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Concept: Islets of Langerhans

184

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Concepts: Cell, Insulin, Glucose, Stem cell, Islets of Langerhans, Pancreas, Cellular differentiation, Embryonic stem cell

178

Diabetes is a major global health issue and the number of individuals with type 1 diabetes (T1D) and type 2 diabetes (T2D) increases annually across multiple populations. Research to develop a cure must overcome multiple immune dysfunctions and the shortage of pancreatic islet β cells, but these challenges have proven intractable despite intensive research effort more than the past decades. Stem Cell Educator (SCE) therapy-which uses only autologous blood immune cells that are externally exposed to cord blood stem cells adhering to the SCE device, has previously been proven safe and effective in Chinese and Spanish subjects for the improvement of T1D, T2D, and other autoimmune diseases. Here, 4-year follow-up studies demonstrated the long-term safety and clinical efficacy of SCE therapy for the treatment of T1D and T2D. Mechanistic studies found that the nature of platelets was modulated in diabetic subjects after receiving SCE therapy. Platelets and their released mitochondria display immune tolerance-associated markers that can modulate the proliferation and function of immune cells. Notably, platelets also expressed embryonic stem cell- and pancreatic islet β-cell-associated markers that are encoded by mitochondrial DNA. Using freshly-isolated human pancreatic islets, ex vivo studies established that platelet-releasing mitochondria can migrate to pancreatic islets and be taken up by islet β cells, leading to the proliferation and enhancement of islet β-cell functions. These findings reveal new mechanisms underlying SCE therapy and open up new avenues to improve the treatment of diabetes in clinics. Stem Cells Translational Medicine 2017.

Concepts: Insulin, Diabetes mellitus type 2, Diabetes mellitus, Mitochondrion, Stem cell, Diabetes mellitus type 1, Islets of Langerhans, Embryonic stem cell

169

Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used for the treatment of mood and anxiety disorders. Here we demonstrate that incubation (2 h) of murine islets or Min6 β cell line with the SSRIs paroxetine, fluoxetine or sertraline inhibited insulin-induced Tyr phosphorylation of insulin receptor substrate (IRS)-2 protein and the activation of its downstream targets Akt and S6K1. Inhibition was dose-dependent with half-maximal effects at ~15-20 μM. It correlated with a rapid phosphorylation and activation of the IRS kinase GSK3β. Introduction of GSK3β-siRNAs eliminated the inhibitory effects of the SSRIs. Inhibition of IRS-2 action by 30 μM SSRIs was associated with a marked inhibition of glucose-stimulated insulin secretion from murine and human pancreatic islets. Secretion induced by basic secretagogues (KCl and Arg) was not affected by these drugs. Prolonged treatment (16h) of Min6 cells with sertraline resulted in the induction of iNOS; activation of an ER stress and the initiation of the Unfolded Protein Response (UPR), manifested by enhanced transcription of ATF4 and CHOP. This triggered an apoptotic process, manifested by enhanced caspase 3/7 activity, that resulted in beta cell death. These findings implicate SSRIs as inhibitors of IRS protein function and insulin action through the activation of GSK3β. They further suggest that SSRIs inhibit insulin secretion; induce the UPR; activate an apoptotic process and trigger beta cell death. Given that SSRIs promote insulin resistance while inhibiting insulin secretion, these drugs might accelerate the transition from an insulin resistant state to overt diabetes.

Concepts: Insulin, Islets of Langerhans, Beta cell, Serotonin, Antidepressant, Selective serotonin reuptake inhibitor, Sertraline, Fluoxetine

168

Loss of function mutations in EIF2AK3, encoding the Pancreatic Endoplasmic Reticulum (ER) Kinase, PERK, are associated with dysfunction of the endocrine pancreas and diabetes. However to date it has not been possible to uncouple the long term developmental effects of PERK deficiency from sensitization to physiological levels of ER unfolded protein stress upon interruption of PERK-modulation of protein synthesis rates. Here we report that a selective PERK inhibitor acutely deregulates protein synthesis in freshly isolated islets of Langerhans, across a range of glucose concentrations. Acute loss of the PERK-mediated strand of the unfolded protein response leads to rapid accumulation of misfolded pro-insulin in cultured beta cells and is associated with a kinetic defect in pro-insulin processing. These in vitro observations uncouple the latent role of PERK in beta cell development from the regulation of unfolded protein flux through the ER and attest to the importance of the latter in beta cell proteostasis.

Concepts: DNA, Protein, Enzyme, Insulin, Islets of Langerhans, Pancreas, Beta cell, Amylin

167

VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature and insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that secretion of VEGF-A, but not VEGF-A gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with an insulin pellet, which resulted in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

Concepts: Protein, Gene, Enzyme, Insulin, Islets of Langerhans, Pancreas, Beta cell, Insulinoma

163

Amylin (islet amyloid polypeptide) and amyloid-beta (Aβ) protein, which are deposited within pancreatic islets of diabetics and brains of Alzheimer’s patients respectively, share many biophysical and physiological properties. Emerging evidence indicates that the amylin receptor is a putative target receptor for the actions of human amylin and Aβ in the brain. The amylin receptor consists of the calcitonin receptor dimerized with a receptor activity-modifying protein and is widely distributed within central nervous system. Both amylin and Aβ directly activate this G protein-coupled receptor and trigger multiple common intracellular signal transduction pathways that can culminate in apoptotic cell death. Moreover, amylin receptor antagonists can block both the biological and neurotoxic effects of human amylin and Aβ. Amylin receptors thus appear to be involved in the pathophysiology of Alzheimer’s disease and diabetes, and could serve as a molecular link between the two conditions that are associated epidemiologically.

Concepts: Protein, Brain, Signal transduction, Insulin, Hormone, Islets of Langerhans, Glucagon, Amylin

160

Zinc is essential for the activities of pancreatic beta-cells, especially insulin storage and secretion. Insulin secretion leads to co-release of zinc which contributes to the paracrine communication in the pancreatic islets. Zinc-transporting proteins (zinc-regulated transporter, iron-regulated transporter-like proteins [ZIPs] and zinc transporters [ZnTs]) and metal-buffering proteins (metallothioneins, MTs) tightly regulate intracellular zinc homeostasis. The present study investigated how modulation of cellular zinc availability affects beta-cell function using INS-1E cells.

Concepts: Enzyme, Insulin, Cell biology, Islets of Langerhans, Glucagon, Pancreas, Beta cell, Amylin

120

Novel interventions that reestablish endogenous insulin secretion and thereby halt progressive end-organ damage and prolong survival of patients with autoimmune Type 1 diabetes mellitus (T1DM) are urgently needed. While this is currently accomplished with allogeneic pancreas or islet transplants, their utility is significantly limited by both the scarcity of organ donors and life-long need for often-toxic antirejection drugs. Coadministering islets with bone marrow-derived mesenchymal stem cells (MSCs) that exert robust immune-modulating, anti-inflammatory, anti-apoptotic, and angiogenic actions, improves intrahepatic islet survival and function. Encapsulation of insulin-producing cells to prevent immune destruction has shown both promise and failures. Recently, stem cell-derived insulin secreting β-like cells induced euglycemia in diabetic animals, although their clinical use would still require encapsulation or anti-rejection drugs. Instead of focusing on further improvements in islet transplantation, we demonstrate here that the intraperitoneal administration of islet-sized “Neo-Islets” (NIs), generated by in vitro coaggregation of allogeneic, culture-expanded islet cells with high numbers of immuno-protective and cyto-protective MSCs, resulted in their omental engraftment in immune-competent, spontaneously diabetic nonobese diabetic (NOD) mice. This achieved long-term glycemic control without immunosuppression and without hypoglycemia. In preparation for an Food and Drug Administration-approved clinical trial in dogs with T1DM, we show that treatment of streptozotocin-diabetic NOD/severe combined immunodeficiency mice with identically formed canine NIs produced durable euglycemia, exclusively mediated by dog-specific insulin. We conclude that this novel technology has significant translational relevance for canine and potentially clinical T1DM as it effectively addresses both the organ donor scarcity (>80 therapeutic NI doses/donor pancreas can be generated) and completely eliminates the need for immunosuppression. Stem Cells Translational Medicine 2017.

Concepts: Insulin, Diabetes mellitus, Diabetes mellitus type 1, Islets of Langerhans, Glucagon, Pancreas, Diabetes, Organ transplant

50

Stem-cell-based therapies can potentially reverse organ dysfunction and diseases, but the removal of impaired tissue and activation of a program leading to organ regeneration pose major challenges. In mice, a 4-day fasting mimicking diet (FMD) induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing β cells, resembling that observed during pancreatic development. FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 expression and insulin production. The effects of the FMD are reversed by IGF-1 treatment and recapitulated by PKA and mTOR inhibition. These results indicate that a FMD promotes the reprogramming of pancreatic cells to restore insulin generation in islets from T1D patients and reverse both T1D and T2D phenotypes in mouse models. PAPERCLIP.

Concepts: Insulin, Diabetes mellitus type 2, Diabetes mellitus, Diabetes mellitus type 1, Obesity, Islets of Langerhans, Pancreas, Liver

45

The arrangement of β cells within islets of Langerhans is critical for insulin release through the generation of rhythmic activity. A privileged role for individual β cells in orchestrating these responses has long been suspected, but not directly demonstrated. We show here that the β cell population in situ is operationally heterogeneous. Mapping of islet functional architecture revealed the presence of hub cells with pacemaker properties, which remain stable over recording periods of 2 to 3 hr. Using a dual optogenetic/photopharmacological strategy, silencing of hubs abolished coordinated islet responses to glucose, whereas specific stimulation restored communication patterns. Hubs were metabolically adapted and targeted by both pro-inflammatory and glucolipotoxic insults to induce widespread β cell dysfunction. Thus, the islet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.

Concepts: Insulin, Diabetes mellitus type 2, Diabetes mellitus, Glucose, Islets of Langerhans, Glucagon, Pancreas, Beta cell