Integrated analysis of the pancreas and islets reveals unexpected findings in human male with type 1 diabetes

Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, A1C 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas in order to understand the cause of the donor’s diabetes. Examination of the donor’s pancreatic tissue found substantial, but reduced, β cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion that is uncommon for type 1 diabetes of this duration. Targeted and whole genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk HLA haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor’s clinical insulin deficiency was most likely the result autoimmune-mediated β cell loss, but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.

Isoform-specific roles of prolyl hydroxylases in the regulation of pancreatic β-cell function

Pancreatic β-cells can secrete insulin via two pathways characterized as KATP channel-dependent and independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP + ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α (HIFα). In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell specific knockout (KO) mice for all three isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the 1 st phase of insulin secretion but enhanced the second-phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP + ratio. All three PHD isoforms are expressed in β-cells, with PHD3 showing the most unique expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation

Aims The exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization. Methods Flow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets. Results We found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls. Conclusion These findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation.

ROCK1 regulates insulin secretion from β-cells

Objective: The endocrine pancreatic β-cells play a pivotal role in the maintenance of whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate b-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homeostasis. Methods: Mice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1loxP/loxP, β-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. Insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1-/- mice or β-cell lines with knockdown of ROCK1 were also evaluated. Proximity ligation assay was performed to determine the physical interactions between PK and ROCK1. Results: Mice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1-/- mice displayed progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS was markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium levels, ATP levels, and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that ROCK1 binding to PK is greatly enhanced by glucose stimulation in β-cells. Conclusions: Our findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and maintenance of glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

Loss of growth hormone signaling in the mouse germline or in adulthood reduces islet mass and alters islet function with notable sex differences

In the endocrine pancreas, growth hormone (GH) is known to promote pancreatic islet growth and insulin secretion. In this study, we show that GH receptor (GHR) loss in the germline and in adulthood impacts islet mass in general but more profoundly in male mice. GHR knockout (GHRKO) mice have enhanced insulin sensitivity and low circulating insulin. We show that the total cross-sectional area of isolated islets (estimated islet mass) was reduced by 72% in male but by only 29% in female GHRKO mice compared to wild type controls. Also, islets from GHRKO mice secreted ~50% less glucose-stimulated insulin compared to size-matched islets from wild type mice. We next used mice with a floxed Ghr gene to knock down the GHR in adult mice at six-months of age (6mGHRKO) and examined the impact on glucose and islet metabolism. By 12-months of age, female 6mGHRKO mice had increased body fat and reduced islet mass but had no change in glucose tolerance or insulin sensitivity. However, male 6mGHRKO mice had nearly twice as much body fat, substantially reduced islet mass, and enhanced insulin sensitivity, but no change in glucose tolerance. Despite large losses in islet mass, glucose-stimulated insulin secretion from isolated islets was not significantly different between male 6mGHRKO and controls while isolated islets from female 6mGHRKO mice showed increased glucose-stimulated insulin release. Our findings demonstrate the importance of GH to islet mass throughout life and that unique sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose metabolism.

Improved in vivo imaging method for individual islets across the mouse pancreas reveals a heterogeneous insulin secretion response to glucose

While numerous techniques can be used to measure and analyze insulin secretion in isolated islets in culture, assessments of insulin secretion in vivo are typically indirect and only semiquantitative. The CpepSfGFP reporter mouse line allows the in vivo imaging of insulin secretion from individual islets after a glucose stimulation, in live, anesthetized mice. Imaging the whole pancreas at high resolution in live mice to track the response of each individual islet over time includes numerous technical challenges and previous reports were only limited in scope and non-quantitative. Elaborating on this previous model—through the development of an improved methodology addressing anesthesia, temperature control and motion blur—we were able to track and quantify longitudinally insulin content throughout a glucose challenge in up to two hundred individual islets simultaneously. Through this approach we demonstrate quantitatively for the first time that while isolated islets respond homogeneously to glucose in culture, their profiles differ significantly in vivo. Independent of size or location, some islets respond sharply to a glucose stimulation while others barely secrete at all. This platform therefore provides a powerful approach to study the impact of disease, diet, surgery or pharmacological treatments on insulin secretion in the intact pancreas in vivo.

Metabolic Effects of Selective Deletion of Group VIA Phospholipase A2 from Macrophages or Pancreatic Islet Beta-Cells

To examine the role of group VIA phospholipase A2 (iPLA2β) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2β deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2β-KO), or in insulin-secreting β-cells (β-Cell-iPLA2β-KO), respectively. MØ-iPLA2β-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2β control mice after consuming a high-fat diet (HFD). MØ-iPLA2β-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2β-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, β-cell-iPLA2β-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. β-Cell-iPLA2β-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. β-Cell-iPLA2β-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2β participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, β-cell iPLA2β plays a role in GSIS and also appears to confer some protection against deterioration in β-cell functions induced by a HFD.

Correction to: Functional Pancreatic Isolated Islets from Cryopreserved Porcine and Human Pancreatic Tissues Based on GLUT2 Receptor Sensitivity

The author has informed the editor to correct the authors list and the acknowledgement in the article(1) as follow: Incorrect authors list in the article: • Authors list: Pimploy Rattanaamnuaychai MSc*, Yaowaluck Maprang Roshorm PhD*, Chumpon Wilasrusmee MD, PhD**, Chairat Supsamutchai MD**, Jakrapan Jirasiritham MD**, Napaphat Porpom MPh**, Shivatra Chutima Talchai PhD*** • Affiliation: * School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand; ** Department of Surgery, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; *** Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand Correct authors list: • Authors list: Shivatra Chutima Talchai PhD*, Pimploy Rattana-amnuaychai MSc**, Yaowaluck Maprang Roshorm PhD**, Chumpon Wilasrusmee MD, PhD***, Chairat Supsamutchai MD***, Jakrapan Jirasiritham MD***, Napaphat Porpom MPh***, Boonsong Ongphiphadhanakul MD**** • Affiliation: * Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand; ** School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand; *** Department of Surgery, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; **** Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Incorrect acknowledgement in the article: This research was supported by King Mongkut’s University of Technology Thonburi and. The authors thank the Ramathibodi hospital for providing materials. Correct acknowledgement: This research is supported by the Institute for the Promotion of Teaching Science and Technology (IPST) under the Research Fund for DPST Graduate with First Placement [Grant no.020/2557]. SCT was funded by the JDRF career transition grant and The King Mongkut’s University of Technology Thonburi. The authors thank the Ramathibodi hospital for providing materials.