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TCF7L2 variants have been associated with type 2 diabetes, body mass index (BMI), and deficits in proinsulin processing and insulin secretion. Here we sought to test whether these effects were apparent in high-risk individuals and modify treatment responses. We examined the potential role of the TCF7L2 rs7903146 variant in predicting resistance to weight loss or a lack of improvement of proinsulin processing during 2.5-years of follow-up participants (N = 2,994) from the Diabetes Prevention Program (DPP), a randomized controlled trial designed to prevent or delay diabetes in high-risk adults. We observed no difference in the degree of weight loss by rs7903146 genotypes. However, the T allele (conferring higher risk of diabetes) at rs7903146 was associated with higher fasting proinsulin at baseline (P<0.001), higher baseline proinsulin:insulin ratio (p<0.0001) and increased proinsulin:insulin ratio over a median of 2.5 years of follow-up (P = 0.003). Effects were comparable across treatment arms. The combination of a lack of impact of the TCF7L2 genotypes on the ability to lose weight, but the presence of a consistent effect on the proinsulin:insulin ratio over the course of DPP, suggests that high-risk genotype carriers at this locus can successfully lose weight to counter diabetes risk despite persistent deficits in insulin production.

Dominant Negative Pathogenesis by Mutant Proinsulin in the Akita Diabetic Mouse Tetsuro Izumi 1 , Hiromi Yokota-Hashimoto 1 , Shengli Zhao 1 , Jie Wang 1 , Philippe A. Halban 2 and Toshiyuki Takeuchi 1 1 Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan 2 Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland Abstract Autosomal dominant diabetes in the Akita mouse is caused by mutation of the insulin 2 gene, whose product replaces a cysteine residue that is engaged in the formation of an intramolecular disulfide bond. These heterozygous mice exhibit severe insulin deficiency despite coexpression of normal insulin molecules derived from three other wild-type alleles of the insulin 1 and 2 genes. Although the results of our previous study suggested that the mutant proinsulin 2 is misfolded and blocked in the transport from the endoplasmic reticulum to the Golgi apparatus, its dominant negative nature has not been fully characterized. In the present study, we investigated the possible pathogenic mechanisms induced by the mutant proinsulin 2. There is no evidence that the mutant proinsulin 2 attenuates the overall protein synthesis rate or promotes the formation of aberrant disulfide bonds. The trafficking of constitutively secreted alkaline phosphatase, however, is significantly decreased in the islets of Akita mice, indicating that the function of early secretory pathways is nonspecifically impaired. Morphological analysis has revealed that secretory pathway organelle architecture is progressively devastated in the β-cells of Akita mice. These findings suggest that the organelle dysfunction resulting from the intracellular accumulation of misfolded proinsulin 2 is primarily responsible for the defect of coexisting wild-type insulin secretion in Akita β-cells. Footnotes Address correspondence and reprint requests to Dr. Tetsuro Izumi, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan. E-mail: tizumi{at}showa.gunma-u.ac.jp . Received for publication 1 August 2002 and accepted in revised form 5 November 2002. Current address for J.W. is Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637. ER, endoplasmic reticulum; SEAP, secretory alkaline phosphatase; TUNEL, transferase-mediated dUTP nick-end labeling. DIABETES

Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell-derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p-/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p-/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p-/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH-releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency.

The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes.

Background Non-coding genetic variants that influence gene transcription in pancreatic islets play a major role in the susceptibility to type 2 diabetes (T2D), and likely also contribute to type 1 diabetes (T1D) risk. For many loci, however, the mechanisms through which non-coding variants influence diabetes susceptibility are unknown. Results We examine splicing QTLs (sQTLs) in pancreatic islets from 399 human donors and observe that common genetic variation has a widespread influence on the splicing of genes with established roles in islet biology and diabetes. In parallel, we profile expression QTLs (eQTLs) and use transcriptome-wide association as well as genetic co-localization studies to assign islet sQTLs or eQTLs to T2D and T1D susceptibility signals, many of which lack candidate effector genes. This analysis reveals biologically plausible mechanisms, including the association of T2D with an sQTL that creates a nonsense isoform in ERO1B , a regulator of ER-stress and proinsulin biosynthesis. The expanded list of T2D risk effector genes reveals overrepresented pathways, including regulators of G-protein-mediated cAMP production. The analysis of sQTLs also reveals candidate effector genes for T1D susceptibility such as DCLRE1B , a senescence regulator, and lncRNA MEG3 . Conclusions These data expose widespread effects of common genetic variants on RNA splicing in pancreatic islets. The results support a role for splicing variation in diabetes susceptibility, and offer a new set of genetic targets with potential therapeutic benefit.

In pancreatic β cells, misfolded proinsulin is a substrate for ER-associated protein degradation (ERAD) via HRD1/SEL1L. Alternately, β cell HRD1 activity is reported to improve, or impair, insulin biogenesis. Further, while β cell SEL1L deficiency causes HRD1 hypofunction and diminishes islet insulin content, reports conflict as to whether β cell ERAD deficiency increases or decreases proinsulin levels. Here, we examined β cell-specific Hrd1-KO mice (chronic deficiency) and rodent (and human islet) β cells treated acutely with HRD1 inhibitor. β-Hrd1-KO mice developed diabetes with decreased islet proinsulin, yet a relative increase of misfolded proinsulin redistributed to the ER. They also showed upregulated biochemical markers of β cell ER stress and autophagy, electron microscopy evidence of ER enlargement and decreased insulin granule content, and increased glucagon-positive islet cells. Misfolded proinsulin was also increased in islets treated with inhibitors of lysosomal degradation. Preceding any loss of total proinsulin, acute HRD1 inhibition triggered increased nonnative proinsulin, increased phospho-eIF2α with inhibited proinsulin synthesis, and increased LC3b-II (the abundance of which requires expression of ΣR1). We posit a subset of proinsulin molecules undergo HRD1-mediated disposal. When HRD1 is unavailable, misfolded proinsulin accumulates, accompanied by increased phospho-eIF2α that limits further proinsulin synthesis, plus ΣR1-dependent autophagy activation, ultimately lowering steady-state β cell proinsulin (and insulin) levels and triggering diabetes.

Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in β cells. This impairs Fe–S cluster biosynthesis, reducing the function of Cdkal1, an Fe–S cluster enzyme that catalyzes methylthiolation of t 6 A37 in tRNA Lys UUU to ms 2 t 6 A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms 2 t 6 A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2 −/− β cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans. Iron metabolism is linked to type 2 diabetes. Here the authors describe a mechanism through which cellular iron deficiency caused by loss of Irp2 impairs Cdkal1 function, resulting in inaccurate proinsulin translation, impaired proinsulin processing and reduced insulin secretion.

Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development. Insulin is a hormone that is crucial for maintaining normal blood sugar levels and is produced by so called beta cells in the pancreas. If the beta cells in the body stop making insulin, blood sugar levels start to rise, which can lead to diabetes. A form of diabetes known as neonatal diabetes, where the body stops making insulin, usually appears during the first six months of life. Infants affected by this early onset of diabetes often have mutations in one copy of the gene that encodes insulin. This means that they can still produce half of the amount of insulin, but it is not enough to keep blood sugar stable. Instead, insulin production stops completely after a few months. Scientists believe that this is because the mutant insulin has a toxic effect on beta cells. Mutations in the insulin gene can affect the structure of insulin. As a result, insulin accumulates inside the beta cells, which stresses them and eventually makes them fail. The mechanisms behind this process are still unclear. Now, Balboa et al. used stem cells (which can turn into other cell types) taken from patients with this rare type of insulin mutation to find out more. They corrected the mutant insulin gene in these stem cells with a technique called CRISPR and then induced the mutant and corrected stem cells to turn into beta cells. The results showed that the mutant beta cells slowed down their rate of cell division but did not die more frequently. When the cells were implanted into mice their growth and development changed. The mutant cells were more stressed and smaller than the cells with the repaired genes. They also had fewer signalling molecules that help cells grow. As a consequence, the cells were struggling to grow and mature. Although this type of diabetes is rare, beta cells come under stress in other forms of the disease. In a separate study, Riahi et al. found that boosting molecular signals for cell growth could protect beta cells in mice with mutant insulin. If this could also work in humans, it may lead to new ways to prevent diabetes.

Three principal ER quality-control mechanisms, namely, the unfolded protein response, ER-associated degradation (ERAD), and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the 3 pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β cells. SEL1L-HRD1 ERAD deficiency in β cells triggers activation of autophagy, at least in part, via IRE1α (an endogenous ERAD substrate). In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β cells leads to diabetes in mice shortly after weaning, with premature death by approximately 11 weeks of age, associated with marked ER retention of proinsulin and β cell loss. Using focused ion beam scanning electron microscopy powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the 3 ER quality-control mechanisms in the dynamic regulation of organellar architecture and β cell function.

Biosynthesis of insulin – critical to metabolic homeostasis – begins with folding of the proinsulin precursor, including formation of three evolutionarily conserved intramolecular disulfide bonds. Remarkably, normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol. In human (or rodent) islets with a perturbed endoplasmic reticulum folding environment, non-native proinsulin enters intermolecular disulfide-linked complexes. In genetically obese mice with otherwise wild-type islets, disulfide-linked complexes of proinsulin are more abundant, and leptin receptor-deficient mice, the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes. Proinsulin-Cys(B19) and Cys(A20) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed, proinsulin Cys(B19)-Cys(B19) covalent homodimers resist reductive dissociation, highlighting a structural basis for aberrant proinsulin complex formation. We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes. Our body fine-tunes the amount of sugar in our blood thanks to specialized ‘beta cells’ in the pancreas, which can release a hormone called insulin. To produce insulin, the beta cells first need to build an early version of the molecule – known as proinsulin – inside a cellular compartment called the endoplasmic reticulum. This process involves the formation of internal staples that keep the molecule of proinsulin folded correctly. Individuals developing type 2 diabetes have spikes of sugar in their blood, and so their bodies often respond by trying to make large amounts of insulin. After a while, the beta cells can fail to keep up, which brings on the full-blown disease. However, scientists have discovered that early in type 2 diabetes, the endoplasmic reticulum of beta cells can already show signs of stress; yet, the exact causes of this early damage are still unknown. To investigate this, Arunagiri et al. looked into whether proinsulin folds correctly during the earliest stages of type 2 diabetes. Biochemical experiments showed that even healthy beta cells contained some misfolded proinsulin molecules, where the molecular staples that should fold proinsulin internally were instead abnormally linking proinsulin molecules together. Further work revealed that the misfolded proinsulin was accumulating inside the endoplasmic reticulum. Finally, obese mice that were in the earliest stages of type 2 diabetes had the highest levels of abnormal proinsulin in their beta cells. Overall, the work by Arunagiri et al. suggests that large amounts of proinsulin molecules stapling themselves to each other in the endoplasmic reticulum of beta cells could be an early hallmark of the disease, and could make it get worse. A separate study by Jang et al. also shows that a protein that limits the misfolding of proinsulin is key to maintain successful insulin production in animals eating a Western-style, high fat diet. Hundreds of millions of people around the world have type 2 diabetes, and this number is rising quickly. Detecting and then fixing early problems associated with the condition may help to stop the disease in its track.