Genetic deletion of miR-204 improves glycemic control despite obesity in db/db mice.

MicroRNAs (miRs) are small non-coding RNAs that regulate the target gene expression. A change in miR profile in the pancreatic islets during diabetes is known, and multiple studies have demonstrated that miRs influence the pancreatic ß-cell function. The miR-204 is highly expressed in the ß-cells and reported to regulate insulin synthesis. Here we investigated whether the absence of miR-204 rescues the impaired glycemic control and obesity in the genetically diabetic (db/db) mice. We found that the db/db mice overexpressed miR-204 in the islets. The db/db mice lacking miR-204 (db/db-204-/-) initially develops hyperglycemia and obesity like the control (db/db) mice but later displayed a gradual improvement in glycemic control despite remaining obese. The db/db-204-/- mice had a lower fasting blood glucose and higher serum insulin level compared to the db/db mice. A homeostatic model assessment (HOMA) suggests the improvement of ß-cell function contributes to the improvement in glycemic control in db/db-204-/- mice. Next, we examined the cellular proliferation and endoplasmic reticulum (ER) stress and found an increased frequency of proliferating cells (PCNA + ve) and a decreased CHOP expression in the islets of db/db-204-/- mice. Next, we determined the effect of systemic miR-204 inhibition in improving glycemic control in the high-fat diet (HFD)-fed insulin-resistant mice. MiR-204 inhibition for 6 weeks improved the HFD-triggered impairment in glucose disposal. In conclusion, the absence of miR-204 improves ß-cell proliferation, decreases islet ER stress, and improves glycemic control with limited change in body weight in obese mice.

Adipose Triglyceride Lipase Is a Key Lipase for the Mobilization of Lipid Droplets in Human ß-Cells and Critical for the Maintenance of Syntaxin 1a Levels in ß-Cells.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse ß-cells, LDs are prominent in human ß-cells. However, the regulation of LD mobilization (lipolysis) in human ß-cells remains unclear. We found that glucose increases lipolysis in nondiabetic human islets but not in islets in patients with type 2 diabetes (T2D), indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets with shRNA targeting ATGL (shATGL) increased triglycerides (TGs) and the number and size of LDs, indicating that ATGL is the principal lipase in human ß-cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS), and insulin secretion to 3-isobutyl-1-methylxanthine and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose-responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL-deficient INS1 cells and human pseudoislets showed reduced SNARE protein syntaxin 1a (STX1A), a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL-deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human ß-cells and supports insulin secretion by stabilizing STX1A. The dysregulated lipolysis may contribute to LD accumulation and ß-cell dysfunction in T2D islets.

Adipose Triglyceride Lipase is a Key Lipase for the Mobilization of Lipid Droplets in Human Beta Cells and Critical for the Maintenance of Syntaxin1a Level in Beta Cells.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse beta cells, LDs are prominent in human beta cells, however, the regulation of LD mobilization (lipolysis) in human beta cells remains unclear. We found that glucose increases lipolysis in non-diabetic human islets, but not in type 2 diabetic (T2D) islets, indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets (shATGL) increased triglycerides, and the number and size of LDs indicating that ATGL is the principal lipase in human beta cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS) and insulin secretion to IBMX and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL deficient INS1 cells and human pseudoislets showed reduced Stx1a, a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human beta cells and supports insulin secretion by stabilizing Stx1a. The dysregulated lipolysis may contribute to LD accumulation and beta cell dysfunction in T2D islets.

Connecting pancreatic islet lipid metabolism with insulin secretion and the development of type 2 diabetes.

Obesity is the major contributing factor for the increased prevalence of type 2 diabetes (T2D) in recent years. Sustained positive influx of lipids is considered to be a precipitating factor for beta cell dysfunction and serves as a connection between obesity and T2D. Importantly, fatty acids (FA), a key building block of lipids, are a double-edged sword for beta cells. FA acutely increase glucose-stimulated insulin secretion through cell-surface receptor and intracellular pathways. However, chronic exposure to FA, combined with elevated glucose, impair the viability and function of beta cells in vitro and in animal models of obesity (glucolipotoxicity), providing an experimental basis for the propensity of beta cell demise under obesity in humans. To better understand the two-sided relationship between lipids and beta cells, we present a current view of acute and chronic handling of lipids by beta cells and implications for beta cell function and health. We also discuss an emerging role for lipid droplets (LD) in the dynamic regulation of lipid metabolism in beta cells and insulin secretion, along with a potential role for LD under nutritional stress in beta cells, and incorporate recent advancement in the field of lipid droplet biology.

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets.

Rodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1 week revealed better preservation of first-phase glucose-stimulated insulin secretion (GSIS) compared with cultured-intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes (IL1B, CCL2, CXCL8). The expression of extracellular matrix markers (ASPN, COL1A1, COL4A1) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first-phase GSIS. Lastly, the lentiviral-mediated delivery of short hairpin RNA targeting glucokinase (GCK) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.

Effect of a mitochondrial-targeted coenzyme Q analog on pancreatic ß-cell function and energetics in high fat fed obese mice.

We recently reported that mitoquinone (mitoQ, 500 µmol/L) added to drinking water of C57BL/6J mice attenuated weight gain and reduced oxidative stress when administered to high-fat (HF) fed mice. Here, we examined the effects of mitoQ administered to HF fed mice on pancreatic islet morphology, dynamics of insulin secretion, and islet mitochondrial metabolism. C57BL/6J mice were fed HF for 130 days while we administered vehicle (cyclodextrin [CD]) or mitoQ added to the drinking water at up to 500 µmol/L. MitoQ-treated mice vs vehicle gained significantly less weight, expended significantly more energy as determined by indirect calorimetry, and trended to consume less (nonsignificant) food. As we and others reported before, mitoQ-treated mice drank less water but showed no difference in percent body fluid by nuclear magnetic resonance. Circulating insulin and glucose-stimulated insulin secretion by isolated islets were decreased in mitoQ-treated mice while insulin sensitivity (plasma insulin x glucose) was greater. Islet respiration as basal oxygen consumption (OCR), OCR directed at ATP synthesis, and maximal uncoupled OCR were also reduced in mitoQ-treated mice. Quantitative morphologic studies revealed that islet size was reduced in the mitoQ-treated mice while visual inspection of histochemically stained sections suggested that mitoQ reduced islet lipid peroxides. MitoQ markedly improved liver function as determined by plasma alanine aminotransferase. In summary, mitoQ treatment reduced the demand for insulin and reduced islet size, likely consequent to the action of mitoQ to mitigate weight gain and improve liver function.