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The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

AbstractThe "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is a major goal for regenerative therapies. Endogenous regeneration of β-cells via β-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous β-cells have been elusive. The regenerative capacity of β-cells declines rapidly with age, due to accumulation of p16(INK4a), resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-β (TGF-β) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication. Importantly, inhibition of TGF-β signaling can result in repression of the Ink4a/Arf locus, resulting in increased β-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-β pathway promote β-cell replication in human islets transplanted into NOD-scid IL-2Rg(null) mice. These data reveal a novel role for TGF-β signaling in the regulation of the Ink4a/Arf locus and highlight the potential of using small molecule inhibitors of TGF-β signaling to promote human β-cell replication.

Aims/hypothesisT cell protein tyrosine phosphatase (TCPTP, encoded by PTPN2) regulates cytokine-induced pancreatic beta cell apoptosis and may contribute to the pathogenesis of type 1 diabetes. However, the role of TCPTP in pancreatic endocrine function and insulin secretion remains largely unknown.MethodsTo investigate the endocrine role of pancreatic TCPTP we generated mice with pancreas Ptpn2/TCPTP deletion (panc-TCPTP KO).ResultsWhen fed regular chow, panc-TCPTP KO and control mice exhibited comparable glucose tolerance. However, when challenged with prolonged high fat feeding panc-TCPTP KO mice exhibited impaired glucose tolerance and attenuated glucose-stimulated insulin secretion (GSIS). The defect in GSIS was recapitulated in primary islets ex vivo and after TCPTP pharmacological inhibition or lentiviral-mediated TCPTP knockdown in the glucose-responsive MIN6 beta cells, consistent with this being cell autonomous. Reconstitution of TCPTP in knockdown cells reversed the defect in GSIS demonstrating that the defect was a direct consequence of TCPTP deficiency. The reduced insulin secretion in TCPTP knockdown MIN6 beta cells was associated with decreased insulin content and glucose sensing. Furthermore, TCPTP deficiency led to enhanced tyrosyl phosphorylation of signal transducer and activator of transcription 1 and 3 (STAT 1/3), and substrate trapping studies in MIN6 beta cells identified STAT 1/3 as TCPTP substrates. STAT3 pharmacological inhibition and small interfering RNA-mediated STAT3 knockdown in TCPTP deficient cells restored GSIS to control levels, indicating that the effects of TCPTP deficiency were mediated, at least in part, through enhanced STAT3 phosphorylation and signalling.Conclusions/interpretationThese studies identify a novel role for TCPTP in insulin secretion and uncover STAT3 as a physiologically relevant target for TCPTP in the endocrine pancreas.

ObjectiveA major determinant of the progression from insulin resistance to the development of overt type 2 diabetes is a failure to mount an appropriate compensatory beta-cell hyperplastic response to maintain normoglycemia. We undertook the present study to directly explore the significance of the cell cycle protein cyclin D2 in the expansion of beta-cell mass in two different models of insulin resistance.Research design and methodsWe created compound knockouts by crossing mice deficient in cyclin D2 (D2KO) with either the insulin receptor substrate 1 knockout (IRS1KO) mice or the insulin receptor liver-specific knockout mice (LIRKO), neither of which develops overt diabetes on its own because of robust compensatory beta-cell hyperplasia. We phenotyped the double knockouts and used RT-qPCR and immunohistochemistry to examine beta-cell mass.ResultsBoth compound knockouts, D2KO/LIRKO and D2KO/IRS1KO, exhibited insulin resistance and hyperinsulinemia and an absence of compensatory beta-cell hyperplasia. However, the diabetic D2KO/LIRKO group rapidly succumbed early compared with a relatively normal lifespan in the glucose-intolerant D2KO/IRS1KO mice.ConclusionsThis study provides direct genetic evidence that cyclin D2 is essential for the expansion of beta-cell mass in response to a spectrum of insulin resistance and points to the cell-cycle protein as a potential therapeutic target that can be harnessed for preventing and curing type 2 diabetes.

Solute Carrier Family 19 Member 2 (SLC19A2) encodes thiamine transporter 1 (THTR1), which facilitates thiamine transport across the cell membrane. SLC19A2 homozygous mutations have been described as a cause of thiamine-responsive megaloblastic anemia (TRMA), an autosomal recessive syndrome characterized by megaloblastic anemia, diabetes, and sensorineural deafness. Here we describe a loss-of-function SLC19A2 mutation (c.A1063C: p.Lys355Gln) in a family with early-onset diabetes and mild TRMA traits transmitted in an autosomal dominant fashion. We show that SLC19A2-deficient β-cells are characterized by impaired thiamine uptake, which is not rescued by overexpression of the p.Lys355Gln mutant protein. We further demonstrate that SLC19A2 deficit causes impaired insulin secretion in conjunction with mitochondrial dysfunction, loss of protection against oxidative stress, and cell cycle arrest. These findings link SLC19A2 mutations to autosomal dominant diabetes and suggest a role of SLC19A2 in β-cell function and survival. [ABSTRACT FROM AUTHOR]

A correcting an error in the affiliations of authors Stephen J. Pandol and Maike Sander in the previously published article titled "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases: Workshop Proceedings."

Type 1 (T1D) or type 2 diabetes (T2D) are caused by a deficit of functional insulin-producing β cells. Thus, the identification of β cell trophic agents could allow the development of therapeutic strategies to counteract diabetes. The discovery of SerpinB1, an elastase inhibitor that promotes human β cell growth, prompted us to hypothesize that pancreatic elastase (PE) regulates β cell viability. Here, we report that PE is up-regulated in acinar cells and in islets from T2D patients, and negatively impacts β cell viability. Using high-throughput screening assays, we identified telaprevir as a potent PE inhibitor that can increase human and rodent β cell viability in vitro and in vivo and improve glucose tolerance in insulin-resistant mice. Phospho-antibody microarrays and single-cell RNA sequencing analysis identified PAR2 and mechano-signaling pathways as potential mediators of PE. Taken together, our work highlights PE as a potential regulator of acinar-β cell crosstalk that acts to limit β cell viability, leading to T2D. [Display omitted] • CELA3B, an exocrine gene, is upregulated in acinar cells of patients with T2D • CELA3B protein deposits in islet milieu and compromises β cell homeostasis in T2D • Telaprevir, a PE inhibitor, improves viability and function of human β cells • PE affects β cell homeostasis by impairing mechano-signaling and PAR2 pathways Basile et al. observed CELA3B genes upregulated in acinar cells linked to augmented CELA3B protein within islet milieu of T2D patients, resulting in reduced β cell homeostasis secondary to impairment of two signaling hubs: mechano-signaling and PAR2 pathways. PE inhibitors rescue β cell homeostasis and point to anti-PE therapies in T2D. [ABSTRACT FROM AUTHOR]

Background The sex of an individual affects glucose homeostasis and the pathophysiology, incidence, and prevalence of diabetes as well as the response to therapy. Scope of the review This review focuses on clinical and experimental sex differences in islet cell biology and dysfunction during development and in adulthood in human and animal models. We discuss sex differences in β-cell and α-cell function, heterogeneity, and dysfunction. We cover sex differences in communication between gonads and islets and islet-cell immune interactions. Finally, we discuss sex differences in β-cell programming by nutrition and other environmental factors during pregnancy. Major conclusions Important sex differences exist in islet cell function and susceptibility to failure. These differences represent sex-related biological factors that can be harnessed for gender-based prevention of and therapy for diabetes. [ABSTRACT FROM AUTHOR]

The endocrine pancreatic β-cells play a pivotal role in maintaining whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate β-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homeostasis. Mice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1loxP/loxP, β-ROCK1−/−) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. An insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1−/− mice or β-cell lines with knockdown of ROCK1 was also evaluated. A proximity ligation assay was performed to determine the physical interactions between PK and ROCK1. Mice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1−/− mice displayed a progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium and ATP levels and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that glucose stimulation in β-cells greatly enhanced ROCK1 binding to PK. Our findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and for glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling. • ROCK1 deletion in β-cells causes hyperglycemia without changes in body weight. • Mice lacking ROCK1 in β-cells are glucose intolerant but insulin tolerant. • Glucose-stimulated insulin secretion is impaired by β-cell ROCK1 deficiency. • Treatment of ROCK1-deficient islets with pyruvate rescues impaired insulin release. • Glucose induces ROCK1 binding to PK in β-cells. [ABSTRACT FROM AUTHOR]

Background Diabetes and metabolic syndromes are chronic, devastating diseases with increasing prevalence. Human pluripotent stem cells are gaining popularity in their usage for human in vitro disease modeling. With recent rapid advances in genome editing tools, these cells can now be genetically manipulated with relative ease to study how genes and gene variants contribute to diabetes and metabolic syndromes. Scope of review We highlight the diabetes and metabolic genes and gene variants, which could potentially be studied, using two powerful technologies – human pluripotent stem cells (hPSCs) and genome editing tools – to aid the elucidation of yet elusive mechanisms underlying these complex diseases. Major conclusions hPSCs and the advancing genome editing tools appear to be a timely and potent combination for probing molecular mechanism(s) underlying diseases such as diabetes and metabolic syndromes. The knowledge gained from these hiPSC-based disease modeling studies can potentially be translated into the clinics by guiding clinicians on the appropriate type of medication to use for each condition based on the mechanism of action of the disease. [ABSTRACT FROM AUTHOR]

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 b 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. [ABSTRACT FROM AUTHOR]

Saturated free fatty acids (FFAs) have complex effects on the islet β-cell, acutely promoting adaptive hyperplasia but chronically impairing insulin release. The acute effects of FFAs remain incompletely defined. To elucidate these early molecular events, we incubated mouse β-cells and islets with palmitate and then studied mRNA translation by polyribosomal profiling and analyzed signaling pathways by immunoblot analysis. We found that palmitate acutely increases polyribosome occupancy of total RNA, consistent with an increase in mRNA translation. This effect on translation was attributable to activation of mammalian target of rapamycin (mTOR) pathways via L-type Ca2+ channels but was independent of insulin signaling. Longer incubations led to depletion of polyribosome-associated RNA, consistent with activation of the unfolded protein response (UPR). Pharmacologic inhibition of mTOR suppressed both the acute effects of palmitate on mRNA translation and the chronic effects on the UPR. Islets from mice fed a high-fat diet for 7 days showed increases in polyribosome-associated RNA and phosphorylation of S6K, both consistent with activation of mTOR. Our results suggest that palmitate acutely activates mRNA translation and that this increase in protein load contributes to the later UPR. [ABSTRACT FROM AUTHOR]

Enhancing β-cell proliferation is a major goal for type 1 and type 2 diabetes research. Unraveling the network of β-cell intracellular signaling pathways that promote β-cell replication can provide the tools to address this important task. In a previous Perspectives in Diabetes article, we discussed what was known regarding several important intracellular signaling pathways in rodent β-cells, including the insulin receptor substrate/phosphatidylinositol-3 kinase/Akt (IRS-PI3K-Akt) pathways, glycogen synthase kinase-3 (GSK3) and mammalian target of rapamycin (mTOR) S6 kinase pathways, protein kinase Cζ (PKCζ) pathways, and their downstream cell-cycle molecular targets, and contrasted that ample knowledge to the small amount of complementary data on human b-cell intracellular signaling pathways. In this Perspectives, we summarize additional important information on signaling pathways activated by nutrients, such as glucose; growth factors, such as epidermal growth factor, platelet-derived growth factor, and Wnt; and hormones, such as leptin, estrogen, and progesterone, that are linked to rodent and human β-cell proliferation. With these two Perspectives, we attempt to construct a brief summary of knowledge for β-cell researchers on mitogenic signaling pathways and to emphasize how little is known regarding intracellular events linked to human β-cell replication. This is a critical aspect in the long-term goal of expanding human β-cells for the prevention and/or cure of type 1 and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is characterized by poor glucose uptake in metabolic tissues and manifests when insulin secretion fails to cope with worsening insulin resistance. In addition to its effects on skeletal muscle, liver, and adipose tissue metabolism, it is evident that insulin resistance also affects pancreatic β-cells. To directly examine the alterations that occur in islet morphology as part of an adaptive mechanism to insulin resistance, we evaluated pancreas samples obtained during pancreatoduodenectomy from nondiabetic subjects who were insulin-resistant or insulin-sensitive. We also compared insulin sensitivity, insulin secretion, and incretin levels between the two groups. We report an increased islet size and an elevated number of band α-cells that resulted in an altered β-cell--to--α-cell area in the insulin- resistant group. Our data in this series of studies suggest that neogenesis from duct cells and transdifferentiation of α-cells are potential contributors to the β-cell compensatory response to insulin resistance in the absence of overt diabetes. [ABSTRACT FROM AUTHOR]

The female steroid, 17β-estradiol (E2), is important for pancreatic β-cell function and acts via at least three estrogen receptors (ER), ERα, ERβ, and the G-protein coupled ER (GPER). Using a pancreas-specific ERα knockout mouse generated using the Cre-lox-P system and a Pdx1-Cre transgenic line (PERαKO−/−), we previously reported that islet ERα suppresses islet glucolipotoxicity and prevents β-cell dysfunction induced by high fat feeding. We also showed that E2 acts via ERα to prevent β-cell apoptosis in vivo. However, the contribution of the islet ERα to β-cell survival in vivo, without the contribution of ERα in other tissues is still unclear. Using the PERαKO−/− mouse, we show that ERα mRNA expression is only decreased by 20% in the arcuate nucleus of the hypothalamus, without a parallel decrease in the VMH, making it a reliable model of pancreas-specific ERα elimination. Following exposure to alloxan-induced oxidative stress in vivo, female and male PERαKO−/− mice exhibited a predisposition to β-cell destruction and insulin deficient diabetes. In male PERαKO−/− mice, exposure to E2 partially prevented alloxan-induced β-cell destruction and diabetes. ERα mRNA expression was induced by hyperglycemia in vivo in islets from young mice as well as in cultured rat islets. The induction of ERα mRNA by hyperglycemia was retained in insulin receptor-deficient β-cells, demonstrating independence from direct insulin regulation. These findings suggest that induction of ERα expression acts to naturally protect β-cells against oxidative injury. [ABSTRACT FROM AUTHOR]

Background: CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas. Methods: We established a monotransgenic floxed (flanking LOX sequences) mouse line carrying the human CEL mutation c.1686delT and crossed it with an elastase-Cre mouse to derive a bitransgenic mouse line with pancreas-specific over-expression of CEL carrying this disease-associated mutation (TgCEL). Following confirmation of murine pancreatic expression of the human transgene by real-time quantitative PCR, we phenotyped the mouse model fed a normal chow and compared it with mice fed a 60% high fat diet (HFD) as well as the effects of short-term and long-term cerulein exposure. Results: Pancreatic exocrine function was normal in TgCEL mice on normal chow as assessed by serum lipid and lipid-soluble vitamin levels, fecal elastase and fecal fat absorption, and the normoglycemic mice exhibited normal pancreatic morphology. On 60% HFD, the mice gained weight to the same extent as controls, had normal pancreatic exocrine function and comparable glucose tolerance even after resuming normal diet and follow up up to 22 months of age. The cerulein-exposed TgCEL mice gained weight and remained glucose tolerant, and there were no detectable mutation-specific differences in serum amylase, islet hormones or the extent of pancreatic tissue inflammation. Conclusions: In this murine model of human CEL-MODY diabetes, we did not detect mutation-specific endocrine or exocrine pancreatic phenotypes, in response to altered diets or exposure to cerulein. [ABSTRACT FROM AUTHOR]

A major goal in diabetes research is to find ways to enhance the mass and function of insulin secreting β-cells in the endocrine pancreas to prevent and/or delay the onset or even reverse overt diabetes. In this Perspectives in Diabetes article, we highlight the contrast between the relatively large body of information that is available in regard to signaling pathways, proteins, and mechanisms that together provide a road map for efforts to regenerate β-cells in rodents versus the scant information in human β-cells. To reverse the state of ignorance regarding human β-cell signaling, we suggest a series of questions for consideration by the scientific community to construct a human β-cell proliferation road map. The hope is that the knowledge from the new studies will allow the community to move faster towards developing therapeutic approaches to enhance human β-cell mass in the long-term goal of preventing and/or curing type 1 and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) is characterized by insulin resistance and pancreatic β-cell dysfunction, the latter possibly caused by a defect in insulin signaling in b-cells. We hypothesized that insulin's effect to potentiate glucose-stimulated insulin secretion (GSIS) would be diminished in insulin-resistant persons. To evaluate the effect of insulin to modulate GSIS in insulin-resistant compared with insulin-sensitive subjects, 10 participants with impaired glucose tolerance (IGT), 11 with T2D, and 8 healthy control subjects were studied on two occasions. The insulin secretory response was assessed by the administration of dextrose for 80 min following a 4-h clamp with either saline infusion (sham) or an isoglycemic-hyperinsulinemic clamp using B28- Asp-insulin (which can be distinguished immunologically from endogenous insulin) that raised insulin concentrations to high physiologic concentrations. Pre-exposure to insulin augmented GSIS in healthy persons. This effect was attenuated in insulin-resistant cohorts, both those with IGT and those with T2D. Insulin potentiates glucose-stimulated insulin secretion in insulin-resistant subjects to a lesser degree than in normal subjects. This is consistent with an effect of insulin to regulate β-cell function in humans in vivo with therapeutic implications. [ABSTRACT FROM AUTHOR]

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K12 position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4×12-VT750) had a high binding affinity (∼3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4×12-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts. [ABSTRACT FROM AUTHOR]

Summary: Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca2+ influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes. [ABSTRACT FROM AUTHOR]

Patients with type 2 diabetes lose β cells, but the underlying mechanisms are incompletely understood. Glucose-6-phosphate dehydrogenase (G6PD) is the principal source of the major intracellular reductant, NADPH, which is required by many enzymes, including enzymes of the antioxidant pathway. Previous work from our laboratory has shown that high glucose impairs G6PD activity in endothelial and kidney cells, which leads to decreased cell survival. Pancreatic β cells are highly sensitive to increased ROS. This study aimed to determine whether G6PD and NADPH play central roles in β-cell survival. Human and mouse islets, MIN6 cell line, and G6PD deficient mice were studied. High glucose inhibited G6PD expression and activity. Inhibition of G6PD with siRNA led to increased ROS and apoptosis, decreased proliferation, and impaired insulin secretion. High glucose decreased insulin secretion, which was improved by overexpressing G6PD. G6PD-deficient mice had smaller islets and impaired glucose tolerance compared with control mice, which suggests that G6PD deficiency per se leads to β-cell dysfunction and death. G6PD plays an important role in β-cell function and survival. High-glucose-mediated decrease in G6PD activity may provide a mechanistic explanation for the gradual loss of β cells in patients with diabetes. [ABSTRACT FROM AUTHOR]

The article focuses on the growth factor control of pancreatic islet and β cell regeneration and regulation in diabetes mellitus. It delineates the growth dynamics, stimuli, and replication of β cell. It highlights the discussions on growth factor signaling and β cell regeneration. Discussions on the synthesis and secretion of insulin and glucagon, as well as the association of β cell to aging are also presented

Interest in the importance of Wnt signaling in diabetes has risen after identification of the transcription factor TCF7L2, a component of this pathway, as a strong risk factor for type 2 diabetes. Here, we review emerging new evidence that Wnt signaling influences endocrine pancreas development and modulates mature β-cell functions including insulin secretion, survival and proliferation. Alterations in Wnt signaling might also impact other metabolic tissues involved in the pathogenesis of diabetes, with TCF7L2 proposed to modulate adipogenesis and regulate GLP-1 production. Together, these studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, highlighting the importance of further investigation of this pathway to develop new therapies for this disease. [Copyright &y& Elsevier]

An appropriate β cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of β cell growth and function (reviewed in ref. 2). To define the roles of these hormones directly, we created a mouse model lacking functional receptors for both insulin and IGF-1 only in β cells (βDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, βDKO mice were born with a normal complement of islet cells, but 3 weeks after birth, they developed diabetes, in contrast to mild phenotypes observed in single mutants. Normoglycemic 2-week-old βDKO mice manifest reduced β cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised β cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating β cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I–dependent pathways are not critical for development of β cells but that a loss of action of these hormones in β cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in β cells might protect against type 2 diabetes. [ABSTRACT FROM AUTHOR]

Insulin and IGF-I activate antiapoptotic pathways via insulin receptor substrate (IRS) proteins in most mammalian cells, including β-cells. IRS-1 knockout (IRS-1KO) mice show growth retardation, hyperinsulinemia, and hyperplastic but dysfunctional islets without developing overt diabetes, whereas IRS-2KOs develop insulin resistance and islet hypoplasia leading to diabetes. Because both models display insulin resistance, it is difficult to differentiate islet response to insulin resistance from islet defects due to loss of proteins in the islets themselves. We used a transplantation approach, as a means of separating host insulin resistance from islet function, to examine alterations in proteins in insulin/IGF-I signaling pathways that may contribute to β-cell proliferation and/or apoptosis in IRS-IKO islets. Islets isolated from wild-type (WT) or IRS-1KO mice were transplanted into WT or insulin-resistant IRS-1KO males under the kidney capsule. The β-cell mitotic rate in transplanted islets in IRS-1KO recipients was increased 1.5-fold compared with WT recipients and was similar to that in endogenous pancreases of IRS-1KOs, whereas β-cell apoptosis was reduced by ∼80% in IRS-1KO grafts in IRS-1KO recipients compared with WT recipients. Immunohistochemistry showed a substantial increase in IRS-2 expression in IRS-1KO islets transplanted into IRS-1KO mice as well as in endogenous islets from IRS-1KOs. Furthermore, enhanced cytosolic forkhead transcription factor (FoxO1) staining in IRS-1KO grafts suggests intact Akt/PKB activity. Together, these data indicate that, even in the absence of insulin resistance, IS-cells deficient in IRS-1 exhibit a compensatory increase in IRS-2, which is associated with islet growth and is characterized by both proliferative and antiapoptotic effects that likely occur via an insulin/IGF-I/IRS-2 pathway. [ABSTRACT FROM AUTHOR]

Inadequate compensatory β cell hyperplasia in insulin-resistant states triggers the development of overt diabetes. The mechanisms that underlie this crucial adaptive response are not fully defined. Here we show that the compensatory islet-growth response to insulin resistance in 2 models - insulin receptor (IR)/IR substrate-1 (IRS-1) double heterozygous mice and liver-specific IR KO (LIRKO) mice - is severely restricted by PDX-i heterozygosity. Six-month-old IR/IRS-1 and LIRKO mice both showed up to a 10-fold increase in β cell mass, which involved epithelial-to-mesenchymal transition. In both models, superimposition of PDX- 1 haploinsufficiency upon the background of insulin resistance completely abrogated the adaptive islet hyperplastic response, and instead the β cells showed apoptosis resulting in premature death of the mice. This study shows that, in postdevelopmental states of β cell growth, PDX-1 is a critical regulator of β cell replication and is required for the compensatory response to insulin resistance. [ABSTRACT FROM AUTHOR]

Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary beta-cells. IRS-1 KO beta-cells exhibited a significantly shorter increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) than controls when briefly stimulated with glucose or glyceraldehyde and when l-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO beta-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca(2+) and O(2) consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca(2+) signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion. [ABSTRACT FROM AUTHOR]

Studies animals in which the insulin receptor gene had been specifically inactivated in β-cells by means of the Cre-loxP system. Evaluation of the adequacy of the intracellular stores of Ca2+; Summary of changes in islet [Ca2+]i.

Determines whether insulin signaling has a functional role in the pancreatic beta cell. Dysfunction of the pancreatic beta cell as an important defect in the pathogenesis of type 2 diabetes; Progressive impairment of glucose tolerance; Creation of the IRIox and the beta cell-specific insulin receptor knockout mice.

The article presents a commentary on a study by Renner and colleagues which sought to identify biomarkers of subclinical diabetes. For this study, the researchers created transgenic pigs by expressing a dominant negative glucose-dependent insulinotropic polypeptide (GIP) receptor restricted to the pancreatic β-cells. It was observed that the model was suitable for identifying potential biomarkers due to the progressive deterioration of glucose homeostasis.

Insulin resistance derives from the pancreas' inability to produce enough insulin to compensate for increased glucose load coupled with decreased peripheral and/or hepatic insulin sensitivity. Mice that lack insulin receptor substrate 1 (irs1) suffer from insulin resistance, but do not develop overt diabetes because of compensatory increase in beta cell mass that leads to hyperinsulinema. To address the molecular basis of the compensatory mechanism that leads to beta cell mass expansion in the face of increased physiologic demand, we deleted both alleles of cyclin D2 in the irs1-/- background. At 10 weeks of age, cyeD2 -/-irs1-/- are smaller than their single knockout or double heterozygous counterparts and show insulin resistance that is accompanied by glucose intolerance. By 25 weeks, however, cycD2-/-irs1-/-, exhibit severe glucose intolerance, hypoinsulinemia, and have with fasting glucose levels over 350mg/dL, indicating severe diabetes. Histological studies confirm that hypoinsulemia in cycD2-/-irs1-/- animals is due to the inability of beta cells to expand. This study shows that cyclin D2 plays an essential role in the expansion of beta cells in response to increasing metabolic demand This work reinforces the idea that cyclin D2 is a specific target for expansion of beta cell mass, and shows potential as a therapeutic for patients suffering from insulin dependent diabetes. [ABSTRACT FROM AUTHOR]

We have recently identified a new form of MODY diabetes (MODY8) associated with exocrine pancreatic insufficiency and due to mutations in the C-terminal region of the carboxyl ester lipase (CEL) gene leading to a reduction in functional enzyme. To investigate the endocrine and exocrine pancreatic phenotype of reduced CEL we have characterized a CEL knock-out mouse model (CELKO). q-RT-PCR of pancreas and isolated islets confirmed the absence of CEL expression in the CELKO mice. In female CELKO mice, the area under the curve (AUC) of an intra-peritoneal glucose tolerance test (IPGTT) in CELKO females was significantly greater than in controls (P=0.02) by age 10 months, and there was a 40% decrease in glucose stimulated insulin release (GSIS), although this did not quite reach statistical significance. L-Arginine potentiation of the GSIS did not differ significantly between groups. CELKO mice also had higher total serum cholesterol levels (146 ±3.3 mg/dL) vs. 74 ±2.2 (P=0.003) and a trend towards higher glucagon levels (65.7 ±13.9 vs. 36.9 ±11.2; P=0.16). By contrast, there were no differences between CELKO and control males in body weight, fasting or random blood glucose, serum insulin or C-peptide, plasma glucagon, GTT, insulin sensitivity, GSIS or Arg-GSIS, total cholesterol, triglycerides, free fatty acids or fecal fat absorption. In contrast to humans, CELKO serum of both sexes also showed no autoimmune reaction when assayed immunohistochemically on normal pancreas sections. Morphological analyses of pancreas sections revealed a relatively normal distribution of insulin-secreting beta-cells and glucagon-secreting alpha-cells, however, the CELKO mice appeared to have an increase in somatostatin cells compared to controls. Thus, whole-body knockout of CEL in mice seems to promote a mild diabetic phenotype in female mice on a chow diet but does not reproduce the phenotype of human MODY8 in males. These data suggest that in humans the full CELKO phenotype may require an interaction between this pathway and dietary or other environmental factors for expression of the disease. [ABSTRACT FROM AUTHOR]

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Although gene-environment interactions have been implicated in the etiology of several disorders, the impact of paternal and/or maternal metabolic syndrome on the clinical phenotypes of offspring and the underlying genetic and epigenetic contributors of NAFLD have not been fully explored. To this end, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique nondietary model manifesting 3 hallmarks that confer high risk for the development of NAFLD: hyperglycemia, insulin resistance, and dyslipidemia. We report that parental metabolic syndrome epigenetically reprograms members of the TGF-β family, including neuronal regeneration-related protein (NREP) and growth differentiation factor 15 (GDF15). NREP and GDF15 modulate the expression of several genes involved in the regulation of hepatic lipid metabolism. In particular, NREP downregulation increases the protein abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and ATP-citrate lyase (ACLY) in a TGF-β receptor/PI3K/protein kinase B-dependent manner, to regulate hepatic acetyl-CoA and cholesterol synthesis. Reduced hepatic expression of NREP in patients with NAFLD and substantial correlations between low serum NREP levels and the presence of steatosis and nonalcoholic steatohepatitis highlight the clinical translational relevance of our findings in the context of recent preclinical trials implicating ACLY in NAFLD progression. [ABSTRACT FROM AUTHOR]

Preservation of insulin-secreting ß-cells is an important goal for therapies aimed at restoring normoglycemia in patients with diabetes. One approach, the inhibition of histone deacetylases (HDACs), has been reported to suppress pancreatic islet inflammation and ß-cell apoptosis in vitro. In this report, we demonstrate the efficacy of HDAC inhibitors (HDACi) in vivo. We show that daily administration of BRD3308, an isoform-selective HDAC3 inhibitor, for 2 weeks to female nonobese diabetic (NOD) mice, beginning at 3 weeks of age, followed by twice-weekly injections until age 25 weeks, protects the animals from diabetes. The preservation of ß-cells was due to a significant decrease in islet infiltration of mononuclear cells. Moreover, the BRD3308 treatment increased basal insulin secretion from islets cultured in vitro. All metabolic tissues tested in vehicle- or BRD3308-treated groups showed virtually no sign of immune cell infiltration, except minimal infiltration in white adipose tissue in animals treated with the highest BRD3308 dose (10 mg/kg), providing additional evidence of protection from immune attack in the treated groups. Furthermore, pancreata from animals treated with 10 mg/kg BRD3308 exhibited significantly decreased numbers of apoptotic ß-cells compared with those treated with vehicle or low-dose BRD3308. Finally, animals treated with 1 or 10 mg/kg BRD3308 had enhanced ß-cell proliferation. These in vivo results point to the potential use of selective HDAC3 inhibitors as a therapeutic approach to suppress pancreatic islet infiltration and prevent ß-cell death with the long-term goal of limiting the progression of type 1 diabetes. [ABSTRACT FROM AUTHOR]

Analogs of the incretin hormones Gip and Glp-1 are used to treat type 2 diabetes and obesity. Findings in experimental models suggest that manipulating several hormones simultaneously may be more effective. To identify small molecules that increase the number of incretin-expressing cells, we established a high-throughput in vivo chemical screen by using the gip promoter to drive the expression of luciferase in zebrafish. All hits increased the numbers of neurogenin 3-expressing enteroendocrine progenitors, Gip-expressing K-cells, and Glp-1-expressing L-cells. One of the hits, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor, additionally decreased glucose levels in both larval and juvenile fish. Knock-down experiments indicated that nfatc4 , a downstream mediator of DYRKs, regulates incretin+ cell number in zebrafish, and that Dyrk1b regulates Glp-1 expression in an enteroendocrine cell line. DYRK inhibition also increased the number of incretin-expressing cells in diabetic mice, suggesting a conserved reinforcement of the enteroendocrine system, with possible implications for diabetes. [Display omitted] • In vivo screen identifies small molecules that increase the number of incretin+ cells • A GnRH agonist and DGAT1 and DYRK inhibitors boost differentiation of incretin+ cells • DYRK1A/B and downstream NFATC4 regulate the differentiation of incretin+ cells • DYRK inhibitors improve glucose control in both zebrafish and diabetic mice Analogs of the incretin hormones Gip and Glp-1 are typically used to treat diabetes. To identify factors directly increasing the number of incretin-expressing cells, Chu et al. establish a zebrafish screen and identify multiple hits, including DYRK inhibitors that increase incretins and improve glucose control in zebrafish and diabetic mice. [ABSTRACT FROM AUTHOR]

Highlights: [•] The glucokinase mutation c.823C> T generates a slightly activated protein (p.R275C). [•] The mutation also causes a misfolded and unstable protein (p.R275C). [•] The mutant protein promotes the formation of dimers and aggregates. [•] The functional impact is an increased rate of cellular degradation. [•] Biochemical effect relates to conformational changes in the F260-L271 loop structure. [Copyright &y& Elsevier]

Abstract: GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically ‘normal’ GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations. [Copyright &y& Elsevier]

Obesity is characterized by hyperinsulinemia, hyperleptinemia, and an increase in islet volume. While the mechanisms that hasten the onset of diabetes in obese individuals are not known, it is possible that the adipose-derived hormone leptin plays a role. In addition to its central actions, leptin exerts biological effects by acting in peripheral tissues including the endocrine pancreas. To explore the impact of disrupting leptin signaling in the pancreas on beta cell growth and/or function, we created pancreas-specific leptin receptor (ObR) KOs using mice expressing Cre recombinase under the control of the pancreatic and duodenal homeobox 1 (Pdx1) promoter. The KOs exhibited improved glucose tolerance due to enhanced early-phase insulin secretion, and a greater beta cell mass secondary to increased beta cell size and enhanced expression and phosphorylation of p70S6K. Similar effects on p70S6K were observed in MIN6 beta cells with knockdown of the ObR gene, suggesting crosstalk between leptin and insulin signaling pathways. Surprisingly, challenging the KOs with a high-fat diet led to attenuated acute insulin secretory response to glucose, poor compensatory islet growth, and glucose intolerance. Together, these data provide direct genetic evidence, from a unique mouse model lacking ObRs only in the pancreas, for a critical role for leptin signaling in islet biology and suggest that altered leptin action in islets is one factor that contributes to obesity-associated diabetes. [ABSTRACT FROM AUTHOR]

The insulin receptor substrate-2 (Irs2) branch of the insulin/IGF signaling system coordinates peripheral insulin action and pancreatic β cell function, so mice lacking Irs2 display similarities to humans with type 2 diabetes. Here we show that β cell-specific expression of Irs2 at a low or a high level delivered a graded physiologic response that promoted β cell growth, survival, and insulin secretion that prevented diabetes in Irs2[SUP-/-] mice, obese mice, and streptozotocin-treated mice; and that upon transplantation, the transgenic islets cured diabetes more effectively than WT islets. Thus, pharmacological approaches that promote Irs2 expression in β cells, especially specific cAMP agonists, could be rational treatments for β cell failure and diabetes. [ABSTRACT FROM AUTHOR]