Case Report: Functional characterization of a missense variant in INSR associated with hypoketotic hypoglycemia.

Hypoketotic hypoglycemia due to dysregulated insulin secretion is the most common cause of persistent hypoglycemia in children. However, this type of hypoglycemia can also result from defects in the insulin signaling pathway. Distinguishing between the two is important for informing treatment decisions. Here we describe the case of a 10-year-old female with fasting and postprandial hypoglycemia who was found to have a missense variant in the INSR gene, which we functionally characterized. The proband presented with fasting and postprandial hypoglycemia at age six. Diagnostic evaluation was consistent with hypoketotic hypoglycemia suspected to be due to hyperinsulinism, and she was treated with diazoxide. Whole exome sequencing identified a maternally inherited heterozygous missense variant in INSR. Phenotypic studies on the mother were consistent with postprandial hypoglycemia. Phosphorylated Akt and ERK1/2 levels were higher at baseline and in response to stimulation with insulin in 3T3-L1 cells expressing mutant INSR compared to cells expressing wild type INSR. Thus, herein we present a heterozygous missense variant in INSR (c.1151A>G, p.Asn384Ser) that results in constitutive and increased activation of the human insulin receptor, leading to both fasting and postprandial hypoglycemia.

Human peripancreatic adipose tissue paracrine signaling impacts insulin secretion, blood flow, and gene transcription.

CONTEXT: Adipose tissue accumulation around non-adipose tissues is associated with obesity and metabolic disease. One relatively unstudied depot is peripancreatic adipose tissue (PAT) that accumulates in obesity and insulin resistance and may impact beta cell function. Pancreatic lipid accumulation and PAT content are negatively related to metabolic outcomes in humans, but these studies are limited by the inability to pursue mechanisms. OBJECTIVE: We obtained PAT from human donors through the Human Pancreas Analysis Program to evaluate differences in paracrine signaling compared to subcutaneous adipose tissue (SAT), as well as effects of the PAT secretome on aortic vasodilation, human islet insulin secretion, and gene transcription using RNAseq. RESULTS: PAT had greater secretion of IFN-γ and most inflammatory eicosanoids compared to SAT. Secretion of adipokines negatively related to metabolic health were also increased in PAT compared to SAT. We found no overall effects of PAT compared to SAT on human islet insulin secretion, however, insulin secretion was suppressed after PAT exposure from men compared to women. Vasodilation was significantly dampened by PAT conditioned media, an effect explained almost completely by PAT from men and not women. Islets treated with PAT showed selective changes in lipid metabolism pathways while SAT altered cellular signaling and growth. RNAseq analysis showed changes in islet gene transcription impacted by PAT compared to SAT, with the biggest changes found between PAT based on sex. CONCLUSION: The PAT secretome is metabolically negative compared to SAT, and impacts islet insulin secretion, blood flow, and gene transcription in a sex dependent manner.

O-GlcNAcylation modulates mTORC1 and autophagy in ß-cells, driving diabetes progression.

Type 2 diabetes (T2D) arises when pancreatic ß-cells fail to produce sufficient insulin to control blood glucose appropriately. Aberrant nutrient sensing by O-GlcNAcylation and mTORC1 is linked to T2D and the failure of insulin-producing ß-cells. However, the nature of their crosstalk in ß-cells remains unexplored. Recently, O-GlcNAcylation, a post-translation modification controlled by enzymes OGT/OGA, emerged as a pivotal regulator for ß-cell health; deficiency in either enzyme causes ß-cell failure. The present study investigates the previously unidentified connection between nutrient sensor OGT and mTORC1 crosstalk to regulate ß-cell mass and function in vivo. We show reduced OGT and mTORC1 activity in islets of preclinical ß-cell dysfunction model and obese human islets. Using loss or gain of function of OGT, we identified that O-GlcNAcylation positively regulates mTORC1 signaling in ß-cells. O-GlcNAcylation negatively modulates autophagy, as the removal of OGT increases autophagy, while the deletion of OGA decreases it. Increasing mTORC1 signaling, via deletion of TSC2, alleviates the diabetic phenotypes by increasing ß-cell mass but not ß-cell function in OGT deficient mice. Downstream phospho-protein signaling analysis reveal diverging impact on MKK4 and calmodulin signaling between islets with OGT, TSC2, or combined deletion. These data provide new evidence of OGT's significance as an upstream regulator of mTORC1 and autophagy, crucial for the regulation of ß-cell function and glucose homeostasis.

The high-fat diet and low-dose streptozotocin type-2 diabetes model induces hyperinsulinemia and insulin resistance in male but not female C57BL/6J mice.

Translation of preclinical findings on the efficacy of dietary interventions for metabolic disease to human clinical studies is challenging due to the predominant use of male rodents in animal research. Our objective was to evaluate a combined high-fat (HF) diet and low-dose streptozotocin (STZ) model for induction of type-2 diabetes (T2D) in male and female C57BL/6J mice. We hypothesized that T2D biomarkers would differ significantly between sexes. Mice were administered either a low-fat (LF) diet (10% kcal from fat), or HF diet (60% kcal from fat) + STZ injections (30 mg/kg/d for 3 days). Both sexes gained weight and developed impaired postprandial oral glucose tolerance on the HF+STZ treatment compared to LF. Only male mice on HF + STZ developed fasting hyperglycemia, fasting hyperinsulinemia and insulin resistance, suggesting that the underlying causes of postprandial hyperglycemia differed between sexes. Principal component analysis of measures such as body weights, glucose and insulin concentrations indicated metabolic derangement for males only on HF+STZ treatment, while LF group males and both groups of females significantly overlapped. Based on our data, we accept our hypothesis that the combined high-fat diet and low-dose STZ model for T2D phenotypes differs significantly in its effect on mice based on sex. The HF diet + low-dose STZ model is not useful for studying insulin resistance in females. Other models are needed to model T2D, and study the effects of dietary interventions in this disease, in females. Sexual dimorphism remains a significant challenge for both preclinical and clinical research.

Role of fatty acids in the pathogenesis of ß-cell failure and Type-2 diabetes.

Pancreatic ß-cells are glucose sensors in charge of regulated insulin delivery to the organism, achieving glucose homeostasis and overall energy storage. The latter function promotes obesity when nutrient intake chronically exceeds daily expenditure. In case of ß-cell failure, such weight gain may pave the way for the development of Type-2 diabetes. However, the causal link between excessive body fat mass and potential degradation of ß-cells remains largely unknown and debated. Over the last decades, intensive research has been conducted on the role of lipids in the pathogenesis of ß-cells, also referred to as lipotoxicity. Among various lipid species, the usual suspects are essentially the non-esterified fatty acids (NEFA), in particular the saturated ones such as palmitate. This review describes the fundamentals and the latest advances of research on the role of fatty acids in ß-cells. This includes intracellular pathways and receptor-mediated signaling, both participating in regulated glucose-stimulated insulin secretion as well as being implicated in ß-cell dysfunction. The discussion extends to the contribution of high glucose exposure, or glucotoxicity, to ß-cell defects. Combining glucotoxicity and lipotoxicity results in the synergistic and more deleterious glucolipotoxicity effect. In recent years, alternative roles for intracellular lipids have been uncovered, pointing to a protective function in case of nutrient overload. This requires dynamic storage of NEFA as neutral lipid droplets within the ß-cell, along with active glycerolipid/NEFA cycle allowing subsequent recruitment of lipid species supporting glucose-stimulated insulin secretion. Overall, the latest studies have revealed the two faces of the same coin.

Embryonic macrophages support endocrine commitment during human pancreatic differentiation.

Organogenesis is a complex process that relies on a dynamic interplay between extrinsic factors originating from the microenvironment and tissue-specific intrinsic factors. For pancreatic endocrine cells, the local niche consists of acinar and ductal cells as well as neuronal, immune, endothelial, and stromal cells. Hematopoietic cells have been detected in human pancreas as early as 6 post-conception weeks, but whether they play a role during human endocrinogenesis remains unknown. To investigate this, we performed single-nucleus RNA sequencing (snRNA-seq) of the second-trimester human pancreas and identified a wide range of hematopoietic cells, including two distinct subsets of tissue-resident macrophages. Leveraging this discovery, we developed a co-culture system of human embryonic stem cell-derived endocrine-macrophage organoids to model their interaction in vitro. Here, we show that macrophages support the differentiation and viability of endocrine cells in vitro and enhance tissue engraftment, highlighting their potential role in tissue engineering strategies for diabetes.

Effect of isoproterenol, a ß-adrenergic agonist, on the differentiation of insulin-producing pancreatic ß cells derived from human pluripotent stem cells.

Research on islet replacement through the differentiation of functionally matured insulin-producing ß-like cells for the treatment of diabetes presents a significant challenge. Neural signals in ß cell differentiation significantly impact the pancreatic microenvironment in glucose metabolism, but they are not fully understood. In this study, isoproterenol, a ß adrenoreceptor agonist, was introduced into pancreatic progenitor cells, derived from human pluripotent stem cells in vitro, undergoing endocrine differentiation, using 2-dimensional (2D) and 3-dimensional (3D) differentiation protocols. This resulted in increased insulin and C-peptide secretion, along with elevated expression of key pancreatic beta cell transcription factors, including PDX-1, NKX6.1, and MAFA, and improved function, demonstrated by increased responsiveness to glucose determined via a glucose-stimulated insulin secretion test. Moreover, RNA transcriptome analysis of isoproterenol-treated endocrine progenitors facilitated the identification of biological pathways and genes that contribute to mature beta cell differentiation efficiency correlated with neural signals, such as adrenoceptor beta 1, calcium/calmodulin dependent protein kinase II alpha, phospholipase C delta 4, and neurotrophic receptor tyrosine kinase 1. Among those genes, calcium/calmodulin dependent protein kinase II alpha was suggested as the most notable gene involved in the isoproterenol mechanism through inhibitor assays. This study illustrates that isoproterenol significantly enhances endocrine differentiation and underscores its effects on stem cell-derived beta cell maturation, emphasizing its therapeutic potential for the treatment of diabetes.

Vemurafenib inhibits the replication of diabetogenic enteroviruses in intestinal epithelial and pancreatic beta cells.

Enteroviruses, which infect via the gut, have been implicated in type 1 diabetes (T1D) development. Prolonged faecal shedding of enterovirus has been associated with islet autoimmunity. Additionally, enteroviral proteins and viral RNA have been detected in the pancreatic islets of individuals with recent-onset T1D, implicating their possible role in beta cell destruction. Despite this, no approved antiviral drugs currently exist that specifically target enterovirus infections for utilisation in disease interventions. Drug repurposing allows for the discovery of new clinical uses for existing drugs and can expedite drug discovery. Previously, the cancer drug Vemurafenib demonstrated unprecedented antiviral activity against several enteroviruses. In the present study, we assessed the efficacy of Vemurafenib and an analogue thereof in preventing infection or reducing the replication of enteroviruses associated with T1D. We tested Vemurafenib in intestinal epithelial cells (IECs) and insulin-producing beta cells. Additionally, we established a protocol for infecting human stem cell-derived islets (SC-islets) and used Vemurafenib and its analogue in this model. Our studies revealed that Vemurafenib exhibited strong antiviral properties in IECs and a beta cell line. The antiviral effect was also seen with the Vemurafenib analogue. SC-islets expressed the viral receptors CAR and DAF, with their highest expression in insulin- and glucagon-positive cells, respectively. SC-islets were successfully infected by CVBs and the antiviral activity of Vemurafenib and its analogue was confirmed in most SC-islet batches. In summary, our observations suggest that Vemurafenib and its analogue warrant further exploration as potential antiviral agents for the treatment of enterovirus-induced diseases, including T1D.

Low-level mosaic GCK mutations in children with diazoxide-unresponsive congenital hyperinsulinism.

CONTEXT: Some children with diazoxide-unresponsive congenital hyperinsulinism (HI) lack any detectable disease-causing mutation in peripheral blood DNA. OBJECTIVE: To examine whether somatic post-zygotic mutations of known HI genes are responsible for disease in children with diazoxide-unresponsive HI requiring surgery with histology not classified as focal or Localized Islet Nuclear Enlargement (LINE), and without detectable mutations by standard genetic testing of peripheral blood DNA. METHODS: Next-generation sequencing (NGS) was performed on specimens of pancreas from 10 children with diazoxide-unresponsive HI. RESULTS: Four unique GCK mutations were identified at low levels of mosaicism ranging from 4.4-10.1% in pancreatic DNA from five of these 10 children. The GCK mutations were not detectable in peripheral blood DNA by NGS in three cases from which peripheral blood DNA was available for testing. All four GCK mutations have been previously published as activating HI mutations. The histology was consistent with diffuse-HI in four of the five cases with mosaic GCK mutations. In one of these, hypomethylation of IC2 on chromosome 11p was identified in pancreatic and peripheral blood DNA. Histology of the fifth case revealed minor islet abnormalities suggestive of Beckwith Wiedemann Spectrum (BWSp) although molecular analysis for 11pUPD was negative in pancreas. CONCLUSION: These results indicate that post-zygotic somatic GCK mutations are responsible for some cases of non-focal diazoxide-unresponsive hyperinsulinism.

Type 1 diabetes: immune pathology and novel therapeutic approaches.

Type 1 diabetes (T1D) is characterized by the progressive destruction of insulin-producing beta cells in the pancreas. Despite improvements in insulin monitoring techniques, there remains no cure for T1D. Individuals with T1D require lifelong insulin therapy and some develop life-threatening complications. T1D is a complex, multifactorial, autoimmune condition. Understanding why people get T1D and how it progresses has advanced our knowledge of the disease and led to the discovery of specific targets that can be therapeutically manipulated to halt or reverse the course of T1D. Scientists investigating the potential of immunotherapy treatment for the treatment have recently had some encouraging results. Teplizumab, an anti-CD3 monoclonal antibody that has been approved by the FDA, delays the onset of clinical T1D in patients ≥ 8 years of age with preclinical T1D and improves beta cell function. Therapies targeting beta cell health, vitality, and function are now thought to be an essential component of successful combination therapy for T1D. The idea that the beta cells themselves may influence their own destruction during the development of T1D is a notion that has recently been gaining acceptance in the field. Researchers have recently made remarkable strides in beta cell replacement therapy and beta cell regeneration techniques. This review offers a detailed exploration of the pathophysiological mechanisms of T1D. It discusses the intricate interplay of factors leading to T1D development and the innovative approaches being explored to discover new treatments and a cure for the millions of people living with T1D worldwide.

Dapagliflozin mitigates cellular stress and inflammation through PI3K/AKT pathway modulation in cardiomyocytes, aortic endothelial cells, and stem cell-derived ß cells.

Dapagliflozin (DAPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is well-recognized for its therapeutic benefits in type 2 diabetes (T2D) and cardiovascular diseases. In this comprehensive in vitro study, we investigated DAPA's effects on cardiomyocytes, aortic endothelial cells (AECs), and stem cell-derived beta cells (SC-ß), focusing on its impact on hypertrophy, inflammation, and cellular stress. Our results demonstrate that DAPA effectively attenuates isoproterenol (ISO)-induced hypertrophy in cardiomyocytes, reducing cell size and improving cellular structure. Mechanistically, DAPA mitigates reactive oxygen species (ROS) production and inflammation by activating the AKT pathway, which influences downstream markers of fibrosis, hypertrophy, and inflammation. Additionally, DAPA's modulation of SGLT2, the Na+/H + exchanger 1 (NHE1), and glucose transporter (GLUT 1) type 1 highlights its critical role in maintaining cellular ion balance and glucose metabolism, providing insights into its cardioprotective mechanisms. In aortic endothelial cells (AECs), DAPA exhibited notable anti-inflammatory properties by restoring AKT and phosphoinositide 3-kinase (PI3K) expression, enhancing mitogen-activated protein kinase (MAPK) activation, and downregulating inflammatory cytokines at both the gene and protein levels. Furthermore, DAPA alleviated tumor necrosis factor (TNFα)-induced inflammation and stress responses while enhancing endothelial nitric oxide synthase (eNOS) expression, suggesting its potential to preserve vascular function and improve endothelial health. Investigating SC-ß cells, we found that DAPA enhances insulin functionality without altering cell identity, indicating potential benefits for diabetes management. DAPA also upregulated MAFA, PI3K, and NRF2 expression, positively influencing ß-cell function and stress response. Additionally, it attenuated NLRP3 activation in inflammation and reduced NHE1 and glucose-regulated protein GRP78 expression, offering novel insights into its anti-inflammatory and stress-modulating effects. Overall, our findings elucidate the multifaceted therapeutic potential of DAPA across various cellular models, emphasizing its role in mitigating hypertrophy, inflammation, and cellular stress through the activation of the AKT pathway and other signaling cascades. These mechanisms may not only contribute to enhanced cardiac and endothelial function but also underscore DAPA's potential to address metabolic dysregulation in T2D.

1. DAPA effectively attenuates ISO-induced cardiomyocyte hypertrophy by reducing cell size and improving cellular structure. 2. DAPA exhibits anti-inflammatory properties in AECs by restoring AKT and PI3K expression, upregulating MAPK activation, and downregulating inflammatory gene expression. 3. DAPA enhances insulin functionality in SC-ß cells without altering cell identity, suggesting potential benefits in diabetes management. 4. DAPA's modulation of SGLT2, NHE1, and GLUT1 expression in cardiomyocytes underscores its role in cellular ion balance and glucose metabolism, contributing to its cardioprotective mechanisms. 5. DAPA alleviates TNFα-induced inflammation and stress responses in AECs, while enhancing eNOS expression, indicating its potential to preserve vascular function. 6. DAPA attenuates NLRP3 activation and reduces NHE1 and GRP78 expression in SC-ß cells, offering novel insights into its anti-inflammatory and stress-modulating effects.

Glucokinase (GCK) in diabetes: from molecular mechanisms to disease pathogenesis.

Glucokinase (GCK), a key enzyme in glucose metabolism, plays a central role in glucose sensing and insulin secretion in pancreatic ß-cells, as well as glycogen synthesis in the liver. Mutations in the GCK gene have been associated with various monogenic diabetes (MD) disorders, including permanent neonatal diabetes mellitus (PNDM) and maturity-onset diabetes of the young (MODY), highlighting its importance in maintaining glucose homeostasis. Additionally, GCK gain-of-function mutations lead to a rare congenital form of hyperinsulinism known as hyperinsulinemic hypoglycemia (HH), characterized by increased enzymatic activity and increased glucose sensitivity in pancreatic ß-cells. This review offers a comprehensive exploration of the critical role played by the GCK gene in diabetes development, shedding light on its expression patterns, regulatory mechanisms, and diverse forms of associated monogenic disorders. Structural and mechanistic insights into GCK's involvement in glucose metabolism are discussed, emphasizing its significance in insulin secretion and glycogen synthesis. Animal models have provided valuable insights into the physiological consequences of GCK mutations, although challenges remain in accurately recapitulating human disease phenotypes. In addition, the potential of human pluripotent stem cell (hPSC) technology in overcoming current model limitations is discussed, offering a promising avenue for studying GCK-related diseases at the molecular level. Ultimately, a deeper understanding of GCK's multifaceted role in glucose metabolism and its dysregulation in disease states holds implications for developing targeted therapeutic interventions for diabetes and related disorders.

Advances and counterpoints in type 2 diabetes. What is ready for translation into real-world practice, ahead of the guidelines.

This review seeks to address major gaps and delays between our rapidly evolving body of knowledge on type 2 diabetes and its translation into real-world practice. Through updated and improved best practices informed by recent evidence and described herein, we stand to better attain A1c targets, help preserve beta cell integrity and moderate glycemic variability, minimize treatment-emergent hypoglycemia, circumvent prescribing to "treatment failure," and prevent long-term complications. The first topic addressed in this review concerns updates in the 2023 and 2024 diabetes treatment guidelines for which further elaboration can help facilitate integration into routine care. The second concerns advances in diabetes research that have not yet found their way into guidelines, though they are endorsed by strong evidence and are ready for real-world use in appropriate patients. The final theme addresses lingering misconceptions about the underpinnings of type 2 diabetes-fundamental fallacies that continue to be asserted in the textbooks and continuing medical education upon which physicians build their approaches. A corrected and up-to-date understanding of the disease state is essential for practitioners to both conceptually and translationally manage initial onset through late-stage type 2 diabetes.

From iPSCs to Pancreatic ß Cells: Unveiling Molecular Pathways and Enhancements with Vitamin C and Retinoic Acid in Diabetes Research.

Diabetes mellitus, a chronic and non-transmissible disease, triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ß-like cells (PßLCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways, directing the differentiation of iPSCs into pancreatic ß cells. Despite significant progress and improved protocols, the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here, we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into PßLCs. Our protocol has shown to be effective, with the resulting cells exhibiting key functional properties of pancreatic ß cells, including the expression of crucial molecular markers (pdx1, nkx6.1, ngn3) and the capability to secrete insulin in response to glucose. Furthermore, the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ß cell genes.

Sulfation pathways in the maintenance of functional beta-cell mass and implications for diabetes.

Diabetes Type 1 and Type 2 are widely occurring diseases. In spite of a vast amount of biomedical literature about diabetic processes in general, links to certain biological processes are only becoming evident these days. One such area of biology is the sulfation of small molecules, such as steroid hormones or metabolites from the gastrointestinal tract, as well as larger biomolecules, such as proteins and proteoglycans. Thus, modulating the physicochemical propensities of the different sulfate acceptors, resulting in enhanced solubility, expedited circulatory transit, or enhanced macromolecular interaction. This review lists evidence for the involvement of sulfation pathways in the maintenance of functional pancreatic beta-cell mass and the implications for diabetes, grouped into various classes of sulfated biomolecule. Complex heparan sulfates might play a role in the development and maintenance of beta-cells. The sulfolipids sulfatide and sulfo-cholesterol might contribute to beta-cell health. In beta-cells, there are only very few proteins with confirmed sulfation on some tyrosine residues, with the IRS4 molecule being one of them. Sulfated steroid hormones, such as estradiol-sulfate and vitamin-D-sulfate, may facilitate downstream steroid signaling in beta-cells, following de-sulfation. Indoxyl sulfate is a metabolite from the intestine, that causes kidney damage, contributing to diabetic kidney disease. Finally, from a technological perspective, there is heparan sulfate, heparin, and chondroitin sulfate, that all might be involved in next-generation beta-cell transplantation. Sulfation pathways may play a role in pancreatic beta-cells through multiple mechanisms. A more coherent understanding of sulfation pathways in diabetes will facilitate discussion and guide future research.

NMDA Suppresses Pancreatic ABCA1 Expression through the MEK/ERK/LXR Pathway in Pancreatic Beta Cells.

Dysfunction or loss of pancreatic ß cells can cause insulin deficiency and impaired glucose regulation, resulting in conditions like type 2 diabetes. The ATP-binding cassette transporter A1 (ABCA1) plays a key role in the reverse cholesterol transport system, and its decreased expression is associated with pancreatic ß cell lipotoxicity, resulting in abnormal insulin synthesis and secretion. Increased glutamate release can cause glucotoxicity in ß cells, though the detailed mechanisms remain unclear. This study investigated the effect of N-methyl-D-aspartic acid (NMDA) on ABCA1 expression in INS-1 cells and primary pancreatic islets to elucidate the signaling mechanisms that suppress insulin secretion. Using Western blotting, microscopy, and biochemical analyses, we found that NMDA activated the mitogen-activated protein kinase (MEK)-dependent pathway, suppressing ABCA1 protein and mRNA expression. The MEK-specific inhibitor PD98059 restored ABCA1 promoter activity, indicating the involvement of the extracellular signal-regulated kinase (MEK/ERK) pathway. Furthermore, we identified the liver X receptor (LXR) as an effector transcription factor in NMDA regulation of ABCA1 transcription. NMDA treatment increased cholesterol and triglyceride levels while decreasing insulin secretion, even under high-glucose conditions. These effects were abrogated by treatment with PD98059. This study reveals that NMDA suppresses ABCA1 expression via the MEK/ERK/LXR pathway, providing new insights into the pathological suppression of insulin secretion in pancreatic ß cells and emphasizing the importance of investigating the role of NMDA in ß cell dysfunction.

Mitochondrial diabetes in mice expressing a dominant-negative allele of nuclear respiratory factor-1 (Nrf1) in pancreatic ß-cells.

Genetic polymorphisms in nuclear respiratory factor-1 (Nrf1), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with ß-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic ß-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in ß-cells. Expression of NRF1 target genes Tfam, Tfb1m and Tfb2m, and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in ß-cells was sufficient to restore ß-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of ß-cell function and establishes a model to study the interplay between regulators of bi-genomic gene transcription in diabetes.

Analysis of beta-cell maturity and mitochondrial morphology in juvenile non-human primates exposed to maternal Western-style diet during development.

Introduction: Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. Methods: We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. Results: There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Discussion: Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

Secreted GDF15 maintains transcriptional responses during DNA damage-mediated senescence in human beta cells.

Type 1 diabetes (T1D) is a chronic metabolic disease resulting from an autoimmune destruction of pancreatic beta cells. Beta cells activate various stress responses during the development of T1D, including senescence, which involves cell cycle arrest, prosurvival signaling, and a proinflammatory secretome termed the senescence-associated secretory phenotype (SASP). We previously identified growth and differentiation factor 15 (GDF15) as a major SASP factor in human islets and human EndoC-ßH5 beta cells in a model of DNA damage-mediated senescence that recapitulates features of senescent beta cells in T1D. Soluble GDF15 has been shown to exert protective effects on human and mouse beta cells during various forms of stress relevant to T1D; therefore, we hypothesized that secreted GDF15 may play a prosurvival role during DNA damage-mediated senescence in human beta cells. We found that elevated GDF15 secretion was associated with endogenous senescent beta cells in an islet preparation from a T1D donor, supporting the validity of our DNA damage model. Using antibody-based neutralization, we found that secreted endogenous GDF15 was not required for senescent human islet or EndoC cell viability. Rather, neutralization of GDF15 led to reduced expression of specific senescence-associated genes, including GDF15 itself and the prosurvival gene BCL2-like protein 1 (BCL2L1). Taken together, these data suggest that SASP factor GDF15 is not required to sustain senescent human islet viability, but it is required to maintain senescence-associated transcriptional responses.NEW & NOTEWORTHY Beta cell senescence is an emerging contributor to the pathogenesis of type 1 diabetes, but candidate therapeutic targets have not been identified in human beta cells. In this study, we examined the role of a secreted factor, GDF15, and found that although it is not required to maintain viability during senescence, it is required to fine-tune gene expression programs involved in the senescence response during DNA damage in human beta cells.