Vitamin D and type 2 diabetes mellitus: mechanisms, clinical evidence, and future perspectives

Recent advances in understanding the anti-diabetic actions of dietary flavonoids

ObjectiveMany studies have reported that stem cell transplantation promotes propagation and protection of pancreatic β-cells in streptozotocin (STZ)-induced diabetic mice without the differentiation of transplanted cells into pancreatic β-cells, suggesting...

Integrin-extracellular matrix interactions are important determinants of beta cell behaviours. The β1 integrin is a well-known regulator of beta cell activities; however, little is known of its associated α subunits. In the present study, αβ1 integrin expression was examined in the rat insulinoma cell line (INS-1) to identify their role in beta cell survival and function. Seven α subunits associated with β1 integrin were identified, including α1-6 and αV. Among these heterodimers, α3β1 was most highly expressed. Common ligands for the α3β1 integrin, including fibronectin, laminin, collagen I and collagen IV were tested to identify the most suitable matrix for INS-1 cell proliferation and function. Cells exposed to collagen I and IV demonstrated significant increases in adhesion, spreading, cell viability, proliferation, and FAK phosphorylation when compared to cells cultured on fibronectin, laminin and controls. Integrin-dependent attachment also had a beneficial effect on beta cell function, increasing Pdx-1 and insulin gene and protein expression on collagens I and IV, in parallel with increased basal insulin release and enhanced insulin secretion upon high glucose challenge. Furthermore, functional blockade of α3β1 integrin decreased cell adhesion, spreading and viability on both collagens and reduced Pdx-1 and insulin expression, indicating that its interactions with collagen matrices are important for beta cell survival and function. These results demonstrate that specific αβ1 integrin-ECM interactions are critical regulators of INS-1 beta cell survival and function and will be important in designing optimal conditions for cell-based therapies for diabetes treatment.

Histone deacytylase inhibitors (HDACis) inhibit the deacetylation of the lysine residue of proteins, including histones, and regulate the transcription of a variety of genes. Recently, HDACis have been used clinically as anti-cancer drugs and possible anti-diabetic drugs. Even though HDACis have been proven to protect the cytokine-induced damage of pancreatic beta cells, evidence also shows that high doses of HDACis are cytotoxic. In the present study, we, therefore, investigated the eff ect of HDACis on insulin secretion in a pancreatic beta cell line. Pancreatic beta cells MIN6 were treated with selected HDACis (trichostatin A, TSA; valproic acid, VPA; and sodium butyrate, NaB) in medium supplemented with 25 mM glucose and 13% heat-inactivated fetal bovine serum (FBS) for indicated time intervals. Protein expression of Pdx1 and Mafa in MIN6 cells was demonstrated by immunohistochemistry and immunocytochemistry, expression of Pdx1 and Mafa genes was measured by quantitative RT-PCR method. Insulin release from MIN6 cells and insulin cell content were estimated by ELISA kit. Superoxide production in MIN6 cells was measured using a Total ROS/Superoxide Detection System. TSA, VPA, and NaB inhibited the expression of Pdx1 and Mafa genes and their products. TSA treatment led to beta cell malfunction, characterized by enhanced insulin secretion at 3 and 9 mM glucose, but impaired insulin secretion at 15 and 25 mM glucose. Th us, TSA induced dysregulation of the insulin secretion mechanism. TSA also enhanced reactive oxygen species production in pancreatic beta cells. Our results showed that HDACis caused failure to suppress insulin secretion at low glucose concentrations and enhance insulin secretion at high glucose concentrations. In other words, when these HDACis are used clinically, high doses of HDACis may cause hypoglycemia in the fasting state and hyperglycemia in the fed state. When using HDACis, physicians should, therefore, be aware of the capacity of these drugs to modulate the insulin secretory capacity of pancreatic beta cells.

Dietary strategies for type 2 diabetes (T2DM) prevention have mainly focused on solid foods and nutrients. Emanating evidence suggests that beverage consumption in adulthood may also influence T2DM development, whereas the role of beverages during adolescence remains unknow. To examine adolescent beverages consumption, and their changes from adolescence to adulthood in relation to T2DM risk in adulthood. This prospective cohort study, conducted within the Nurses' Health Study II (NHS II), enrolled 41,317 women who completed a food-frequency questionnaire (FFQ) regarding their diet in high school and had no diabetes, cardiovascular disease, or cancer at baseline (1997). Beverage consumption including coffee, tea, regular or diet soda, fruit juice or milk, was assessed using the FFQ. Cox proportional hazards models were utilized to estimate hazard ratios (HRs) for the association between beverage consumption in adolescence and risk of incident type 2 diabetes (T2DM) in adulthood, adjusting for potential confounders. During 725,650 person-years of follow-up, 2,844 participants developed T2DM. After adjustment for demographic, lifestyle and dietary risk factors, comparing ≥ 1 serving/day with non-consumers, adolescent coffee [HR, 0.86 (95% confidence interval: 0.75 to 0.98); P-trend = 0.02)] and orange juice [HR, 0.83 (0.71 to 0.96); P-trend = 0.0008)] consumption was associated with lower T2DM risk, whereas, regular soda [HR, 1.37 (1.20 to 1.57); P-trend < 0.0001)] and iced tea [HR, 1.41 (1.21 to 1.65); P-trend < 0.0001)] intake was associated with higher T2DM risk. Increased coffee intake from adolescence to adulthood in 1991 was associated with a lower T2DM risk [HR, 0.70 (0.61 to 0.80); P-trend < 0.0001), comparing ≥ + 3 servings/day with no change], whereas the opposite was observed for increased regular soda [HR, 1.20 (1.06 to 1.35); P-trend = 0.004), comparing ≥ + 1 or more servings/week with no change)] and diet soda consumption [HR, 1.59 (1.41 to 1.80); P-trend = 0.0002), comparing ≥ + 2 servings/day with no change]. Adolescent consumption of coffee or orange juice intake was associated with a lower risk of T2DM, whereas the opposite was observed for intake of regular soda or iced tea. In addition, increased coffee intake was associated with a lower diabetes risk, whereas the opposite was observed for regular or diet soda intake. These data highlight a potentially important role of beverage intake at early life in the etiology of diabetes during adulthood.

microRNAs (miRNAs) are critical regulators of glucose metabolism and contribute to the pathogenesis of Type 2 Diabetes (T2D). Recently, we reported that high-density lipoproteins (HDL) transport and deliver functional miRNAs to recipient cells. Here, we report that miR-375 is decreased on HDL in two models of chronic hyperglycemia -- T2D human subjects and Zucker Diabetic Fatty (ZDF) rats. Since miR-375 expression in the islets is 10X greater than in other organs, we tested whether pancreatic beta cells have the ability to export miR-375 to HDL through in vitro export assays, incubating HDL with INS1 beta cells or primary human islets. Indeed, we found miR-375 to be readily exported to HDL from INS1 cells and primary islets in vitro . To determine if cholesterol transporters contribute to HDL-miR-375 export from beta cells, Abca1, Abcg1 and Scarb1 (SR-BI) were inhibited using siRNAs; however, we found that knockdown of each of these transporters failed to affect the beta cell’s ability to export miR-375 to HDL. Nonetheless, enhancing insulin secretion with tolbutamide resulted in the suppression of HDL-miR-375 export, suggesting that miRNA export and insulin secretion are inversely regulated. To determine the roles of Argonaute (Ago) family proteins in HDL-miRNA export, INS1 cells were transfected with siRNAs against Eif2c1-4 to knockdown Ago1-4. We found HDL-miR-375 export to be suppressed when Ago1, but not Ago2-4, were inhibited, suggesting that miRNA export is downstream of miRNA processing by Ago1. We are currently investigating the relationship between HDL-miR-375 export, insulin secretion, and miRNA processing in pancreatic beta cells to elucidate the mechanism(s) controlling HDL-miR-375 export. Collectively, results suggest that a large fraction of HDL-miRNAs originate from pancreatic beta cells and HDL-miRNAs are exported independent of cholesterol transporters.

Time to rejuvenate ultra-low dose whole-body radiotherapy of cancer.

Glycolytic enzyme Enolase-1 regulates insulin gene expression in pancreatic β-cell

The pancreatic islets are composed of discrete hormone-producing cells that orchestrate systemic glucose homeostasis. Here we identify subsets of beta cells using a single-cell transcriptomic approach. One subset of beta cells marked by high CD63 expression is enriched for the expression of mitochondrial metabolism genes and exhibits higher mitochondrial respiration compared with CD63lo beta cells. Human and murine pseudo-islets derived from CD63hi beta cells demonstrate enhanced glucose-stimulated insulin secretion compared with pseudo-islets from CD63lo beta cells. We show that CD63hi beta cells are diminished in mouse models of and in humans with type 2 diabetes. Finally, transplantation of pseudo-islets generated from CD63hi but not CD63lo beta cells into diabetic mice restores glucose homeostasis. These findings suggest that loss of a specific subset of beta cells may lead to diabetes. Strategies to reconstitute or maintain CD63hi beta cells may represent a potential anti-diabetic therapy.

Type 2 diabetes results from inadequate insulin production to control blood glucose. To address this need for replacing lost insulin-producing beta cells, most therapeutic development strategies are focused on enhancing functional beta cell number. Fairbanks Pharmaceuticals is pursuing therapies that enhance beta cell function and regeneration and is based on prior research in which inactivation of follistatin-like-3 (FSTL3) resulted in enlarged islets, increased beta cell number, and enhanced insulin secretion. At least some of these new beta cells arose from alpha to beta cell transdifferentiation in FSTL3 knockout mice. Fairbanks has produced a monoclonal antibody (FP-101) that can completely block FSTL3 from binding activin A and B, GDF11, and myostatin that could also disrupt pre-formed and otherwise irreversible complexes. FP-101 was also able to restore glucose-responsive insulin secretion in otherwise non-functional islets taken from HFD-fed mice. To determine if FSTL3 neutralization could be effective in humans, human islets from normal donors were first exposed to elevated glucose (25 nM) for 48-72 hours, a commonly utilized model for glucotoxicity. As expected, this caused loss of glucose response to subsequent GSIS. However, when treated for 24 hours with FP-101, GSIS was restored, indicating that neutralization of FSTL3 might be an effective strategy for restoring function to human non-functional islets. This observation agrees with previous research in which activin treatment directly restored GSIS in human islets from diabetic donors. Taken together with the potential for induction of alpha to beta cell transdifferentiation, these results suggest that neutralization of FSTL3 may have a dual benefit with acute restoration of GSIS in non-functional islets and chronic treatment leading to restoration of beta cell number through enhancement of alpha to beta cell transdifferentiation. In vivo studies will be initiated shortly. Disclosure A. Schneyer: Stock/Shareholder; Self; Fairbanks Pharmaceuticals, Inc.. Stock/Shareholder; Spouse/Partner; Fairbanks Pharmaceuticals, Inc.. Employee; Self; Fairbanks Pharmaceuticals, Inc.. M. Brown: None.

The receptor tyrosine kinase, c-Kit, and its ligand, stem cell factor, control a variety of cellular processes, including pancreatic beta cell survival and differentiation as revealed in c-Kit ( Wv ) mice, which have a point mutation in the c-Kit allele leading to loss of kinase activity and develop diabetes. The present study further investigated the intrinsic role of c-Kit in beta cells, especially the underlying mechanisms that influence beta cell function. We generated a novel transgenic mouse model with c-KIT overexpression specifically in beta cells (c-KitβTg) to further examine the physiological and functional roles of c-Kit in beta cells. Isolated islets from these mice were used to investigate the underlying molecular pathway of c-Kit in beta cells. We also characterised the ability of c-Kit to protect animals from high-fat-diet-induced diabetes, as well as to rescue c-Kit ( Wv ) mice from early onset of diabetes. c-KitβTg mice exhibited improved beta cell function, with significantly improved insulin secretion, and increased beta cell mass and proliferation in response to high-fat-diet-induced diabetes. c-KitβTg islets exhibited upregulation of: (1) insulin receptor and IRSs; (2) Akt and glycogen synthase kinase 3β phosphorylation; and (3) transcription factors important for islet function. c-KIT overexpression in beta cells also rescued diabetes observed in c-Kit ( Wv ) mice. These findings demonstrate that c-Kit plays a direct protective role in beta cells, by regulating glucose metabolism and beta cell function. c-Kit may therefore represent a novel target for treating diabetes.

BackgroundIslet transplantation is a recommended treatment for type 1 diabetes but is limited by donor organ shortage. This study introduces an innovative approach for improving the differentiation and functionality of insulin-producing cells (IPCs) from iPSCs using 3D spheroid formation and hydrogel matrix as an alternative pancreatic islet source. The extracellular matrix (ECM) is crucial for pancreatic islet functionality, but finding the ideal matrix for β-cell differentiation has been challenging. We aimed to advance IPC differentiation and maturation through an esterified collagen hydrogel, comparing its effectiveness with conventional basement membrane extract (BME) hydrogels.MethodsiPSCs were differentiated into IPCs using a small molecule-based sequential protocol, followed by spheroid formation in concave microwells. Rheological analysis, scanning electron microscopy, and proteomic profiling were used to characterize the chemical and physical properties of each matrix. IPCs, both in single-cell form and as spheroids, were embedded in either ionized collagen or BME hydrogels, which was followed by assessments of morphological changes, pancreatic islet-related gene expression, insulin secretion, and pathway activation using comprehensive analytical techniques.ResultsEsterified collagen hydrogels markedly improved the structural integrity, insulin expression, and cell-cell interactions in IPC spheroids, forming densely packed insulin-expressing clusters, in contrast to the dispersed cells observed in BME cultures. Collagen hydrogel significantly enhanced the mRNA expression of crucial endocrine markers and maturation factors, with IPC spheroids showing accelerated differentiation from day 5, suggesting a faster differentiation compared to single cells in hydrogel encapsulation. Insulin secretion in response to glucose in collagen environments, with a GSIS index of 2.46 ± 0.05, exceeded those in 2D and BME, demonstrating superior pancreatic islet functionality. Pathway analysis highlighted enhanced insulin secretion capabilities, evidenced by the upregulation of genes like Secretogranin III and Chromogranin A in collagen cultures. In vivo transplantation results showed that collagen hydrogel enhanced cluster integrity, tissue integration, and insulin secretion compared to non-embedded IPCs and BME groups.ConclusionEsterified collagen hydrogels demonstrated superior efficacy over 2D and BME in promoting IPC differentiation and maturation, possibly through upregulation of the expression of key secretion pathway genes. Our findings suggest that using collagen hydrogels presents a promising approach to enhance insulin secretion efficiency in differentiating pancreatic β-cells, advancing cell therapy in diabetes cell therapy.

Gestational diabetes mellitus (GDM) is one of the most common pregnancy complications characterized by insulin resistance and pancreatic β-cell dysfunction. Increasing evidence suggests that microRNAs (miRNAs) play key roles in the diverse types of diabetes, including GDM. However, the underlying mechanisms remain largely unknown. The purpose of this study is to investigate the role of microRNAs in GDM. The microarray data of dysregulated miRNAs in blood and placenta was retrieved in the GEO dataset under the accession number GSE19649. Quantitative reverse transcription PCR (qRT-PCR) was performed to analyze the expression levels of miR-494 in peripheral blood from twenty pairs of gestational diabetes (GDM) women and healthy women. Then, we investigated the effects of miR-494 on the insulin secretion of pancreatic β-cells. Moreover, the role of this miR-494 in regulating the proliferation and apoptosis of pancreatic β-cells were determined by MTT assay and flow cytometry, respectively in INS1 cells transfected with a miR-494 mimic or inhibitor. In addition, the direct target of miR-494 was confirmed using 3' untranslated region (UTR) luciferase reporter assay. Our data demonstrated that the miR-494 level was significantly decreased in the blood of GDM patients, and the low level was associated with a high concentration of blood glucose. Furthermore, overexpression of miR-494 improved pancreatic β-cell dysfunction by enhancing insulin secretion and total insulin content, inducing cell proliferation, and inhibiting cell apoptosis, whereas miR-494 knockdown exhibited decreased insulin secretion and proliferation, as well as stimulated apoptosis. In addition, phosphatase and tensin homolog (PTEN) which has been shown to play a pivotal role in apoptosis, was proved to be a direct target of miR-494 in pancreatic β-cells. More importantly, siRNA-induced downregulation of PTEN reversed the effects of miR-494 knockdown on insulin secretion, cell proliferation, and apoptosis of pancreatic β-cells.

Eugenosedin-A improves obesity-related hyperglycemia by regulating ATP-sensitive K+ channels and insulin secretion in pancreatic β cells

Role of the Sik2-p35-Pja2 Complex in Pancreatic Beta Cell Functional Compensation