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The β cell Golgi apparatus serves as a significant store of intracellular Ca2+ and an important site of proinsulin maturation. However, the contribution of Golgi Ca2+ to diabetes pathophysiology is unknown. The Golgi primarily utilizes the Secretory Pathway Ca2+ ATPase (SPCA1) to maintain intraluminal Ca2+ stores, and loss of SPCA1 has been linked to impaired Golgi function in other cell types. Here, we demonstrated that SPCA1 expression is decreased in islets from diabetic mice and human organ donors with type 2 diabetes, suggesting SPCA1 may impact diabetes development. INS-1 β cells lacking SPCA1 (SPCA1KO) showed reduced intraluminal Golgi Ca2+ levels, reduced glucose-stimulated insulin secretion (GSIS), and increased insulin content. Islets from SPCA1 haploinsufficient mice (SPCA1+/-) exhibited reduced GSIS, altered glucose-induced Ca2+ oscillations, and altered insulin granule maturation. Autophagy can regulate granule homeostasis, therefore we induced autophagy with Torin1 and found that SPCA1KO cells and SPCA1+/- islets had reduced levels of the autophagosome marker LC3-II. Furthermore, SPCA1KO LC3-II were unchanged after blocking autophagy initiation or autophagolysosome fusion and acidification. Thus, we concluded that β cell SPCA1 plays an important role in the maintenance of Golgi Ca2+ homeostasis and reduced Golgi Ca2+ impairs autophagy initiation and may impact insulin granule homeostasis.

Diabetes is a common complication among persons with cystic fibrosis (CF), affecting nearly 20% of adolescents and 50% of all adults with CF. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout (KO) ferret is a well-characterized animal model of CF that exhibits distinct periods of dysglycemia and pancreatic remodeling that are similar to patterns observed in humans. After the first month of life (normoglycemia/Phase I), hyperglycemia develops during the second month of life (Phase II) with reduced islet mass, followed by a period of near normoglycemia (Phase III) during which the islet mass regenerates, followed by persistent hyperglycemia/CFRD at the 4th month of life (Phase IV). While dynamic changes in islet hormone mRNA levels have been described across these phases, changes in protein expression patterns have not been characterized. To this end, immunofluorescence was performed to characterize islet hormone expression patterns in age-matched CFTR KO and wild type (WT) ferrets, focusing on the regenerative Phase III. The relative intensity of immunofluorescent staining per islet area was calculated. Compared to WT ferrets, the relative staining intensity of insulin and glucagon increased (4.687 vs. 3.228, p Disclosure H. M. Ismail: None. R. G. Mirmira: Advisory Panel; Self; Hibercell Inc., Sigilon Therapeutics, Inc., Veralox Therapeutics, Employee; Spouse/Partner; Beta Bionics, Inc. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc. R. N. Bone: None.

Offspring of obese mothers suffer higher risks of type 2 diabetes due to increased adiposity and decreased β cell function. To date, the sex-differences in offspring islet insulin secretion during early life has not been evaluated extensively, particularly prior to weaning at postnatal day 21 (P21). To determine the role of maternal obesity on offspring islet insulin secretion, C57BL/6J female dams were fed chow or western diet from 4 weeks prior to mating to induce maternal obesity. First, offspring of chow-fed and obese dams were evaluated on postnatal day 21 (P21) prior to weaning for body composition, glucose and insulin tolerance, and islet phasic insulin-secretion. Compared to same-sex controls, both male and female P21 offspring born to obese dams (MatOb) had higher body adiposity and exhibited sex-specific differences in glucose tolerance and insulin secretion. The male MatOb offspring developed the highest extent of glucose intolerance and lowest glucose-induced insulin secretion. In contrast, P21 female offspring of obese dams had unimpaired insulin secretion. Using SAX-HPLC, we found that male MatOb had a decrease in pancreatic heparan sulfate glycosaminoglycan, which is a macromolecule critical for islet health. Notably, 8-weeks-old offspring of obese dams continued to exhibit a similar pattern of sex-differences in glucose intolerance and decreased islet insulin secretion. Overall, our study suggests that maternal obesity induces sex-specific changes to pancreatic HSG in offspring and a lasting effect on offspring insulin secretion, leading to the sex-differences in glucose intolerance.

A bidirectional and complex relationship exists between bone and glycemia. Persons with type 2 diabetes (T2D) are at risk for bone loss and fracture, however, heightened osteoanabolism may ameliorate T2D-induced deficits in glycemia as bone-forming osteoblasts contribute to energy metabolism via increased glucose uptake and cellular glycolysis. Mice globally lacking Nuclear Matrix Protein 4 (Nmp4), a transcription factor expressed in all tissues and conserved between humans and rodents, are healthy and exhibit enhanced bone formation in response to anabolic osteoporosis therapies. To test whether loss of Nmp4 similarly impacted bone deficits caused by diet induced obesity, male wild type (WT) and Nmp4(−/−) mice (8wks) were fed either low-fat diet (LFD) or high-fat diet (HFD) for 12wks. Endpoint parameters included bone architecture, structural and estimated tissue level mechanical properties, body weight/composition, glucose-stimulated insulin secretion, glucose tolerance, insulin tolerance and metabolic cage analysis. HFD diminished bone architecture and ultimate force and stiffness equally in both genotypes. Unexpectedly, the Nmp4(−/−) mice exhibited deficits in pancreatic β-cell function and were modestly glucose intolerant under normal diet conditions. Despite the β-cell deficits, the Nmp4(−/−) mice were less sensitive to HFD-induced weight gain, increases in % fat mass, and decreases in glucose tolerance and insulin sensitivity. We conclude that Nmp4 supports pancreatic β-cell function but suppresses peripheral glucose utilization, perhaps contributing to its suppression of induced skeletal anabolism. Selective disruption of Nmp4 in peripheral tissues may provide a strategy for improving both induced osteoanabolism and energy metabolism in comorbid patients.

Insulin resistance impairs postprandial glucose uptake through glucose transporter type 4 (GLUT4) and is the primary defect preceding type 2 diabetes. We previously generated an insulin-resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in the muscle, adipose, and neuronal subpopulations. However, the rate of diabetes in GIRKO mice remained low prior to 6 months of age on normal chow diet (NCD), suggesting that additional factors/mechanisms are responsible for adverse metabolic effects driving the ultimate progression of overt diabetes. In this study, we characterized the metabolic phenotypes of the adult GIRKO mice acutely switched to high-fat diet (HFD) feeding in order to identify additional metabolic challenges required for disease progression. Distinct from other diet-induced obesity (DIO) and genetic models (e.g., db/db mice), GIRKO mice remained leaner on HFD feeding, but developed other cardinal features of insulin resistance syndrome. GIRKO mice rapidly developed hyperglycemia despite compensatory increases in β-cell mass and hyperinsulinemia. Furthermore, GIRKO mice also had impaired oral glucose tolerance and a limited glucose-lowering benefit from exendin-4, suggesting that the blunted incretin effect contributed to hyperglycemia. Secondly, GIRKO mice manifested severe dyslipidemia while on HFD due to elevated hepatic lipid secretion, serum triglyceride concentration, and lipid droplet accumulation in hepatocytes. Thirdly, GIRKO mice on HFD had increased inflammatory cues in the gut, which were associated with the HFD-induced microbiome alterations and increased serum lipopolysaccharide (LPS). In conclusion, our studies identified important gene/diet interactions contributing to diabetes progression, which might be leveraged to develop more efficacious therapies.

Store-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in β-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by β-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued β-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve β-cell health and function.

ObjectivesEpidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic β-cell function using rodent and in vitro models.MethodsBeginning at 8 weeks of age, C57BL/6J mice were concurrently fed high fat-diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD exposure. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured β-cells (INS-1) and primary islets were exposed ex vivo to CS extract (CSE), and β-cell function and viability were tested. Since CS increases ceramide in lungs cells and these bioactive sphingolipids have been implicated in pancreatic β-cell dysfunction in diabetes, islet and β-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry.ResultsCompared to HFD-fed ambient air-exposed mice, HFD-fed and CS- exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and a significantly greater increase in glucose intolerance compared to non-smoking control mice. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of β-cell dysfunction, significantly lower β-cell mass (p=0.02), and reduced β-cell proliferation (p=0.006), and increased islet ceramide accumulation. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide accumulation, reduce insulin gene expression and glucose-stimulated insulin secretion, and increase β-cell oxidative and ER stress. Treatment with the antioxidant N-acetylcysteine, markedly attenuated the effects of CSE on ceramide levels, restored β-cell function and survival, and increased cyclin D2 expression, while also reducing activation of β-cell ER and oxidative stress.ConclusionsOur results indicate that CS exposure inhibits insulin production, processing, and secretion and reduced β-cell viability and proliferation. These effects were linked to increased β-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of mechanisms by which CS exposure impairs β-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.

The β-cell Golgi, an important site of proinsulin maturation, is a significant store of intracellular Ca2+. However, the contribution of Golgi Ca2+ to diabetes pathophysiology is unknown. The Golgi primarily utilizes the Secretory Pathway Ca2+ ATPase (SPCA1) to maintain intra-Golgi Ca2+ stores. SPCA1 expression was measured in islets from Akita and db/db mice and cadaveric human islets from donors with T1D and T2D. In all cases, SPCA1 levels were significantly lower compared to nondiabetic controls. Proteomics and Reverse Phase Protein Array performed in INS-1 β-cells lacking SPCA1 (SPCA1KO) indicated dysregulated cell death, cell growth, and proliferation pathways compared to WT INS-1 cells. Consistent with this, SPCA1KO cells exhibited increased caspase-3/7 following tunicamycin treatment. SPCA1KO cells also had elevated basal cytosolic Ca2+ levels, decreased organelle Ca2+ reuptake following organelle Ca2+ store depletion, and accelerated Golgi collapse. Despite a significant reduction in GSIS, SPCA1KO cells exhibited a 12-fold insulin content increase. Autophagy has been reported as a key regulator in maintaining β-cell insulin homeostasis. Immunoblot revealed reduced expression of the autophagy marker LC3-II and the autophagosome Atg5/Atg12 complex in SPCA1KO cells, following autophagy induction with Torin. Islets from mice haploinsufficient for SPCA1 (SPCA1+/-) exhibited elevated cytosolic Ca2+ levels and impaired Ca2+ oscillations as reflected by increased phase 1 amplitude and period, compared to WT littermates. In islets, restoration of SPCA1 to normal levels rescued the oscillatory defect. Interestingly, islets from SPCA1+/- mice did not exhibit increased insulin content, however, SPCA1+/- islets had decreased insulin secretion and increased numbers of immature insulin granules. Taken together, we find that β-cell SPCA1 plays an important role in the maintenance of Golgi Ca2+ levels, while loss of SPCA1 led to dysregulated β-cell Ca2+ homeostasis. Disclosure R.N. Bone: None. T. Kono: None. C. Evans-Molina: None.

Type 1 diabetes (T1D) is an autoimmune disease marked by β-cell destruction. Immunotherapies for T1D have been investigated since the 1980s and have focused on restoration of tolerance, T cell or B cell inhibition, regulatory T cell (Treg) induction, suppression of innate immunity and inflammation, immune system reset, and islet transplantation. The purpose of this review is to provide an overview and lessons learned from single immunotherapy trials, describe recent and ongoing combination immunotherapy trials, and provide perspectives on strategies for future combination clinical interventions aimed at preserving insulin secretion in T1D. Combination immunotherapies have had mixed results in improving short-term glycemic control and insulin secretion in recent-onset T1D. A handful of studies have successfully reached their primary end-point of improved insulin secretion in recent-onset T1D. However, long-term improvements glycemic control and the restoration of insulin independence remain elusive. Future interventions should focus on strategies that combine immunomodulation with efforts to alleviate β-cell stress and address the formation of antigens that activate autoimmunity.

Autoimmune β-cell death leads to type 1 diabetes, and with findings that Ca2+-independent phospholipase A2β (iPLA2β) activation contributes to β-cell death, we assessed the effects of iPLA2β inhibition on diabetes development. Administration of FKGK18, a reversible iPLA2β inhibitor, to NOD female mice significantly reduced diabetes incidence in association with 1) reduced insulitis, reflected by reductions in CD4+ T cells and B cells; 2) improved glucose homeostasis; 3) higher circulating insulin; and 4) β-cell preservation. Furthermore, FKGK18 inhibited production of tumor necrosis factor-α (TNF-α) from CD4+ T cells and antibodies from B cells, suggesting modulation of immune cell responses by iPLA2β-derived products. Consistent with this, 1) adoptive transfer of diabetes by CD4+ T cells to immunodeficient and diabetes-resistant NOD.scid mice was mitigated by FKGK18 pretreatment and 2) TNF-α production from CD4+ T cells was reduced by inhibitors of cyclooxygenase and 12-lipoxygenase, which metabolize arachidonic acid to generate bioactive inflammatory eicosanoids. However, adoptive transfer of diabetes was not prevented when mice were administered FKGK18-pretreated T cells or when FKGK18 administration was initiated with T-cell transfer. The present observations suggest that iPLA2β-derived lipid signals modulate immune cell responses, raising the possibility that early inhibition of iPLA2β may be beneficial in ameliorating autoimmune destruction of β-cells and mitigating type 1 diabetes development.

Type 1 Diabetes is characterized by an absolute insulin deficiency due to the autoimmune destruction of insulin producing β-cells in the pancreatic islets. Akt1/Protein Kinase B is the direct downstream target of PI3 Kinase activation, and has shown potent anti-apoptotic and proliferation-inducing activities. This study was designed to explore whether gene transfer of constitutively active Akt1 (CA-Akt1) would promote β-cell survival and proliferation, thus be protective against experimental diabetes. In the study, a fiber-modified infectivity-enhanced adenoviral vector, Ad5RGDpK7, was used to deliver rat insulin promoter (RIP)-driven CA-Akt1 into β-cells. Our data showed this vector efficiently delivered CA-Akt1 into freshly isolated pancreatic islets, and promoted islet cell survival and β-cell proliferation in vitro. The therapeutic effect of the vector in vivo was assessed using streptozotocin (STZ)-induced diabetes mice. Two means of vector administration were explored: intravenous and intra-bile ductal injections. While direct vector administration into pancreas via bile-ductal injection resulted in local adverse effect, intravenous injection of the vectors offered therapeutic benefits. Further analysis suggests systemic vector administration caused endogenous Akt expression and activation in islets, which may be responsible, at least in part, for the protective effect of the infectivity-enhanced CA-Akt1 gene delivery vector. Taken together, our data suggest CA-Akt1 is effective in promoting β-cell survival and proliferation in vitro, but direct in vivo use is compromised by the efficacy of transgene delivery into β-cells. Nonetheless, the vector evoked the expression and activation of endogenous Akt in the islets, thus offering beneficial bystander effect against STZ-induced diabetes.

Ongoing studies suggest an important role for iPLA2β in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA2βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA2β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA2 than of cPLA2 or sPLA2, leading to its designation as a "specific" inhibitor of iPLA2, it has been shown to also inhibit non-PLA2 enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA2β and membrane-associated iPLA2γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA2β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA2β with a greater potency (100-fold) than iPLA2γ, (b) inhibition of iPLA2β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA2β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA2β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA2β role in biological functions.

The non-β endocrine cells in pancreatic islets play an essential counterpart and regulatory role to the insulin-producing β-cells in the regulation of blood-glucose homeostasis. While significant progress has been made towards the understanding of β-cell regeneration in adults, very little is known about the regeneration of the non-β endocrine cells such as glucagon-producing α-cells and somatostatin producing δ-cells. Previous studies have noted the increase of α-cell composition in diabetes patients and in animal models. It is thus our hypothesis that non-β-cells such as α-cells and δ-cells in adults can regenerate, and that the regeneration accelerates in diabetic conditions. To test this hypothesis, we examined islet cell composition in a streptozotocin (STZ)-induced diabetes mouse model in detail. Our data showed the number of α-cells in each islet increased following STZ-mediated β-cell destruction, peaked at Day 6, which was about 3 times that of normal islets. In addition, we found δ-cell numbers doubled by Day 6 following STZ treatment. These data suggest α- and δ-cell regeneration occurred rapidly following a single diabetes-inducing dose of STZ in mice. Using in vivo BrdU labeling techniques, we demonstrated α- and δ-cell regeneration involved cell proliferation. Co-staining of the islets with the proliferating cell marker Ki67 showed α- and δ-cells could replicate, suggesting self-duplication played a role in their regeneration. Furthermore, Pdx1(+)/Insulin(-) cells were detected following STZ treatment, indicating the involvement of endocrine progenitor cells in the regeneration of these non-β cells. This is further confirmed by the detection of Pdx1(+)/glucagon(+) cells and Pdx1(+)/somatostatin(+) cells following STZ treatment. Taken together, our study demonstrated adult α- and δ-cells could regenerate, and both self-duplication and regeneration from endocrine precursor cells were involved in their regeneration.