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Identifying the mechanisms behind the b-cell adaptation-to-failure is important to develop strategies to manage type 2 diabetes (T2D). Using db/db mice at early stages of the disease process, we took advantage of unbiased RNAseq to identify genes/pathways regulated by insulin resistance in b-cells. We demonstrate herein that islets from 4-week-old non-obese and non-diabetic leptin-receptor deficient db/db mice exhibited downregulation of several genes involved in cell-cycle regulation and DNA repair. We identified the transcription factor Yin Yang 1 (YY1) as a common gene between both pathways. The expression of YY1 and its targeted genes was decreased in the db/db islets. We confirmed the reduction in YY1 expression in b-cells from diabetic db/db mice, mice fed high fat diet (HFD) and individuals with T2D. ChIP-seq profiling in EndocBH1 cells, a human pancreatic b-cell line, indicated that YY1 binding regions regulate cell-cycle control, DNA damage recognition and repair. We then generated mouse models with constitutive and inducible YY1 deficiency in b-cells. YY1 deficient mice developed diabetes early in life due to b-cell loss. b-cells from these mice exhibited higher DNA damage, cell cycle arrest and cell death as well as decreased maturation markers. Tamoxifen-induced YY1 deficiency in mature b-cells impaired b-cell function and induced DNA damage. In summary, we identified YY1 as a critical factor for b-cell DNA repair and cell-cycle progression.

Maternal low-protein diet (LP) throughout gestation affects pancreatic β-cell fraction of the offspring at birth, thus increasing their susceptibility to metabolic dysfunction and type 2 diabetes in adulthood. The present study sought to strictly examine the effects of LP during the last week of gestation (LP12.5) alone as a developmental window for β-cell programming and metabolic dysfunction in adulthood. Islet morphology analysis revealed normal β-cell fraction in LP12.5 newborns. Normal glucose tolerance was observed in 6- to 8-wk-old male and female LP12.5 offspring. However, male LP12.5 offspring displayed glucose intolerance and reduced insulin sensitivity associated with β-cell dysfunction with aging. High-fat diet exposure of metabolically normal 12-wk-old male LP12.5 induced glucose intolerance due to increased body weight, insulin resistance, and insufficient β-cell mass adaptation despite higher insulin secretion. Assessment of epigenetic mechanisms through microRNAs (miRs) by a real-time PCR-based microarray in islets revealed elevation in miRs that regulate insulin secretion (miRs 342, 143), insulin resistance (miR143), and obesity (miR219). In the islets, overexpression of miR143 reduced insulin secretion in response to glucose. In contrast to the model of LP exposure throughout pregnancy, islet protein levels of mTOR and pancreatic and duodenal homeobox 1 were normal in LP12.5 islets. Collectively, these data suggest that LP diet during the last week of pregnancy is critical and sufficient to induce specific and distinct developmental programming effects of tissues that control glucose homeostasis, thus causing permanent changes in specific set of microRNAs that may contribute to the overall vulnerability of the offspring to obesity, insulin resistance, and type 2 diabetes.

AIMS/HYPOTHESIS: Islet vascular fibrosis may play an important role in the progression of type 2 diabetes, but there are no mouse models allowing detailed mechanistic studies to understand how a dysfunctional islet microvasculature contributes to diabetes pathogenesis. Here we report that the transgenic AktTg mouse, unlike other mouse strains, shows an increased deposition of extracellular matrix (ECM) proteins in perivascular regions, allowing us to study the cellular mechanisms that lead to islet vascular fibrosis. METHODS: Using immunohistochemistry, we labelled the islet microvasculature and ECM in pancreas sections of AktTg mice and human donors and performed lineage tracing to follow the fate of islet pericytes. We compared islet microvascular responses in living pancreas slices from wild-type and AktTg mice. RESULTS: We found that vascular pericytes proliferate extensively, convert into profibrotic myofibroblasts and substantially contribute to vascular fibrosis in the AktTg mouse model. The increased deposition of collagen I, fibronectin and periostin within the islet is associated with diminished islet perfusion as well as impaired capillary responses to noradrenaline (norepinephrine) and to high glucose in living pancreas slices. CONCLUSIONS/INTERPRETATION: Our study thus illustrates how the AktTg mouse serves to elucidate a cellular mechanism in the development of islet vascular fibrosis, namely a change in pericyte phenotype that leads to vascular dysfunction. Because beta cells in the AktTg mouse are more numerous and larger, and secrete more insulin, in future studies we will test the role beta cell secretory products play in determining the phenotype of pericytes and other cells residing in the islet microenvironment under physiological and pathophysiological conditions.

G protein–coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gβ5–R7, a protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit β 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M–stimulated glucose–stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5(−/−) islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K(+)-evoked membrane depolarization. These results indicate that Gβ5–R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gβ5–R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout β-cells from female, but not male mice, whereas only male βInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female βInsrKO and βInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter β-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include β-cell insulin resistance, which predicts that β-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female βInsrKO and βInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that β-cell insulin resistance in the form of reduced β-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

The risk of obesity in adulthood is subject to programming in the womb. Maternal obesity contributes to programming of obesity and metabolic disease risk in the adult offspring. With the increasing prevalence of obesity in women of reproductive age there is a need to understand the ramifications of maternal high-fat diet (HFD) during pregnancy on offspring’s metabolic heath trajectory. In the present study, we determined the long-term metabolic outcomes on adult male and female offspring of dams fed with HFD during pregnancy. C57BL/6J dams were fed either Ctrl or 60% Kcal HFD for 4 weeks before and throughout pregnancy, and we tested glucose homeostasis in the adult offspring. Both Ctrl and HFD-dams displayed increased weight during pregnancy, but HFD-dams gained more weight than Ctrl-dams. Litter size and offspring birthweight were not different between HFD-dams or Ctrl-dams. A significant reduction in random blood glucose was evident in newborns from HFD-dams compared to Ctrl-dams. Islet morphology and alpha-cell fraction were normal but a reduction in beta-cell fraction was observed in newborns from HFD-dams compared to Ctrl-dams. During adulthood, male offspring of HFD-dams displayed comparable glucose tolerance under normal chow. Male offspring re-challenged with HFD displayed glucose intolerance transiently. Adult female offspring of HFD-dams demonstrated normal glucose tolerance but displayed increased insulin resistance relative to controls under normal chow diet. Moreover, adult female offspring of HFD-dams displayed increased insulin secretion in response to high-glucose treatment, but beta-cell mass were comparable between groups. Together, these data show that maternal HFD at pre-conception and during gestation predisposes the female offspring to insulin resistance in adulthood.

Mammalian target of rapamycin (mTOR) kinase is an essential hub where nutrients and growth factors converge to control cellular metabolism. mTOR interacts with different accessory proteins to form complexes 1 and 2 (mTORC), and each complex has different intracellular targets. Although mTORC1's role in β-cells has been extensively studied, less is known about mTORC2's function in β-cells. Here, we show that mice with constitutive and inducible β-cell-specific deletion of RICTOR (; βRicKO; and i; βRicKO; mice, respectively) are glucose intolerant due to impaired insulin secretion when glucose is injected intraperitoneally. Decreased insulin secretion in βRicKO islets was caused by abnormal actin polymerization. Interestingly, when glucose was administered orally, no difference in glucose homeostasis and insulin secretion were observed, suggesting that incretins are counteracting the mTORC2 deficiency. Mechanistically, glucagon-like peptide-1 (GLP-1), but not gastric inhibitory polypeptide (GIP), rescued insulin secretion in vivo and in vitro by improving actin polymerization in; βRicKO; islets. In conclusion, mTORC2 regulates glucose-stimulated insulin secretion by promoting actin filament remodeling.; NEW & NOTEWORTHY; The current studies uncover a novel mechanism linking mTORC2 signaling to glucose-stimulated insulin secretion by modulation of the actin filaments. This work also underscores the important role of GLP-1 in rescuing defects in insulin secretion by modulating actin polymerization and suggests that this effect is independent of mTORC2 signaling.

O-GlcNAc transferase (OGT), a nutrient sensor sensitive to glucose flux, is highly expressed in the pancreas. However, the role of OGT in the mitochondria of β-cells is unexplored. In this study, we identified the role of OGT in mitochondrial function in β-cells. Constitutive deletion of OGT (βOGTKO) or inducible ablation in mature β-cells (iβOGTKO) causes distinct effects on mitochondrial morphology and function. Islets from βOGTKO, but not iβOGTKO, mice display swollen mitochondria, reduced glucose-stimulated oxygen consumption rate, ATP production, and glycolysis. Alleviating endoplasmic reticulum stress by genetic deletion of Chop did not rescue the mitochondrial dysfunction in βOGTKO mice. We identified altered islet proteome between βOGTKO and iβOGTKO mice. Pancreatic and duodenal homeobox 1 (Pdx1) was reduced in in βOGTKO islets. Pdx1 overexpression increased insulin content and improved mitochondrial morphology and function in βOGTKO islets. These data underscore the essential role of OGT in regulating β-cell mitochondrial morphology and bioenergetics. In conclusion, OGT couples nutrient signal and mitochondrial function to promote normal β-cell physiology.

Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in β-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in β-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human β-cells from type 2 diabetes donors. Using mice with conditional (βTGS1KO) and inducible (MIP-Cre

The dynamic regulation of autophagy in β-cells by cycles of fasting-feeding and its effects on insulin secretion are unknown. In β-cells, mechanistic target of rapamycin complex 1 (mTORC1) is inhibited while fasting and is rapidly stimulated during refeeding by a single amino acid, leucine, and glucose. Stimulation of mTORC1 by nutrients inhibited the autophagy initiator ULK1 and the transcription factor TFEB, thereby preventing autophagy when β-cells were continuously exposed to nutrients. Inhibition of mTORC1 by Raptor knockout mimicked the effects of fasting and stimulated autophagy while inhibiting insulin secretion, whereas moderate inhibition of autophagy under these conditions rescued insulin secretion. These results show that mTORC1 regulates insulin secretion through modulation of autophagy under different nutritional situations. In the fasting state, autophagy is regulated in an mTORC1-dependent manner, and its stimulation is required to keep insulin levels low, thereby preventing hypoglycemia. Reciprocally, stimulation of mTORC1 by elevated leucine and glucose, which is common in obesity, may promote hyperinsulinemia by inhibiting autophagy.

Elevation of glucagon levels and increase in a-cell mass are associated with states of hyperglycemia in diabetes. Our previous studies have highlighted the role of nutrient signaling via mTOR Complex 1 (mTORC1) regulation that controls glucagon secretion and a-cell mass. The current studies investigated the effects of activation of nutrient signaling by conditional deletion of the mTORC1 inhibitor, TSC2, in a-cells (aTSC2KO). We showed that activation of mTORC1 signaling is sufficient to induce chronic hyperglucagonemia as a result of a-cell proliferation, cell size and mass expansion. Hyperglucagonemia in aTSC2KO was associated with an increase in glucagon content and enhanced glucagon secretion. This model allowed us to identify the effects of chronic hyperglucagonemia on glucose homeostasis by inducing insulin secretion and resistance to glucagon in the liver. Liver glucagon resistance in aTSC2KO mice were characterized by reduced expression of the glucagon receptor (GCGR), phosphoenolpyruvate carboxykinase (PEPCK) and genes involved in amino acid metabolism and urea production. Glucagon resistance in aTSC2KO mice was associated with improved glucose levels in Streptozotocin (STZ)-induced β-cell destruction and HFD-induced glucose intolerance. These studies demonstrate that chronic hyperglucagonemia can improve glucose homeostasis by inducing glucagon resistance in the liver.

We previously demonstrated that exposing mouse dams to metformin during gestation results in increased beta-cell mass at birth and increased beta-cell insulin secretion in adult male offspring. Given these favorable changes after a gestational maternal metformin exposure, we wanted to understand the long-term metabolic impact on offspring after exposing dams to metformin during the postnatal window. The newborn period provides a feasible clinical window for intervention and is important for beta-cell proliferation and metabolic tissue development. Using a C57BL/6 model, we administered metformin to dams from the day of birth to postnatal day 21. We monitored maternal health and offspring growth during the lactation window, as well as adult glucose homeostasis through in vivo testing. At necropsy we assessed pancreas and adipocyte morphology using histological and immunofluorescent staining techniques. We found that metformin exposure programmed male and female offspring to be leaner with a higher proportion of small adipocytes in the gonadal white adipose tissue (GWAT). Male, but not female, offspring had an improvement in glucose tolerance as young adults concordant with a mild increase in insulin secretion in response to glucose in vivo. These data demonstrate long-term metabolic programming of offspring associated with maternal exposure to metformin during lactation.

Mechanistic target of rapamycin complex 1 (mTORC1) deficiency or chronic hyperactivation in pancreatic β-cells leads to diabetes. mTORC1 complexes with La-related protein 1 (LARP1) to specifically regulate the expression of 5′ terminal oligopyrimidine tract (5′TOP) mRNAs which encode proteins of the translation machinery and ribosome biogenesis. Here we show that LARP1 is the most expressed LARP in mouse islets and human β-cells, being 2–4-fold more abundant than LARP1B, a member of the family that also interacts with mTORC1. Interestingly, β-cells from diabetic patients have higher LARP1 and LARP1B expression. However, specific deletion of Larp1 gene in β-cells (β-Larp1KO mice) did not impair insulin secretion and glucose metabolism in male and female mice. High fat or high branched-chain amino acid (BCAA) diets did not disturb glucose homeostasis compared to control littermates up to 8 weeks; BCAA diet slightly impaired glucose tolerance in the β-Larp1KO mice at 16 weeks. However, no differences in plasma insulin levels, non-fasting glycemia and β-cell mass were observed in the β-Larp1KO mice. In conclusion, LARP1 is the most abundant LARP in mouse islets and human β-cells, and it is upregulated in diabetic subjects. However, genetically disruption of Larp1 gene did not impact glucose homeostasis in basal and diabetogenic conditions, suggesting no major role for LARP1 in β-cells.

Summary Diabetic kidney disease (DKD) increases the risk for mortality and is the leading cause of end-stage renal disease. Treatment with sodium-glucose cotransporter 2 inhibitors (SGLT2i) attenuates the progression of DKD, especially in patients with advanced kidney disease. Herein, we show that in diabetes, mTORC1 activity is increased in renal proximal tubule cells (RPTCs) along with enhanced tubule-interstitial fibrosis; this is prevented by SGLT2i. Constitutive activation of mTORC1 in RPTCs induces renal fibrosis and failure and abolishes the renal-protective effects of SGLT2i in diabetes. On the contrary, partial inhibition of mTORC1 in RPTCs prevents fibrosis and the decline in renal function. Stimulation of mTORC1 in RPTCs turns on a pro-fibrotic program in the renal cortex, whereas its inhibition in diabetes reverses the alterations in gene expression. We suggest that RPTC mTORC1 is a critical node that mediates kidney dysfunction in diabetes and the protective effects of SGLT2i by regulating fibrogenesis., Graphical Abstract, Highlights • In diabetes, mTORC1 activity is increased in renal proximal tubule cells (RPTCs) • Diabetes and SGLT2i regulate mTORC1 by modulating nutrient transport to RPTCs • Inhibition of mTORC1 in RPTCs prevents fibrosis and the decline in renal function • RPTC mTORC1 mediates renal fibrosis in diabetes and the beneficial effects of SGLT2i, Kogot-Levin et al. show that treatment with sodium-glucose cotransporter 2 inhibitors (SGLT2i) attenuates the progression of diabetic kidney disease (DKD), which is the leading cause of end-stage renal disease. The nutrient sensor mTORC1 is a critical node that mediates kidney dysfunction in diabetes and the protective effects of SGLT2i by regulating fibrogenesis.

Deoxyhypusine synthase (DHPS) utilizes the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly-defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that enabled the inducible, postnatal deletion of Dhps specifically in postnatal islet β cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces β-cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation and protein secretion, reduced production of the cell cycle molecule cyclin D2, impaired β-cell proliferation, and induced overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-ζ and was required for c-Myc-induced proliferation. Our studies reveal a requirement for DHPS in β cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis.

Regulation of glucose homeostasis by insulin depends on β-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in β-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in β-cells. We examined glucose homeostasis and β-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in β-cell function without alterations in β-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their β-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and β-cell insulin secretion defect but showed no changes in β-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to β-cell growth but is involved in regulating β-cell function in normal and diabetogenic conditions.

MicroRNA 199 (miR-199) negatively impacts pancreatic β-cell function and its expression is highly increased in islets from diabetic mice as well as in plasma of diabetic patients. Here we investigated how miR-199 expression is regulated in β-cells by assessing expression of miR-199 precursors (primiR-199a1, primiR-199a2, and primiR-199b) and mature miR-199 (miR-199-3p and miR-199-5p) and promoter transcriptional activity assays in mouse islets and mouse insulinoma cells (MIN6) under different stimuli. We found that mouse islets equally express miR-199-3p and miR-199-5p. However, the primiRNA expression levels differed; although primiR-199a1 expression was about 30% greater than that of primiR-199a2, primiR-199b is barely detected in islets. We observed a 2-fold increase in primiR-199a1 and primiR-199a2 mRNA levels in mouse islets cultured in 10 mm glucose compared with 5.5 mm glucose. Similar responses to glucose were observed in MIN6 cells. Exposure to 30 mm KCl to induce membrane depolarization and calcium influx increased expression of primiR-199a2 but not of primiR-199a1 in MIN6 cells, indicating that calcium influx was involved. Transcriptional activity studies in MIN6 cells also revealed that primiR-199a2 promoter activity was enhanced by glucose and reduced by 2-deoxy-D-glucose–induced starvation. KCl and the potassium channel blocker tolbutamide also stimulated primiR-199a2 promoter activity. Calcium channel blockade by nifedipine reduced primiR-199a2 promoter activity in MIN6 cells, and diazoxide-mediated calcium influx inhibition blunted glucose up-regulation of miR-199-3p in islets. In conclusion, we uncover that glucose acutely up-regulates miR-199 family expression in β-cells. Glucose metabolism and calcium influx are involved in primiR-199a2 expression but not primiR-199a1 expression.

Branched-chain amino acid (BCAAs: leucine, isoleucine, and valine) contribute to the development of obesity-associated insulin resistance in the context of consumption of a high-fat diet (HFD) in humans and rodents. Maternal diet is a major determinant of offspring health, and there is strong evidence that maternal HFD alters hypothalamic developmental programming and disrupts offspring energy homeostasis in rodents. In this study, we exposed pregnant and lactating C57BL/6JB female mice to either HFD, HFD with supplemented BCAA (HFD+BCAA), or standard diet (SC), and we studied offspring metabolic phenotypes. Both maternal HFD and HFD supplemented with BCAA had similar effect rendering the offspring metabolic imbalance and impairing their ability to cope with HFD when challenged during aging. The metabolic effects of HFD challenge were more profound in females, worsening female offspring ability to cope with an HFD challenge by activating hypothalamic inflammation in aging. Moreover, the sex differences in hypothalamic estrogen receptor α (ER-α) expression levels were lost in female offspring upon HFD challenge, supporting a link between ER-α levels and hypothalamic inflammation in offspring and highlighting the programming potential of hypothalamic inflammatory responses and maternal nutrition.

The need for chronic immune suppression (IS) is one of the hurdles precluding widespread use of islet cell transplantation to restore glycemic control in patients with type 1 diabetes. We report the case of a healthy nonhuman primate (NHP) treated on and off for over 2.5 years with steroid-free IS, consisting of daclizumab induction and maintenance therapy with rapamycin and low dose tacrolimus. Treatment for 1 year resulted in a striking destabilization of glycemic control, with concomitant decreases in fasting c-peptide and insulin levels. Although these changes gradually reversed during a wash out period of 7 months, retreatment with the same therapy led to accelerated deterioration in glycemic control. Intravenous glucose tolerance and percentage of glycosylated hemoglobin testing further supported a dramatic effect on metabolic control. IS also led to decreases in weight during treatment. Histological evaluation of the pancreas revealed islet hyperplasia, with varying sizes and endocrine cell ratios that differed from normal islet composition, and parenchymal infiltration with adipose tissue. These deleterious effects of IS on glucose control and endocrine components in the native pancreas of a healthy NHP suggest that IS agents commonly utilized for islet transplantation may contribute to failure in islet allograft function in long-term transplant patients.

Pancreatic beta cells (β-cells) differentiate during fetal life, but only postnatally acquire the capacity for glucose-stimulated insulin secretion (GSIS). How this happens is not clear. In exploring what molecular mechanisms drive the maturation of β-cell function, we found that the control of cellular signaling in β-cells fundamentally switched from the nutrient sensor target of rapamycin (mTORC1) to the energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK), and that this was critical for functional maturation. Moreover, AMPK was activated by the dietary transition taking place during weaning, and this in turn inhibited mTORC1 activity to drive the adult β-cell phenotype. While forcing constitutive mTORC1 signaling in adult β-cells relegated them to a functionally immature phenotype with characteristic transcriptional and metabolic profiles, engineering the switch from mTORC1 to AMPK signaling was sufficient to promote β-cell mitochondrial biogenesis, a shift to oxidative metabolism, and functional maturation. We also found that type 2 diabetes, a condition marked by both mitochondrial degeneration and dysregulated GSIS, was associated with a remarkable reversion of the normal AMPK-dependent adult β-cell signature to a more neonatal one characterized by mTORC1 activation. Manipulating the way in which cellular nutrient signaling pathways regulate β-cell metabolism may thus offer new targets to improve β-cell function in diabetes.

The mammalian target of rapamycin complex 1 (mTORC1) regulates several biological processes, although the key downstream mechanisms responsible for these effects are poorly defined. Using mice with deletion of eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), we determine that this downstream target is a major regulator of glucose homeostasis and β-cell mass, proliferation, and survival by increasing insulin receptor substrate 2 (IRS2) levels and identify a novel feedback mechanism by which mTORC1 signaling increases IRS2 levels. In this feedback loop, we show that 4E-BP2 deletion induces translation of the adaptor protein SH2B1 and promotes the formation of a complex with IRS2 and Janus kinase 2, preventing IRS2 ubiquitination. The changes in IRS2 levels result in increases in cell cycle progression, cell survival, and β-cell mass by increasing Akt signaling and reducing p27 levels. Importantly, 4E-BP2 deletion confers resistance to cytokine treatment in vitro. Our data identify SH2B1 as a major regulator of IRS2 stability, demonstrate a novel feedback mechanism linking mTORC1 signaling with IRS2, and identify 4E-BP2 as a major regulator of proliferation and survival of β-cells.

The fetal environment plays a decisive role in modifying the risk for developing diabetes later in life. Developing novel methodology for noninvasive imaging of β-cell development in vivo under the controlled physiological conditions of the host can serve to understand how this environment affects β-cell growth and differentiation. A number of culture models have been designed for pancreatic rudiment but none match the complexity of the in utero or even normal physiological environment. Speier et al. recently developed a platform of noninvasive in vivo imaging of pancreatic islets using the anterior chamber of the eye where islets get vascularized, grow and respond to physiological changes. The same methodology was adapted for the study of pancreatic development. E13.0, still undifferentiated rudiments with fluorescent lineage tracing were implanted in the AC of the eye, allowing the longitudinal study of their growth and differentiation. Within 48 h the anlages get vascularized and grow but their mesenchyme displays a selective growth advantage. The resulting imbalance leads to alteration in the differentiation pattern of the progenitors. Reducing the mesenchyme to its bare minimum before implantation allows the restoration of a proper balance and a development that mimics the normal pancreatic development. These groundbreaking observations demonstrate that the anterior chamber of the eye provides a good system for noninvasive in vivo fluorescence imaging of the developing pancreas under the physiology of the host and can have important implications for designing strategies to prevent or reverse the deleterious effects of hyperglycemia on altering β-cell function later in life.

Androgen excess predisposes women to type 2 diabetes (T2D), but the mechanism of this is poorly understood. We report that female mice fed a Western diet and exposed to chronic androgen excess using dihydrotestosterone (DHT) exhibit hyperinsulinemia and insulin resistance associated with secondary pancreatic β cell failure, leading to hyperglycemia. These abnormalities are not observed in mice lacking the androgen receptor (AR) in β cells and partially in neurons of the mediobasal hypothalamus (MBH) as well as in mice lacking AR selectively in neurons. Accordingly, i.c.v. infusion of DHT produces hyperinsulinemia and insulin resistance in female WT mice. We observe that acute DHT produces insulin hypersecretion in response to glucose in cultured female mouse and human pancreatic islets in an AR-dependent manner via a cAMP- and mTOR-dependent pathway. Acute DHT exposure increases mitochondrial respiration and oxygen consumption in female cultured islets. As a result, chronic DHT exposure in vivo promotes islet oxidative damage and susceptibility to additional stress induced by streptozotocin via AR in β cells. This study suggests that excess androgen predisposes female mice to T2D following AR activation in neurons, producing peripheral insulin resistance, and in pancreatic β cells, promoting insulin hypersecretion, oxidative injury, and secondary β cell failure.

This is the third in a series of Perspectives on intracellular signaling pathways coupled to proliferation in pancreatic β-cells. We contrast the large knowledge base in rodent β-cells with the more limited human database. With the increasing incidence of type 1 diabetes and the recognition that type 2 diabetes is also due in part to a deficiency of functioning β-cells, there is great urgency to identify therapeutic approaches to expand human β-cell numbers. Therapeutic approaches might include stem cell differentiation, transdifferentiation, or expansion of cadaver islets or residual endogenous β-cells. In these Perspectives, we focus on β-cell proliferation. Past Perspectives reviewed fundamental cell cycle regulation and its upstream regulation by insulin/IGF signaling via phosphatidylinositol-3 kinase/mammalian target of rapamycin signaling, glucose, glycogen synthase kinase-3 and liver kinase B1, protein kinase Cζ, calcium-calcineurin–nuclear factor of activated T cells, epidermal growth factor/platelet-derived growth factor family members, Wnt/β-catenin, leptin, and estrogen and progesterone. Here, we emphasize Janus kinase/signal transducers and activators of transcription, Ras/Raf/extracellular signal–related kinase, cadherins and integrins, G-protein–coupled receptors, and transforming growth factor β signaling. We hope these three Perspectives will serve to introduce these pathways to new researchers and will encourage additional investigators to focus on understanding how to harness key intracellular signaling pathways for therapeutic human β-cell regeneration for diabetes.

Pancreatic β-cells are exquisitely sensitive to developmental nutrient stressors, and alterations in nutrient sensing pathways may underlie changes observed in these models. Here we developed a mouse model of in utero exposure to the anti-diabetic agent metformin. We have previously shown that this exposure increases offspring pancreatic β-cell mass at birth. We hypothesized that adult offspring would have improved metabolic parameters as a long-term outcome of metformin exposure. Virgin dams were given 5 mg/mL metformin in their water from E0.5 to delivery at E18.5. Body weight, glucose tolerance, insulin tolerance and glucose stimulated insulin secretion were analyzed in the offspring. When male offspring of dams given metformin during gestation were tested as adults they had improved glucose tolerance and enhanced insulin secretion in vivo as did their islets in vitro. Enhanced insulin secretion was accompanied by changes in intracellular free calcium responses to glucose and potassium chloride, possibly mediated by increased L channel expression. Female offspring exhibited improved glucose tolerance at advanced ages. In conclusion, in this model in utero metformin exposure leads to improved offspring metabolism in a gender-specific manner. These findings suggest that metformin applied during gestation may be an option for reprogramming metabolism in at risk groups.

Glucagon plays a major role in the regulation of glucose homeostasis during fed and fasting states. However, the mechanisms responsible for the regulation of pancreatic α cell mass and function are not completely understood. In the current study, we identified mTOR complex 1 (mTORC1) as a major regulator of α cell mass and glucagon secretion. Using mice with tissue-specific deletion of the mTORC1 regulator Raptor in α cells (αRaptorKO), we showed that mTORC1 signaling is dispensable for α cell development, but essential for α cell maturation during the transition from a milk-based diet to a chow-based diet after weaning. Moreover, inhibition of mTORC1 signaling in αRaptorKO mice and in WT animals exposed to chronic rapamycin administration decreased glucagon content and glucagon secretion. In αRaptorKO mice, impaired glucagon secretion occurred in response to different secretagogues and was mediated by alterations in KATP channel subunit expression and activity. Additionally, our data identify the mTORC1/FoxA2 axis as a link between mTORC1 and transcriptional regulation of key genes responsible for α cell function. Thus, our results reveal a potential function of mTORC1 in nutrient-dependent regulation of glucagon secretion and identify a role for mTORC1 in controlling α cell-mass maintenance.

Objective Poor fetal nutrition increases the risk of type 2 diabetes in the offspring at least in part by reduced embryonic β-cell growth and impaired function. However, it is not entirely clear how fetal nutrients and growth factors impact β-cells during development to alter glucose homeostasis and metabolism later in life. The current experiments aimed to test the impact of fetal nutrients and growth factors on endocrine development and how these signals acting on mTOR signaling regulate β-cell mass and glucose homeostasis. Method Pancreatic rudiments in culture were used to study the role of glucose, growth factors, and amino acids on β-cell development. The number and proliferation of pancreatic and endocrine progenitor were assessed in the presence or absence of rapamycin. The impact of mTOR signaling in vivo on pancreas development and glucose homeostasis was assessed in models deficient for mTOR or Raptor in Pdx1 expressing pancreatic progenitors. Results We found that amino acid concentrations, and leucine in particular, enhance the number of pancreatic and endocrine progenitors and are essential for growth factor induced proliferation. Rapamycin, an mTORC1 complex inhibitor, reduced the number and proliferation of pancreatic and endocrine progenitors. Mice lacking mTOR in pancreatic progenitors exhibited hyperglycemia in neonates, hypoinsulinemia and pancreatic agenesis/hypoplasia with pancreas rudiments containing ductal structures lacking differentiated acinar and endocrine cells. In addition, loss of mTORC1 by deletion of raptor in pancreatic progenitors reduced pancreas size with reduced number of β-cells. Conclusion Together, these results suggest that amino acids concentrations and in particular leucine modulates growth responses of pancreatic and endocrine progenitors and that mTOR signaling is critical for these responses. Inactivation of mTOR and raptor in pancreatic progenitors suggested that alterations in some of the components of this pathway during development could be a cause of pancreatic agenesis/hypoplasia and hyperglycemia., Graphical abstract Image 1, Highlights • Leucine concentrations modulate proliferation of pancreatic progenitors. • mTOR signaling plays a critical role in regulating pancreatic and endocrine growth. • Inactivation of mTOR in pancreatic progenitors leads to pancreatic agenesis. • Inactivation of Raptor in pancreatic progenitors leads to pancreatic hypoplasia.

Deregulation of mTOR complex 1 (mTORC1) signalling increases the risk for metabolic diseases, including type 2 diabetes. Here we show that β-cell-specific loss of mTORC1 causes diabetes and β-cell failure due to defects in proliferation, autophagy, apoptosis and insulin secretion by using mice with conditional (βraKO) and inducible (MIP-βraKOf/f) raptor deletion. Through genetic reconstitution of mTORC1 downstream targets, we identify mTORC1/S6K pathway as the mechanism by which mTORC1 regulates β-cell apoptosis, size and autophagy, whereas mTORC1/4E-BP2-eIF4E pathway regulates β-cell proliferation. Restoration of both pathways partially recovers β-cell mass and hyperglycaemia. This study also demonstrates a central role of mTORC1 in controlling insulin processing by regulating cap-dependent translation of carboxypeptidase E in a 4EBP2/eIF4E-dependent manner. Rapamycin treatment decreases CPE expression and insulin secretion in mice and human islets. We suggest an important role of mTORC1 in β-cells and identify downstream pathways driving β-cell mass, function and insulin processing., Deregulation of mTORC1 pathway has been associated with several human diseases including diabetes, neurodegeneration and cancer. Here Blandino-Rosano et al. show that mTORC1 signalling controls insulin secretion and β-cell maintenance by regulation of β-cell proliferation, apoptosis and autophagy and insulin processing.

A maternal diet that is low in protein increases the susceptibility of offspring to type 2 diabetes by inducing long-term alterations in β cell mass and function. Nutrients and growth factor signaling converge through mTOR, suggesting that this pathway participates in β cell programming during fetal development. Here, we revealed that newborns of dams exposed to low-protein diet (LP0.5) throughout pregnancy exhibited decreased insulin levels, a lower β cell fraction, and reduced mTOR signaling. Adult offspring of LP0.5-exposed mothers exhibited glucose intolerance as a result of an insulin secretory defect and not β cell mass reduction. The β cell insulin secretory defect was distal to glucose-dependent Ca2+ influx and resulted from reduced proinsulin biosynthesis and insulin content. Islets from offspring of LP0.5-fed dams exhibited reduced mTOR and increased expression of a subset of microRNAs, and blockade of microRNA-199a-3p and -342 in these islets restored mTOR and insulin secretion to normal. Finally, transient β cell activation of mTORC1 signaling in offspring during the last week of pregnancy of mothers fed a LP0.5 rescued the defect in the neonatal β cell fraction and metabolic abnormalities in the adult. Together, these findings indicate that a maternal low-protein diet alters microRNA and mTOR expression in the offspring, influencing insulin secretion and glucose homeostasis.

Decreasing glucagon action lowers the blood glucose and may be useful therapeutically for diabetes. However, interrupted glucagon signaling leads to α-cell proliferation. To identify postulated hepatic-derived, circulating factor(s) responsible for α-cell proliferation, we used transcriptomics/proteomics/metabolomics in three models of interrupted glucagon signaling and found that proliferation of mouse, zebrafish, and human α-cells was mTOR- and FoxP transcription factor-dependent. Changes in hepatic amino acid (AA) catabolism gene expression predicted the observed increase in circulating AA. Mimicking these AA levels stimulated α-cell proliferation in a newly developed in vitro assay with L-glutamine being a critical AA. α-cell expression of the AA transporter Slc38a5 was markedly increased in mice with interrupted glucagon signaling and played a role in α-cell proliferation. These results indicate a hepatic-α-islet cell axis where glucagon regulates serum AA availability and AA, especially L-glutamine, regulates α-cell proliferation and mass via mTOR-dependent nutrient sensing.

Notch signaling is known to control early pancreatic differentiation through Ngn3 repression. In later stages, downstream of Notch, the Presenilins are still required to maintain the endocrine fate allocation. Amongst their multiple targets, it remains unclear which one actually controls the maintenance of the fate of the early islets. Conditional deletions of the Notch effector RBP-Jκ with lineage tracing in Presenilin-deficient endocrine progenitors, demonstrated that this factor is central to the control of the fate through a non-canonical Notch mechanism. RBP-Jκ mice exhibit normal islet morphogenesis and function, however, a fraction of the progenitors fails to differentiate and develop into disorganized masses resembling acinar to ductal metaplasia and chronic pancreatitis. A subsequent deletion of RBP-Jκ in forming β-cells led to the transdifferentiation into the other endocrine cells types, indicating that this factor still mediates the maintenance of the fate within the endocrine lineage itself. These results highlight the dual importance of Notch signaling for the endocrine lineage. Even after Ngn3 expression, Notch activity is required to maintain both fate and maturation of the Ngn3 progenitors. In a subset of the cells, these alterations of Notch signaling halt their differentiation and leads to acinar to ductal metaplasia.

OBJECTIVE Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain. RESEARCH DESIGN AND METHODS Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. RESULTS All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain. CONCLUSIONS Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.

OBJECTIVEThe purpose of this study was to evaluate the role of the S6K arm of mammalian target of rapamycin complex 1 (mTORC1) signaling in regulation of β-cell mass and function. Additionally, we aimed to delineate the importance of in vivo S6K activation in the regulation of insulin signaling and the extent to which alteration of insulin receptor substrate (IRS) signaling modulates β-cell mass and function.RESEARCH DESIGN AND METHODSThe current experiments describe the phenotype of transgenic mice overexpressing a constitutively active form of S6K under the control of the rat insulin promoter.RESULTSActivation of S6K signaling in these mice improved insulin secretion in the absence of changes in β-cell mass. The lack of β-cell mass expansion resulted from decreased G1-S progression and increased apoptosis. This phenotype was associated with increased p16 and p27 and decreased Cdk2 levels. The changes in cell cycle were accompanied by diminished survival signals because of impaired IRS/Akt signaling.CONCLUSIONSThis work defines the importance of S6K in regulation of β-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces β-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in β-cell failure and diabetes in conditions of nutrient overload.

The serine-threonine kinase Akt regulates multiple biological processes. An important strategy to study Akt signaling in different tissues is targeted activation of this pathway in vivo. The current studies describe the generation of a mouse model that combines a double reporter system with activation of a constitutively active form of Akt1 (caAkt) in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development as well as in most adult tissues. After Cre-mediated excision of the LacZ reporter, functionality of the transgene was demonstrated by expression of the caAkt mutant along with the second reporter, EGFP in different pancreatic compartments and in the nervous system. This animal model provides a critical reagent for assessing the effects of Akt activation in specific tissues. The lineage-tracing properties provide a useful tool to study the role of Akt signaling in regulation of differentiation programs during development and plasticity of mature tissues.

OBJECTIVE—β-Cells are particularly susceptible to fatty acid–induced apoptosis associated with decreased insulin receptor/phosphatidylinositol-3 kinase/Akt signaling and the activation of stress kinases. We examined the mechanism of fatty acid–induced apoptosis of mouse β-cells especially as related to the role played by endoplasmic reticulum (ER) stress–induced Foxo1 activation and whether decreasing Foxo1 activity could enhance cell survival.RESEARCH DESIGN AND METHODS—Mouse insulinoma (MIN6) cells were administered with fatty acids, and the role of Foxo1 in mediating effects on signaling pathways and apoptosis was examined by measuring Foxo1 activity and using dominant-negative Foxo1.RESULTS—Increasing fatty acid concentrations (100–400 μmol/l palmitate or oleate) led to early Jun NH2-terminal kinase (JNK) activation that preceded induction of ER stress markers and apoptosis. Foxo1 activity was increased with fatty acid administration and by pharmacological inducers of ER stress, and this increase was prevented by JNK inhibition. Fatty acids induced nuclear localization of Foxo1 at 4 h when Akt activity was increased, indicating that FoxO1 activation was not mediated by JNK inhibition of Akt. In contrast, fatty acid administration for 24 h was associated with decreased insulin signaling. A dominant-negative Foxo1 adenovirus (Adv-DNFoxo) conferred cells with protection from ER stress and fatty acid–mediated apoptosis. Microarray analysis revealed that fatty acid induction of gene expression was in most cases reversed by Adv-DNFoxo, including the proapoptotic transcription factor CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein).CONCLUSIONS—Early induction of JNK and Foxo1 activation plays an important role in fatty acid–induced apoptosis. Expressing a dominant-negative allele of Foxo1 reduces expression of apoptotic and ER stress markers and promotes β-cell survival from fatty acid and ER stress, identifying a potential therapeutic target for preserving β-cells in type 2 diabetes.

Glycogen synthase kinase-3 (GSK3) has been implicated in the pathophysiology of several prevalent diseases, including diabetes. However, despite recent progress in our understanding of the role of GSK3 in the regulation of glucose metabolism in peripheral tissues, the involvement of GSK3 in islet beta cell growth and function in vivo is unknown. We therefore sought to determine whether over-activation of GSK3beta would lead to alterations in islet beta cell mass and/or function.Transgenic mice overexpressing a constitutively active form of human GSK3beta (S9A) under the control of the rat insulin promoter (RIP-GSK3betaCA) were created. Studies using mouse insulinoma cells (MIN6) were conducted to investigate the regulation of GSK3beta activity and its impact on pancreas/duodenum homeobox protein-1 (PDX-1) levels.We demonstrated that phosphorylation of GSK3beta was decreased, indicating increased GSK3beta activity in two animal models of diabetes, Lepr(-/- ) mice and Ins2 (Akita/+) mice. In MIN6 cells, the activity of GSK3beta was regulated by glucose, in a fashion largely dependent on phosphatidylinositol 3-kinase. RIP-GSK3betaCA transgenic mice showed impaired glucose tolerance after 5 months of age. Histological studies revealed that transgenic mice had decreased beta cell mass and decreased beta cell proliferation, with a 50% decrease (p0.05) in the level of PDX-1.We showed direct evidence that GSK3beta activity is associated with beta cell failure in diabetic mouse models and that its overactivation resulted in decreased pancreatic beta cell proliferation and mass. GSK3 modulates PDX-1 stability in both cultured insulinoma cells and islets in vivo. These results may ultimately facilitate the development of potential therapeutic interventions targeting type 2 diabetes and/or islet transplantation.

The insulin receptor substrate-2/phosphoinositide 3-kinase (PI3K) pathway plays a critical role in the regulation of beta-cell mass and function, demonstrated both in vitro and in vivo. The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of beta cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of beta-cell mass and function by Akt.

SummaryNutrient levels dictate the activity of O-linked N-acetylglucosamine transferase (OGT) to regulate O-GlcNAcylation, a post-translational modification mechanism to “fine-tune” intracellular signaling and metabolic status. However, the requirement of O-GlcNAcylation for maintaining glucose homeostasis by regulating pancreatic β cell mass and function is unclear. Here, we reveal that mice lacking β cell OGT (βOGT-KO) develop diabetes and β cell failure. βOGT-KO mice demonstrated increased ER stress and distended ER architecture, and these changes ultimately caused the loss of β cell mass due to ER-stress-induced apoptosis and decreased proliferation. Akt1/2 signaling was also dampened in βOGT-KO islets. The mechanistic role of these processes was demonstrated by rescuing the phenotype of βOGT-KO mice with concomitant Chop gene deletion or genetic reconstitution of Akt2. These findings identify OGT as a regulator of β cell mass and function and provide a direct link between O-GlcNAcylation and β cell survival by regulation of ER stress responses and modulation of Akt1/2 signaling.

Activation of pancreatic β-cell proliferation has been proposed as an approach to replace reduced functional β-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic β-cell proliferation. We hypothesized that cyclin C overexpression would induce β-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional β-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [3H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected β-cell death by TUNEL, β-cell differentiation by RT-PCR, and β-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human β-cell proliferation. This augmented proliferation did not induce β-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing β-cell regeneration., This work was supported by grants from ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00671); Programa Ramón y Cajal (RYC-2011-08101) Ministerio de Economía y Competitividad, Spain; and Europe-FP7 Marie Curie grant (IRG-247835) to I. Cózar-Castellano, by Europe-FP7 Marie Curie grant (IRG-256369) to G. Perdomo, by National Institute of Diabetes and Digestive and Kidney Diseases Grants (RO1-DK-073716;, DK-084236) and Juvenile Diabetes Research Foundation (17-2013-416) to E. Bernal-Mizrachi, and by a Minority Postdoctoral Fellowship from the American Diabetes Association to M. Jiménez-Palomares.

Insulin is both a hormone regulating energy metabolism and a growth factor. We and others have shown that physiological doses of insulin initiate complex signals in primary human and mouse β-cells, but the functional significance of insulin's effects on this cell type remains unclear. In the present study, the role of insulin in β-cell apoptosis was examined. Exogenous insulin completely prevented apoptosis induced by serum withdrawal when given at picomolar or low nanomolar concentrations but not at higher concentrations, indicating that physiological concentrations of insulin are antiapoptotic and that insulin signaling is self-limiting in islets. Insulin treatment was associated with the nuclear localization of Pdx1 and the prosurvival effects of insulin were largely absent in islets 50% deficient in Pdx1, providing direct evidence that Pdx1 is a signaling target of insulin. Physiological levels of insulin did not increase Akt phosphorylation, and the protective effects of insulin were only partially altered in islets lacking 80% of normal Akt activity, suggesting the presence of additional insulin-regulated antiapoptotic pathways. Proteomic analysis of insulin-treated human islets revealed significant changes in multiple proteins. Bridge-1, a Pdx1-binding partner and regulator of β-cell survival, was increased significantly at low insulin doses. Together, these data suggest that insulin can act as a master regulator of islet survival by regulating Pdx1.

beta-Cell cycle progression and proliferation are critical to maintain beta-cell mass in adult mice. Of the cell cycle inhibitors, p27Kip1 is thought to be the primary modulator of the proliferative status in most cell types. p27 plays a role in beta-cell adaptation in genetic models of insulin resistance. To study the role of p27 in beta-cells during physiological conditions and at different stages of beta-cell differentiation, we studied mice deficient of or overexpressing p27. Experiments in p27-deficient mice showed improved glucose tolerance and hyperinsulinemia. These changes were associated with increased islet mass and proliferation. The experiments overexpressing p27 in beta-cells were performed using a doxycycline-inducible model. Interestingly, overexpression of p27 for 16 weeks in beta-cells from adult mice had no effect on glucose tolerance, beta-cell mass, or proliferation. In contrast, induction of p27 expression during beta-cell development or early neonatal period resulted in severe glucose intolerance and reduced beta-cell mass by decreased proliferation. These changes were reversible upon discontinuation of doxycycline. These experiments suggest that p27 is a critical molecule for beta-cell proliferation during beta-cell development and early postnatal life but not for maintenance of adult mass.

CD38 is a multifunctional enzyme capable of generating metabolites that release Ca 2+ from intracellular stores, including nicotinic acid adenine dinucleotide phosphate (NAADP). A number of studies have led to the controversial proposal that CD38 mediates an alternate pathway for glucose-stimulated insulin release and contributes to the pathogenesis of diabetes. It has recently been shown that NAADP mediates Ca 2+ mobilization by insulin in human pancreatic β-cells. In the present study, we report altered Ca 2+ homeostasis and reduced responsiveness to insulin, but not glucose, in Cd38 −/− β-cells. In keeping with the antiapoptotic role of insulin signaling, Cd38 −/− islets were significantly more susceptible to apoptosis compared with islets isolated from littermate controls. This finding correlated with disrupted islet architecture and reduced β-cell mass in Cd38 −/− mice, both in the context of a normal lab diet and a high-fat diet. Nevertheless, we did not find robust differences in glucose homeostasis in vivo or glucose signaling in vitro in Cd38 −/− mice on the C57BL/6 genetic background, in contrast to previous studies by others of Cd38 knockout mice on the ICR background. Thus, our results suggest that CD38 plays a role in novel antiapoptotic signaling pathways but does not directly control glucose signaling in pancreatic β-cells.

Proliferation is the major component for maintenance of beta-cell mass in adult animals. Activation of phosphoinositide 3-kinase/Akt-kinase pathway is a critical regulator of beta-cell mass. Pancreatic beta-cell overexpression of constitutively active Akt in mice (caAkt(Tg)) resulted in marked expansion of beta-cell mass by increase in beta-cell proliferation and size. The current studies provide new insights into the molecular mechanisms involved in beta-cell proliferation by Akt. Proliferation of beta-cells in caAkt(Tg) was associated with increased cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 (cdk4) activity. To determine the role of cdk4 in beta-cell proliferation induced by Akt, we generated caAkt(Tg) mice that were homozygous, heterozygous, or nullizygous for cdk4. The results of these studies showed that deletion of one cdk4 allele significantly reduced beta-cell expansion in caAkt(Tg) mice by decreased proliferation. CaAkt(Tg) mice deficient in cdk4 developed beta-cell failure and diabetes. These experiments suggest that Akt induces beta-cell proliferation in a cdk4-dependent manner by regulation of cyclin D1, cyclin D2, and p21 levels. These data also indicate that alteration in levels of these cell cycle components could affect the maintenance of beta-cell mass in basal states and the adaptation of beta-cells to pathological states resulting in diabetes.

Fractalkine is a chemokine, which has been shown to play important roles in metabolic disease in both animal models and humans. Fractalkine is a key player in the accumulation of atherosclerotic plaques, and fractalkine receptor (CX3CR1) mutations have been implicated in obesity. Serum fractalkine levels have been found to be elevated in type 2 diabetic patients, but the role of fractalkine signaling on the pancreatic β cell was unclear. Recently published findings in April 2013 issue of the journal Cell by Lee and Olefsky et al. have implicated fractalkine in β-cell insulin secretion. They demonstrate that Cx3cr1 knockout mice have impaired glucose tolerance resulting from decreased insulin secretion. In addition, fractalkine administration improved glucose tolerance and induced insulin secretion. This modulation of insulin secretion was proposed to result from an increase in intracellular calcium and potentiation of insulin secretion, which occurs in a Gαi and MEK-dependent manner. They also found that Cx3cr1 knockout animals had transcriptional repression of genes important for β-cell function, specifically NeuroD, via induction of ICER-1. One important issue that remains unresolved is how CX3CR1 signaling regulates the potentiation of calcium influx and the distal events in insulin exocytosis. Finally, testing the effects of fractalkine treatment on proliferation and survival in vivo during regenerative conditions would be critical to determine the potential use of this chemokine in diabetes. While these exciting results open the possibility for new therapeutics, there are some concerns about a potential risk for exacerbation of atherosclerosis.

AIMS/HYPOTHESIS: Wolfram syndrome is an autosomal recessive disorder characterised by childhood diabetes mellitus, optic atrophy and severe neurodegeneration, resulting in premature death. The aim of this study was to investigate the mechanisms responsible for the phenotype of carbohydrate intolerance and loss of pancreatic beta cells in this disorder. MATERIALS AND METHODS: To study the role of the Wolfram gene (Wfs1) in beta cells, we developed a mouse model with conditional deletion of Wfs1 in beta cells by crossing floxed Wfs1 exon 8 animals with mice expressing Cre recombinase under the control of a rat insulin promoter (RIP2-Cre). Complementary experiments using RNA interference of Wfs1 expression were performed in mouse insulinoma (MIN6) cell lines (WfsKD). RESULTS: Male knockout mice (betaWfs(-/-)) began developing variable and progressive glucose intolerance and concomitant insulin deficiency, compared with littermate controls, by 12 weeks of age. Analysis of islets from betaWfs(-/-) mice revealed a reduction in beta cell mass, enhanced apoptosis, elevation of a marker of endoplasmic reticulum stress (immunoglobulin heavy chain-binding protein [BiP]), and dilated endoplasmic reticulum with decreased secretory granules by electron microscopy. WfsKD cell lines had significantly increased apoptosis and elevated expression of the genes encoding BiP and C/EBP-homologous protein (CHOP), two markers of endoplasmic reticulum stress. CONCLUSIONS/INTERPRETATION: These results indicate that (1) lack of expression of Wfs1 in beta cells was sufficient to result in the diabetes mellitus phenotype; (2) beta cell death occurred by an accelerated process of apoptosis; and (3) lack of Wfs1 was associated with dilated endoplasmic reticulum and increased markers of endoplasmic reticulum stress, which appears to be a significant contributor to the reduction in beta cell survival.

Summary Sir2 NAD-dependent deacetylases connect transcription, metabolism, and aging. Increasing the dosage or activity of Sir2 extends life span in yeast, worms, and flies and promotes fat mobilization and glucose production in mammalian cells. Here we show that increased dosage of Sirt1, the mammalian Sir2 ortholog, in pancreatic β cells improves glucose tolerance and enhances insulin secretion in response to glucose in be ta cell-specific S ir t 1- o verexpressing (BESTO) transgenic mice. This phenotype is maintained as BESTO mice age. Pancreatic perfusion experiments further demonstrate that Sirt1 enhances insulin secretion in response to glucose and KCl. Microarray analyses of β cell lines reveal that Sirt1 regulates genes involved in insulin secretion, including uncoupling protein 2 ( Ucp2 ). Isolated BESTO islets also have reduced Ucp2, increased ATP production, and enhanced insulin secretion during glucose and KCl stimulation. These findings establish the importance of Sirt1 in β cell function in vivo and suggest therapeutic interventions for type 2 diabetes.

An imbalance between the rate of protein synthesis and folding capacity of the endoplasmic reticulum (ER) results in stress that has been increasingly implicated in pancreatic islet beta-cell apoptosis and diabetes. Because insulin/IGF/Akt signaling has been implicated in beta-cell survival, we sought to determine whether this pathway is involved in ER stress-induced apoptosis. Mouse insulinoma cells treated with pharmacological agents commonly used to induce ER stress exhibited apoptosis within 48 h. ER stress-induced apoptosis was inhibited by cotreatment of the cells with IGF-1. Stable cell lines were created by small-interfering RNA (siRNA) with graded reduction of insulin receptor expression, and these cells had enhanced susceptibility to ER stress-induced apoptosis and reduced levels of phospho-glycogen synthase kinase 3beta (GSK3beta). In control cells, ER stress-induced apoptosis was associated with a reduction in phospho-Akt and phospho-GSK3beta. To further assess the role of GSK3beta in ER stress-induced apoptosis, stable cell lines were created by siRNA with up to 80% reduction in GSK3beta expression. These cells were found to resist ER stress-induced apoptosis. These results illustrate that ER stress-induced apoptosis is mediated at least in part by signaling through the phosphatidylinositol 3-kinase/Akt/GSK3beta pathway and that GSK3beta represents a novel target for agents to promote beta-cell survival.

The role of insulin signaling in pancreatic beta cells has become increasingly apparent. Stably transformed insulinoma cell lines (MIN6) were created with small interfering RNA resulting in the reduction of insulin receptor (IR) expression up to 80% (insulin receptor knockdown, IRKDDelta80). Functionally perturbed IR signaling was confirmed with the absence of insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation. Additionally, Akt phosphorylation was reduced and responded poorly to glucose stimulation. Gene expression profiling revealed that reduced IR expression was associated with alterations in expression of >1,500 genes with diverse functions. IRKD cells exhibited low rate of proliferation due to delay in transition from G0/G1 to S phase, whereas susceptibility to apoptosis did not differ from that of control cells. Insulin content was reduced in proportion to the reduction of IR. IRKD cells maintained glucose responsiveness as measured by NADPH generation, whereas Ca2+ responses and insulin secretion were enhanced. IRKDDelta80 and control cells were treated with glucose (25 mm) or insulin (100 nm) for 45 min, and gene expression profiles were assessed. Transcriptional activation of several hundred early response genes common to both glucose and insulin stimulation was observed in control cells. In IRKDDelta80 cells, insulin failed to activate any genes as anticipated. Importantly, glucose stimulation of gene expression in IRKDDelta80 cells showed that most genes previously activated by glucose were no longer activated, suggesting a major autocrine/paracrine effect of insulin on glucose-regulated gene expression. On the other hand, there were a number of glucose-regulated genes in the IRKDDelta80 cells that were not previously observed in control cells, suggesting a feedback regulation of insulin signaling on glucose-regulated gene expression. These results demonstrate important roles of the insulin receptor in islet beta cell gene expression and function and may serve to elucidate molecular defects in animal models with diminished beta cell insulin signaling.

The purpose of the current study was to determine whether nuclear factor-kappaB (NF-kappaB) activation is a component of the depolarization/Ca(2+)-dependent signaling in beta-cells. MIN6 cells were transfected with a plasmid containing five tandem repeats of NF-kappaB binding sites linked to a luciferase reporter. The results of these experiments showed that KCl induced depolarization-activated NF-kappaB-dependent transcription (3.8-fold at 45 mmol/l, P < 0.01) in a concentration-dependent manner. Tumor necrosis factor-alpha (TNF-alpha), a known inducer of NF-kappaB signaling, activated this construct by 3.4-fold (P < 0.01). The response of NF-kappaB to depolarization was inhibited by the Ca(2+)-channel blocker verapamil and by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (70 and 62%, respectively). TNF-alpha, glucose, and KCl treatment resulted in inhibitory kappaBalpha degradation by Western blot analysis. TNF-alpha treatment and depolarization activation of NF-kappaB differed significantly in that TNF-alpha activation was not blocked by PD98059. Transfection with PKA, MEK, and MEK kinase induced NF-kappaB-dependent transcription by 20-, 90-, and 300-fold, respectively, suggesting that these pathways contribute to the activation in the depolarization response. These findings demonstrate that depolarization/Ca(2+) influx, as well as TNF-alpha treatment, can activate NF-kappaB-dependent transcription in pancreatic beta-cells, but by different signaling pathways. The current studies show that Ca(2+) signals in pancreatic beta-cells can activate transcription factors involved in the regulation of cell cycle and apoptosis. These findings now add NF-kappaB to the list of depolarization-induced transcription factors in pancreatic beta-cells.