Type 2 Diabetes Human Islets Research Articles

Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets.

Recent evidence suggests that circadian clocks ensure temporal orchestration of lipid homeostasis and play a role in pathophysiology of metabolic diseases in humans, including type 2 diabetes (T2D). Nevertheless, circadian regulation of lipid metabolism in human pancreatic islets has not been explored. Employing lipidomic analyses, we conducted temporal profiling in human pancreatic islets derived from 10 nondiabetic (ND) and 6 T2D donors. Among 329 detected lipid species across 8 major lipid classes, 5% exhibited circadian rhythmicity in ND human islets synchronized in vitro. Two-time point-based lipidomic analyses in T2D human islets revealed global and temporal alterations in phospho- and sphingolipids. Key enzymes regulating turnover of sphingolipids were rhythmically expressed in ND islets and exhibited altered levels in ND islets bearing disrupted clocks and in T2D islets. Strikingly, cellular membrane fluidity, measured by a Nile Red derivative NR12S, was reduced in plasma membrane of T2D diabetic human islets, in ND donors' islets with disrupted circadian clockwork, or treated with sphingolipid pathway modulators. Moreover, inhibiting the glycosphingolipid biosynthesis led to strong reduction of insulin secretion triggered by glucose or KCl, whereas inhibiting earlier steps of de novo ceramide synthesis resulted in milder inhibitory effect on insulin secretion by ND islets. Our data suggest that circadian clocks operative in human pancreatic islets are required for temporal orchestration of lipid homeostasis, and that perturbation of temporal regulation of the islet lipid metabolism upon T2D leads to altered insulin secretion and membrane fluidity. These phenotypes were recapitulated in ND islets bearing disrupted clocks.

Deciphering regulatory protein activity in human pancreatic islets via reverse engineering of single-cell sequencing data.

The loss of functional β cell mass contributes to development and progression of type 2 diabetes (T2D). However, the molecular mechanisms differentiating islet dysfunction in T2D from nondiabetic states remain elusive. In this issue of the JCI, Son et al. applied reverse engineering to obtain the activity of gene expression regulatory proteins from single-cell RNA sequencing data of nondiabetic and T2D human islets. The authors identify unique patterns of regulatory protein activities associated with T2D. Furthermore, BACH2 emerged as a potential transcription factor that drives activation of T2D-associated regulatory proteins in human islets.

A requirement for PAK1 to support mitochondrial function and maintain cellular redox balance via electron transport chain proteins to prevent β-cell apoptosis

Objectivep21 (Cdc42/Rac1) activated Kinase 1 (PAK1) is a candidate susceptibility factor for type 2 diabetes (T2D). PAK1 is depleted in the islets from T2D donors, compared to control individuals. In addition, whole-body PAK1 knock out (PAK1-KO) in mice worsens the T2D-like effects of high-fat diet. The current study tested the effects of modulating PAK1 levels only in β-cells. Materials/methodsβ-cell-specific inducible PAK1 KO (βPAK1-iKO) mice were generated and used with human β-cells and T2D islets to evaluate β-cell function. ResultsβPAK1-iKO mice exhibited glucose intolerance and elevated β-cell apoptosis, but without peripheral insulin resistance. β-cells from βPAK-iKO mice also contained fewer mitochondria per cell. At the cellular level, human PAK1-deficient β-cells showed blunted glucose-stimulated insulin secretion and reduced mitochondrial function. Mitochondria from human PAK1-deficient β-cells were deficient in the electron transport chain (ETC) subunits CI, CIII, and CIV; NDUFA12, a CI complex protein, was identified as a novel PAK1 binding partner, and was significantly reduced with PAK1 knockdown. PAK1 knockdown disrupted the NAD+/NADH and NADP+/NADPH ratios, and elevated ROS. An imbalance of the redox state due to mitochondrial dysfunction leads to ER stress in β-cells. PAK1 replenishment in the β-cells of T2D human islets ameliorated levels of ER stress markers. ConclusionsThese findings support a protective function for PAK1 in β-cells. The results support a new model whereby the PAK1 in the β-cell plays a required role upstream of mitochondrial function, via maintaining ETC protein levels and averting stress-induced β-cell apoptosis to retain healthy functional β-cell mass.

DOC2b Protects Functional Beta‐cell Mass via A Novel Tyrosine Phosphorylation Dependent Mechanism

Type 2 diabetes (T2D) has reached the stage of a global pandemic. Approximately 10% of the total US population (~30 million people) are impacted by T2D and 86 million more are in the stage of prediabetes. Accumulating evidence suggests that intrinsic beta‐cell stresses, secretory dysfunction, and inflammation are the early players during the progression of the T2D and leads to the loss of functional beta‐cell mass. Hence, reduction of cell stress and enhancement of beta‐cell secretory function hold promise towards delayed disease progression and prevention. Our lab has revealed the crucial role of the SNARE‐regulator protein DOC2b (double C2 domain‐containing protein) on stimulating the glucose‐stimulated insulin secretion (GSIS) and protecting the beta‐cell from the diabetogenic stresses. The objective of this study is to develop DOC2b as a therapeutic target for the treatment of T2D by elucidating the mechanism by which DOC2b preserves functional beta‐cell mass. We hypothesize that tyrosine phosphorylation of DOC2b’s functionally indispensable C2AB domain is required for DOC2b’s beneficial function during the pathological conditions related to T2D.To test this hypothesis we have used human and rodent clonal beta‐cell lines and primary human islets procured from non‐diabetic and T2D donors. Our results showed a marked loss of DOC2b mRNA and protein (~50% reduction) in the T2D human islets and a resurrected first phase of GSIS in otherwise dysfunctional T2D islets following DOC2b overexpression. Multiple studies confirmed elevated expressions of the CXCL9 and 10 (chemokine receptor 3 ligands) in diabetics. Our qPCR and biochemical analysis revealed significant attenuation of cytokine‐induced elevation of CXCL9 and 10 gene expressions following DOC2b overexpression in the clonal and primary beta‐cells. These results again signified the beneficial effect of DOC2b enrichment in the beta‐cell. In islet beta‐cells, glucose‐stimulated tyrosine phosphorylation events regulate SNARE complex assembly and GSIS. Using biochemical approaches, we determined that DOC2b is phosphorylated following glucose stimulation and pervanadate (a tyrosine phosphatase inhibitor) treatment in the clonal beta‐cells. In silico evaluation from the crystal structure of the C2B domain of DOC2b protein revealed three putative tyrosine phosphorylation sites: Y301 (within the loop region), and Y305, Y309 (within the ordered domain) and our results using point mutants of each putative site (Y to F) indicated the requirement for all three phosphorylation sites for the boosting effect of DOC2b on GSIS in the beta‐cells. We further identified that the mutations in at least Y301 and Y305 residues significantly blunted DOC2b’s ability to protect beta‐cells from cytokine‐induced beta‐cell stress and apoptosis. These results unveil a novel mechanism by which DOC2b enrichment boosts GSIS and protects beta‐cells against diabetogenic stimuli, the knowledge of which would be crucial for pursuing DOC2b as a novel drug target to prevent and/treat diabetes.Support or Funding Information5R01DK112917‐02

Expression of miR-206 in human islets and its role in glucokinase regulation.

Inappropriate insulin secretion from β-cells is considered as an early sign of impaired glucose tolerance and type 2 diabetes (T2D). Glucokinase (GCK) is an important enzyme that regulates glucose metabolism and ensures that the normal circulating glucose concentrations are maintained. GCK expression is induced by glucose and regulated via transcription factors and regulatory proteins. Recently, microRNA-206 (miR-206) was reported to regulate GCK and alter glucose tolerance in normal and high-fat diet-fed mice. Although the study findings have implications for human diabetes, studies in human islets are lacking. Here, we analyze human islets from individuals without or with T2D, using TaqMan-based real-time qPCR at the tissue (isolated islet) level as well as at single cell resolution, to assess the relationship between miR-206 and GCK expression in normal and T2D human islets. Our data suggest that, unlike mouse islets, human islets do not exhibit any correlation between miR-206 and GCK transcripts. These data implicate the need for further studies aimed toward exploring its potential role(s) in human islets.