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We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

Metformin is becoming a popular treatment before and during pregnancy but current literature on in utero exposure to metformin lacks long-term clinical trials and mechanistic studies. Current literature on the effects of metformin on mature pancreatic β cells highlighted its dual, opposing, protective or inhibitory, effects depending on metabolic environments. However, the impact of metformin on developing human pancreatic β cells remains unknown. Here, we investigated the potential effects of metformin exposure on human pancreatic β cell development and function in vitro. In the absence of metabolic challenges such as high levels of glucose and fatty acids, metformin exposure impaired the development and function of pancreatic β cells, with downregulation of pancreatic genes and dysfunctional mitochondrial respiration. It also affected the insulin secretion function of pancreatic β cells. These findings call for further in-depth evaluation of the exposure of human embryonic and fetal tissue during pregnancy to metformin, and its implications on long-term offspring health.

Neonatal diabetes affects 1 in 90,000-200,000 live births. Permanent neonatal diabetes mellitus (PNDM) presents itself in the first few months of life, and patients require treatment henceforth. PNDM can be caused by mutations in the insulin gene, INS, most of which are heterozygous. This study involves two INS gene mutations: C109Y and G32V. The first mutation was reported in a Singaporean family, and the latter has not been reported yet. Patients with either mutation are unable to secrete insulin despite having one wild type (WT) allele. We hypothesize that both C109Y and G32V mutations result in misfolding of the proinsulin protein, leading to β-cell dysfunction. The misfolded proinsulin is then able to exert its dominant negative effect on WT proinsulin, either directly through heteromerization, or indirectly through altering endoplasmic reticulum (ER) integrity, preventing insulin secretion and eventually insulin synthesis and/or even β-cell death. We computationally modelled both C109Y and G32V proinsulin structures, and found that both mutations result in conformational changes that likely affect further insulin processing. We also overexpressed C109Y and G32V preproinsulin in a mouse insulinoma cell line, MIN6. Analysis of electron microscopy images revealed differences in the size and number of insulin granules. In addition, we obtained skin fibroblasts from patients with the C109Y mutation and their family members, reprogrammed them into induced pluripotent stem cells (iPSCs), and characterized them. We differentiated the iPSCs into β-like cells and saw no difference in size between WT and mutant cells. Current efforts are underway to determine the impact of the mutant insulin gene in the iPSC-derived β-like cells. Our findings will reveal mechanistic insights into this neonatal diabetes condition caused by the mutant insulin gene and potentially identify therapeutic approaches to reinstate the function of the existing wild type insulin gene. Disclosure N. Amirruddin: None. Y. Tan: None. C.S. Verma: Other Relationship; Self; sinopsee therapeutics. D. Gardner: None. Y. Bee: None. S. Hoon: None. A. Teo: None. Funding Institute of Molecular and Cell Biology; Khoo Teck Puat Hospital; National University Health System; Agency for Science, Technology and Research; National Medical Research Council; Lee Foundation; Skin Research Institute of Singapore