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.

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.

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 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.

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.

Redundant role of the cytochrome c-mediated intrinsic apoptotic pathway in pancreatic ß-cells.

Cytochrome c is one of the central mediators of the mitochondrial or the intrinsic apoptotic pathway. Mice harboring a 'knock-in' mutation of cytochrome c, impairing only its apoptotic function, have permitted studies on the essential role of cytochrome c-mediated apoptosis in various tissue homeostasis. To this end, we examined the role of cytochrome c in pancreatic ß-cells under homeostatic conditions and in diabetes models, including those induced by streptozotocin (STZ) and c-Myc. Previous studies have shown that both STZ- and c-Myc-induced ß-cell apoptosis is mediated through caspase-3 activation; however, the precise mechanism in these modes of cell death was not characterized. The results of our study show that lack of functional cytochrome c does not affect glucose homeostasis or pancreatic ß-cell mass under basal conditions. Moreover, the cytochrome c-mediated intrinsic apoptotic pathway is required for neither STZ- nor c-Myc-induced ß-cell death. We also observed that the extrinsic apoptotic pathway mediated through caspase-8 was not essential in c-Myc-induced ß-cell destruction. These findings suggest that cytochrome c is not required for STZ-induced ß-cell apoptosis and, together with the caspase-8-mediated extrinsic pathway, plays a redundant role in c-Myc-induced ß-cell apoptosis.

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

Type 2 diabetes is hallmarked by insulin resistance and insufficient ß-cell function. Islets of type 2 diabetes patients have been shown to have decreased hypoxia-inducible factor (HIF)-1α/ß 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 ß-cells would increase ß-cell mass and function. We generated ß-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 ß-cell function. VHL deletion in the pancreatic ß-cells led to impaired glucose tolerance due to defects in glucose-stimulated insulin secretion and ß-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 ß-cell defects that occurred with VHL loss. We observed improved glucose tolerance and restoration of GLUT2 expression in VHL-deficient ß-cells in response to rHuEPO. Contrary to our hypothesis, loss of VHL and increased transcription of HIF-target genes resulted in impaired ß-cell function and mass, which can be overcome with exogenous EPO. Our results indicate a critical role for VHL in ß-cell function and mass, and that EPO administration improved ß-cell function making it a potential strategy for diabetes treatment.

Epigallocatechin gallate (EGCG) and rutin suppress the glucotoxicity through activating IRS2 and AMPK signaling in rat pancreatic beta cells.

Pancreatic beta cell failure is one critical metabolic disorder in the development of type 2 diabetes. Decreased viability and dysfunction of beta cells would accelerate the diabetic pathogenesis associated with higher mortality. In this study, the tea polyphenol EGCG (epigallocatechin gallate) and the buckwheat flavonoid Rutin were investigated to attenuate the induced glucotoxicity in beta cells. EGCG and Rutin could preserve the insulin secretory machinery and stimulate insulin receptor substrate 2 (IRS2) signaling in rat pancreatic beta cells, RIN-m5F. These findings further demonstrated the reduced glucolipotoxic effects of EGCG and Rutin through activating AMP-activated protein kinase (AMPK) signaling to inhibit the activities of lipogenic enzymes and ameliorating mitochondrial function. Consequently, the cell viability was retained after attenuating the glucotoxicity through the broad effect of EGCG and Rutin. The intrinsic protective effects of EGCG and Rutin in preserving the insulin signaling and regulating lipogenesis, manipulating cell cycling, and maintaining mitochondrial function to achieve the integrity of beta cells, which highlight the possibilities of EGCG and Rutin as novel strategies for the prevention of type 2 diabetes.