FGFR-mediated ERK1/2 signaling contributes to mesendoderm and definitive endoderm formation in vitro.

The differentiation of human pluripotent stem cells into the SOX17+ definitive endoderm (DE) germ layer is important for generating tissues for regenerative medicine. Multiple developmental and stem cell studies have demonstrated that Activin/Nodal signaling is the primary driver of definitive endoderm formation. Here, we uncover that the FGF2-FGFR-ERK1/2 signaling contributes to mesendoderm and SOX17+ DE formation. Without ERK1/2 signaling, the Activin/Nodal signaling is insufficient to drive mesendoderm and DE formation. Besides FGF2-FGFR-mediated signaling, IGF1R signaling possibly contributes to the ERK1/2 signaling for DE formation. We identified a temporal relationship between Activin/Nodal-SMAD2 and FGF2-FGFR-ERK1/2 signaling in which Activin/Nodal-SMAD2 participates in the initiation of mesendoderm and DE specification that is followed by increasing activity of FGF2-FGFR-ERK1/2 to facilitate and permit the successful generation of SOX17+ DE. Overall, besides the role of Activin/Nodal signaling for DE formation, our findings shed light on the contribution of ERK1/2 signaling for mesendoderm and DE formation.

Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction.

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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 Perturbs Pancreatic Differentiation From Human Embryonic Stem Cells.

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 ß-cells highlights its dual, opposing, protective, or inhibitory effects, depending on metabolic environment. However, the impact of metformin on developing human pancreatic ß-cells remains unknown. In this study, we investigated the potential effects of metformin exposure on human pancreatic ß-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 ß-cells, with downregulation of pancreatic genes and dysfunctional mitochondrial respiration. It also affected the insulin secretion function of pancreatic ß-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 for long-term offspring health.

The type 2 diabetes gene product STARD10 is a phosphoinositide-binding protein that controls insulin secretory granule biogenesis.

OBJECTIVE: Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the ß-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates ß-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the ß-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. METHODS: We used isolated islets from mice deleted selectively in the ß-cell for Stard10 (ßStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. RESULTS: ßStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3' position. Lipidomic analysis of ßStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in ßStard10KO islets. CONCLUSION: Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing.

New insights into human beta cell biology using human pluripotent stem cells.

Pancreatic ß-cells are responsible for maintaining glucose homeostasis. Therefore, their dysregulation leads to diabetes. Pancreas or islet transplants can be used to treat diabetes but these human tissues remain in short supply. Significant progress has now been made in differentiating human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into pancreatic ß-like cells for potential cell replacement therapy. Additionally, these hPSC-derived ß-like cells represent a new invaluable model for studying diabetes disease mechanisms. Here, we review the use of hPSC-derived ß-like cells as a platform to model various types of defects in human ß-cells in diabetes, comparing them against existing animal models, ex vivo human islets and human ß-cell line. We also discuss how hPSC-derived ß-like cells are being used as a platform for screening novel therapeutic compounds. Last but not least, we evaluate the strengths and limitations of this human cell-based platform as an avenue to study and reveal new insights into human ß-cell biology.

Tools for Bioimaging Pancreatic ß Cells in Diabetes.

When diabetes is diagnosed, the majority of insulin-secreting pancreatic ß cells are already dysfunctional or destroyed. This ß cell dysfunction/destruction usually takes place over many years, making timely detection and clinical intervention difficult. For this reason, there is immense interest in developing tools to bioimage ß cell mass and/or function noninvasively to facilitate early diagnosis of diabetes as well as to assist the development of novel antidiabetic therapies. Recent years have brought significant progress in ß cell imaging that is now inching towards clinical applicability. We explore here the need to bioimage human ß cells noninvasively in various types of diabetes, and we discuss current and emerging tools for bioimaging ß cells. Further developments in this field are expected to facilitate ß cell imaging in diabetes.