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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 β-cell function. The miR-204 is highly expressed in the β-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 β-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 β-cell proliferation, decreases islet ER stress, and improves glycemic control with limited change in body weight in obese mice.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse β-cells, LDs are prominent in human β-cells. However, the regulation of LD mobilization (lipolysis) in human β-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 β-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 β-cells and supports insulin secretion by stabilizing STX1A. The dysregulated lipolysis may contribute to LD accumulation and β-cell dysfunction in T2D islets.

Perilipin 2 (PLIN2) is a lipid droplet (LD) protein in β cells that increases under nutritional stress. Downregulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was downregulated in mouse β cells, INS1 cells, and human islet cells. β Cell–specific deletion of PLIN2 in mice on a high-fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Downregulation of PLIN2 in INS1 cells blunted GSIS after 24-hour incubation with 0.2 mM palmitic acid. Downregulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Downregulation of PLIN2 decreased specific OXPHOS proteins in all 3 models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2-deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress, as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells has an important role in preserving insulin secretion, β cell metabolism, and mitochondrial function under nutritional stress.

Perilipin 2 (PLIN2) is the lipid droplet (LD) protein in β cells that increases under nutritional stress. Down-regulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was down-regulated in mouse β cells, INS1 cells, and human islet cells. β cell specific deletion of PLIN2 in mice on a high fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Down-regulation of PLIN2 in INS1 cells blunted GSIS after 24 h incubation with 0.2 mM palmitic acids. Down-regulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Down-regulation of PLIN2 decreased specific OXPHOS proteins in all three models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2 deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells have an important role in preserving insulin secretion, β cell metabolism and mitochondrial function under nutritional stress.

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.

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.

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.

The loss of functional beta cell mass is the central pathology for both type 1 and type 2 diabetes (Kahn 2001; Atkinson et al. 2014; Chen et al. 2017). As the three‐dimensional structure of pancreatic islets supports viability and function of beta cells through cell‐cell and cell‐matrix communications (Rutter and Hodson 2015; Arous and Wehrle‐Haller 2017; Reissaus and Piston 2017; Briant et al. 2018), it is critical to address beta cell pathophysiology in pancreatic islets. Rodent islets are readily available, cost effective, can be easily genetically manipulated, and can be compared with syngeneic animals to connect in vitro observations to in vivo phenotype. However, human islets differ substantially from their rodent counterparts anatomically and functionally. Humans and mice show distinct islet innervation, cell distribution, and ratio of beta to alpha cells (Arrojo e Drigo et al. 2015). Glucose transporters, ion channels, the ratio of first/second phase of glucose‐stimulated insulin secretion (GSIS), and amyloid deposition also differ between human and mouse islets (Arrojo e Drigo et al. 2015; Dai et al. 2016; Skelin Klemen et al. 2017). Thus, studies of human islets from organ donors are important for understanding the regulation of islet function and beta cell viability in humans. However, human islets are limited in availability, costly, difficult to maintain in culture, and challenging to genetically modify. Islet function, including GSIS, reduces over time in culture (Paraskevas et al. 2000; Arzouni et al. 2018). Genetic manipulation of intact islets by liposomal or viral‐mediated vehicles has low efficiency and typically requires partial dispersion or enzyme digestion that compromises insulin secretion and removes cell‐cell communication. The enormous heterogeneity of islet sizes also introduces high variability in assays. To overcome the variability of isolated human islets, islet spheroids or pseudoislets composed of dissociated reaggregated islet cells has been used. Reaggregated islet cells form uniformly sized pseudoislets that maintain similar spatial distribution of beta and alpha cells with better first phase GSIS compared with dispersed cells (Hopcroft et al. 1985; Halban et al. 1987) and original islets (Zuellig et al. 2017; Yu et al. 2018). Pseudoislets also lend themselves to more efficient gene modification (Caton et al. 2003; Arda et al. 2016; Peiris et al. 2018). Caton et al. reported that lentiviral‐mediated overexpression of connexin cDNA does not interfere with pseudoislet formation and allows for the transduction of a large proportion of cells (Caton et al. 2003). Thus, pseudoislets appear to offer a unique and useful model to assess human islet function after the modulation of gene expression. Using readily available reagents and resources, we first characterized pseudoislets for insulin secretion in response to secretagogues by perifusion and analyzed their expression of markers for islet cell types, beta cell function, cell stress, and extracellular matrix (ECM). We compared transduction efficiency between adenovirus and lentivirus side by side and determine the impact of transduction on GSIS by perifusion. Following characterization of the pseudoislet platform, we tested short hairpin RNA (shRNA) delivered with a lentiviral vector targeting glucokinase (GCK) as a model target to test dynamism of insulin secretion by perifusion. Our data demonstrate that lentiviral‐mediated gene downregulation combined with a simple protocol to form human pseudoislets is a useful tool that enables assessment of the impact of gene function on islet GSIS.

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.