (3,4). Although the intracellular targets of PKC-ζ that induce mitogenesis are being actively explored in many tissues and include the extracellular signal–regulated kinases (ERK)1/2 and -5, glycogen synthase kinase 3 (GSK-3), mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K) (10–14), whether these targets are activated by PKC-ζ in β-cells is unknown.Studies using a variety of PKC inhibitors have suggested that glucose-stimulated insulin secretion (GSIS) is in part dependent on atypical PKCs activation in rat islets (6). In addition, inhibition of glucose-mediated activation of PKC-ζ correlates with decreased sulphonylurea receptor 1 (SUR1), inward rectifier K⁺ channel subunit (Kir6.2), and forkhead box A2 (Foxa2) expression and diminished GSIS (7). Interestingly, it has been suggested that PKC-ζ could be involved in glucose-mediated DNA-binding activity of pancreatic and duodenal homeobox 1 (Pdx-1) to the insulin gene in MIN6 cells (5). Taken together, these in vitro studies strongly suggest that PKC-ζ activation in β-cells could lead to increased β-cell function, proliferation, and mass and improved glucose homeostasis in vivo. However, this has never been explored.To analyze the effects of PKC-ζ activation in the β-cell in vivo, we generated transgenic (TG) mice with CA-PKC-ζ expression in the β-cell by using the rat insulin-II promoter (RIP). TG mice show increased β-cell replication, size, and mass concomitant with enhanced insulin secretion and improved glucose tolerance. These studies also uncover mTOR as a downstream key regulator of PKC-ζ effects in the β-cell. Our results clearly indicate that PKC-ζ activation could have therapeutic potential to expand β-cell mass and function for the treatment of diabetes.     

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(+)fak(fl/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 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 β-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking.     

Loss of HGF/c-Met signaling in pancreatic β-cells leads to incomplete maternal β-cell adaptation and gestational diabetes mellitus

Hepatocyte growth factor (HGF) is a mitogen and insulinotropic agent for the β-cell. However, whether HGF/c-Met has a role in maternal β-cell adaptation during pregnancy is unknown. To address this issue, we characterized glucose and β-cell homeostasis in pregnant mice lacking c-Met in the pancreas (PancMet KO mice). Circulating HGF and islet c-Met and HGF expression were increased in pregnant mice. Importantly, PancMet KO mice displayed decreased β-cell replication and increased β-cell apoptosis at gestational day (GD)15. The decreased β-cell replication was associated with reductions in islet prolactin receptor levels, STAT5 nuclear localization and forkhead box M1 mRNA, and upregulation of p27. Furthermore, PancMet KO mouse β-cells were more sensitive to dexamethasone-induced cytotoxicity, whereas HGF protected human β-cells against dexamethasone in vitro. These detrimental alterations in β-cell proliferation and death led to incomplete maternal β-cell mass expansion in PancMet KO mice at GD19 and early postpartum periods. The decreased β-cell mass was accompanied by increased blood glucose, decreased plasma insulin, and impaired glucose tolerance. PancMet KO mouse islets failed to upregulate GLUT2 and pancreatic duodenal homeobox-1 mRNA, insulin content, and glucose-stimulated insulin secretion during gestation. These studies indicate that HGF/c-Met signaling is essential for maternal β-cell adaptation during pregnancy and that its absence/attenuation leads to gestational diabetes mellitus.     

Betatrophin: A liver-derived hormone for the pancreatic β-cell proliferation

The pancreatic β-cell failure which invariably accompanies insulin resistance in the liver and skeletal muscle is a hallmark of type-2 diabetes mellitus (T2DM). The persistent hyperglycemia of T2DM is often treated with anti-diabetic drugs with or without subcutaneous insulin injections, neither of which mimic the physiological glycemic control seen in individuals with fully functional pancreas. A sought after goal for the treatment of T2DM has been to harness the regenerative potential of pancreatic β-cells that might obviate a need for exogenous insulin injections. A new study towards attaining this aim was reported by Yi et al, who have characterized a liver-derived protein, named betatrophin, capable of inducing pancreatic β-cell proliferation in mice. Using a variety of in vitro and in vivo methods, Yi et al, have shown that betatrophin was expressed mainly in the liver and adipose tissue of mice. Exogenous expression of betatrophin in the liver led to dramatic increase in the pancreatic β-cell mass and higher output of insulin in mice that also concomitantly elicited improved glucose tolerance. The authors discovered that betatrophin was also present in the human plasma. Surprisingly, betatrophin has been previously described by three other names, i.e., re-feeding-induced fat and liver protein, lipasin and atypical angiopoeitin-like 8, by three independent laboratories, as nutritionally regulated liver-enriched factors that control serum triglyceride levels and lipid metabolism. Yi et al demonstration of betatrophin, as a circulating hormone that regulates β-cell proliferation, if successfully translated in the clinic, holds the potential to change the course of current therapies for diabetes.     

Normal glucagon signaling and β-cell function after near-total α-cell ablation in adult mice

OBJECTIVE

To evaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects β-cell function and blood glucose homeostasis.

RESEARCH DESIGN AND METHODS

We generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after β-cell loss.

RESULTS

Adult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. β-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and β-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive β-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions.

CONCLUSIONS

An extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme β-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating β-cells by reprogramming adult α-cells.In rodents, glucagon-producing α-cells are the second most abundant endocrine cell type in pancreatic islets of Langerhans, after the insulin-producing β-cells. In human islets, α-cells are nearly as abundant as β-cells (1,2). They secrete glucagon in response to reduced blood glucose to promote gluconeogenesis and glycogenolysis in the liver (3). Proper control of blood glucose level thus relies on insulin action and the counter-regulation mediated by glucagon signaling. Besides this, insulin and glucagon reciprocally regulate α- and β-cell function through local, intraislet paracrine signaling (4).Excess of plasma glucagon (hyperglucagonemia) is frequently reported in diabetic patients, a deregulation that exacerbates hyperglycemia and triggers ketoacidosis, a major complication of diabetes (5). A recent study showed that streptozotocin (STZ)-induced diabetes is prevented in glucagon receptor–knockout (GcgR^−/−) mice, suggesting that in diabetes, hyperglycemia is largely due to glucagon action (6). Therefore, reducing the α-cell mass to limit glucagon production may represent an interesting approach to prevent glucagon excess in diabetes. Nevertheless, it is unknown how a severe decrease in pancreatic α-cell mass would be tolerated in physiologic or in diabetic conditions. Indeed, whereas a strong deficit in β-cell mass triggers diabetes, less is known regarding the requirement of maintaining intact the adult α-cell population.Complete disruption of glucagon signaling in GcgR^−/− mice is associated with defects in endocrine cell differentiation and increased embryonic lethality (7–9). In addition, mice that are unable to process proglucagon in its mature and active form due to prohormone convertase 2 (PC2) inactivation also display altered islet cell differentiation (10,11). GcgR^−/− and PC2^−/− adult mutant mice both exhibit an expanded α-cell mass, which is associated with lower blood glucose levels.Mice constitutively lacking aristaless-related homeobox (ARX) in the pancreas from early development display an α-cell deficit associated with abnormally increased non–α-cell numbers and hypoglycemia (12). In this situation, the altered metabolic status of adult individuals may reflect adaptation phenomena subsequent to developmental defects. Collectively, these studies that focused on the effect of glucagon deficiency did not directly address whether functional α-cells are essential per se for proper β-cell function and blood glucose homeostasis in physiologic conditions in adulthood.In the current study, we have analyzed a model of inducible, selective α-cell loss that we previously used in studies involving β-cell regeneration (13). Here, we examine the short- and long-term influence on blood glucose homeostasis of an acute, rapid, near-total α-cell ablation induced in healthy normal adult mice. We show that a very limited number of adult α-cells, some 2% of the normal α-cell mass, is sufficient to preserve a normal counter-response to insulin in basal conditions, without affecting β-cell function or longevity. Furthermore, we report that extreme α-cell removal, contrary to what happens in GcgR^−/− mice (6), does not prevent hyperglycemia after β-cell loss, indicating that the few remaining α-cells still mediate normal glucagon signaling in diabetes.     

Prevention of autoimmune diabetes by ectopic pancreatic β-cell expression of interleukin-35

Interleukin (IL)-35 is a newly identified inhibitory cytokine used by T regulatory cells to control T cell-driven immune responses. However, the therapeutic potential of native, biologically active IL-35 has not been fully examined. Expression of the heterodimeric IL-35 cytokine was targeted to β-cells via the rat insulin promoter (RIP) II. Autoimmune diabetes, insulitis, and the infiltrating cellular populations were analyzed. Ectopic expression of IL-35 by pancreatic β-cells led to substantial, long-term protection against autoimmune diabetes, despite limited intraislet IL-35 secretion. Nonobese diabetic RIP-IL35 transgenic mice exhibited decreased islet infiltration with substantial reductions in the number of CD4(+) and CD8(+) T cells, and frequency of glucose-6-phosphatase catalytic subunit-related protein-specific CD8(+) T cells. Although there were limited alterations in cytokine expression, the reduced T-cell numbers observed coincided with diminished T-cell proliferation and G1 arrest, hallmarks of IL-35 biological activity. These data present a proof of principle that IL-35 could be used as a potent inhibitor of autoimmune diabetes and implicate its potential therapeutic utility in the treatment of type 1 diabetes.     

Identification and differentiation of PDX1 β-cell progenitors within the human pancreatic epithelium

AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,progenitor cells within the pancreatic epithelium cannot be characterized in vitro,thoughβ-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition(EMT)may generateβ-cell progenitors.Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth(dexamethasone,epidermal growth factor,3,5,3’-triiodo-l-thyronine,bovine brain extract).Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation.Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter.In combination with lentiviral tracing,cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry(BD fluorescence activated cell sorting),immunostaining and real-time polymerase chain reaction(PCR)(7900HT Fast Realtime PCR System).RESULTS:In the presence of 10%serum MSCs rapidly expand in vitro while the epithelial cell population declines.The percentage of vimentin+cells increased from 22%±5.83%to 80.43%±3.24%(14 d)and99.00%±0.0%(21 d),and the percentage of epithelial cells decreased from 74.71%±8.34%to 26.57%±9.75%(14 d)and 4.00%±1.53%(21 d),P0.01 for all time points.Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT.Cells expanded in our epithelial medium contained significantly less mesenchymal cells(vimentin+)compared to controls(44.87%±4.93%vs 95.67%±1.36%;P0.01).During cell differentiation lentiviral reporting demonstrated that,PDX1+and insulin+cells were localized within adherent epithelial cell aggregates compared to controls.Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1,insulin,PAX4 and RFX(P0.05).CONCLUSION:PDX1+cells were confined to adherent epithelial cell aggregates and not vimentin+cells(mesenchymal),suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.     

Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling

In this study we investigated if Wnt/β-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of β-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in β-catenin^Prx1ER conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (μCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the β-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through β-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in β-catenin^Prx1ER conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.     

PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic β-cell apoptosis via regulation of the BH3-only protein Bim

OBJECTIVE

Genome-wide association studies allowed the identification of several associations between specific loci and type 1 diabetes (T1D). However, the mechanisms by which most candidate genes predispose to T1D remain unclear. We presently evaluated the mechanisms by which PTPN2, a candidate gene for T1D, modulates β-cell apoptosis after exposure to type I and II interferons (IFNs), cytokines that contribute to β-cell loss in early T1D.

RESEARCH DESIGN AND METHODS

Small interfering RNAs were used to inhibit PTPN2, STAT1, Bim, and Jun NH₂-terminal kinase 1 (JNK1) expression. Cell death was assessed by Hoechst and propidium iodide staining. BAX translocation, Bim phosphorylation, cytochrome c release, and caspases 9 and 3 activation were measured by Western blot or immunofluorescence.

RESULTS

PTPN2 knockdown exacerbated type I IFN–induced apoptosis in INS-1E, primary rat, and human β-cells. PTPN2 silencing and exposure to type I and II IFNs induced BAX translocation to the mitochondria, cytochrome c release, and caspase 3 activation. There was also an increase in Bim phosphorylation that was at least in part regulated by JNK1. Of note, both Bim and JNK1 knockdown protected β-cells against IFN-induced apoptosis in PTPN2-silenced cells.

CONCLUSIONS

The present findings suggest that local IFN production may interact with a genetic factor (PTPN2) to induce aberrant proapoptotic activity of the BH3-only protein Bim, resulting in increased β-cell apoptosis via JNK activation and the intrinsic apoptotic pathway. This is the first indication of a direct interaction between a candidate gene for T1D and the activation of a specific downstream proapoptotic pathway in β-cells.Type 1 diabetes (T1D) is a chronic autoimmune disease during which pancreatic β-cells are specifically damaged by an aberrant immune response. Susceptibility to T1D is linked to genetic factors, but T1D-predisposing genes have low penetrance and only a small proportion of individuals genetically at risk will develop the disease. During the past few years, genome-wide association studies allowed the identification of a large number of robust associations between specific chromosomal loci and T1D development (1). Well-known susceptibility genes include HLA-DR, CTLA-4, IFIH1 (MDA5), and PTPN22 (2). However, these genes only account for part of interindividual differences in disease predisposition or phenotypic diversity, and the pathophysiologic mechanisms by which most candidate genes predispose to T1D remain unclear.It is likely that an interplay between T1D susceptibility genes and environmental factors contributes to the triggering and progression of the disease (3). In this context, a better understanding of the functional effects of the susceptibility genes and their interaction with putative environmental causalities would help to understand the pathogenesis of T1D (4). In animal models for other autoimmune diseases (e.g., Crohn’s disease), there is a striking interaction between a mutation in the Atg16L1 candidate gene and the Murine norovirus, resulting in pathologic abnormalities similar to Crohn’s disease (5). In the context of T1D, a single nucleotide polymorphism (SNP) in the susceptibility gene PTPN22 and early introduction of cow’s milk in the diet are associated with the induction of islet autoantibodies and diabetes development in the Finnish population (6).Approximately 30% of the T1D candidate genes are expressed in β-cells (7) (M.L.C., F.M., D.L.E., unpublished data), suggesting that β-cells play a role in their own demise in T1D. The reduction of β-cell mass in T1D is preceded by an inflammatory process (insulitis) driven in part by a “dialog” between β-cells and infiltrating immune cells, mediated by the local release of cytokines and chemokines (8). Viruses are potential environmental factors contributing to the triggering of insulitis (9,10). During viral infections, β-cells release several chemokines and cytokines, including type I interferons (IFNs) (IFNα and IFNβ) (7,11), which contribute to T1D pathogenesis (12,13). In this respect, the T1D candidate gene IFIH1 (MDA5) is involved in the recognition of double-stranded RNA (dsRNA), a by-product of viral replication (14), and we previously observed that knocking down MDA5 in pancreatic β-cells prevents dsRNA-induced expression of key cytokines and chemokines (7).PTPN2 (also known as TC-PTP or PTP-S2) is another candidate gene for T1D (1). Known risk alleles for T1D in the PTPN2 gene are noncoding, and noncoding variants that may affect splicing have been identified by resequencing (1). One T1D risk variant of PTPN2 is associated with decreased PTPN2 expression in CD4⁺ T cells and transformed B-cell lines (15). The PTPN2 gene encodes a phosphatase that is ubiquitously expressed (16). The cytokines IFNγ and tumor necrosis factor-α increase PTPN2 expression in human colonic intraepithelial cells, and an upregulation of PTPN2 expression has been observed in intestinal biopsies from patients with active celiac disease (17,18). PTPN2 is highly expressed in immune-related cells, and its expression is modified in CD4⁺ T cells from patients with T1D when compared with CD4⁺ T cells from healthy control subjects (15).PTPN2 is an important negative regulator of the Janus kinase-STAT signaling pathway that is activated downstream of type I (IFNα and IFNβ) and II (IFNγ) IFN receptors. We recently described that this phosphatase is induced by IFNγ and a synthetic dsRNA, polyinosinic-polycitidilic acid (PIC), in β-cells and exacerbates IFNγ- and PIC-induced β-cell apoptosis by modulating STAT1 activation (7,19). However, the mechanisms connecting this candidate gene to actual β-cell death remain unclear.We presently observed that PTPN2 also regulates type I IFN–induced apoptosis in β-cells. By systematically knocking down genes putatively involved in the apoptosis pathway of β-cells (20), we identified the apoptotic mechanisms by which PTPN2 knockdown exacerbates type I and II IFN–induced β-cell death. This clarifies the interaction between a candidate gene for T1D and the activation of specific proapoptotic pathways in β-cells and broadens our understanding of the molecular mechanisms involved in the gene/environment interactions triggering insulitis and β-cell apoptosis.