Islet Cell Lines Research Articles

P11-043-23 High 5-Methyltetrahydrofolate Concentrations Impair Glucose-Stimulated Insulin Secretion in a Pancreatic Islet Cell Line

P11-043-23 High 5-Methyltetrahydrofolate Concentrations Impair Glucose-Stimulated Insulin Secretion in a Pancreatic Islet Cell Line

Role of Thyrotropin-Releasing Hormone in Regulating Fibroblast Growth Factor 21 in Mouse Pancreatic β Cells.

Background: Thyrotropin-releasing hormone (TRH) is primarily produced in the hypothalamus and regulates the thyrotropin secretion from the pituitary. TRH is distributed ubiquitously in the extrahypothalamic region, especially in pancreatic islets, while its physiological role remains nebulous. We have previously established a TRH-deficient mouse model, and showed impaired glucose tolerance and downregulated expression of fibroblast growth factor 21 (FGF21) in islets. Recent studies have demonstrated the physiological roles of pancreatic FGF21. Therefore, in this study, we elucidate the direct functions of TRH in pancreatic islets via the regulation of FGF21. Methods: To explore the functions of TRH in pancreatic islets, a microarray analysis using isolated islets from TRH-knockout mice was conducted. The regulatory mechanism of TRH in pancreatic FGF21 was investigated using islet cell lines; reverse transcription-quantitative polymerase chain reaction and Western blotting were used to determine the mRNA and protein expression levels of FGF21 in pancreatic islets and islet cell lines. Induction of FGF21 expression by TRH treatment was examined in vitro. To identify the transcription factors binding to the region responsible for TRH-induced stimulation of the FGF21 promoter, electromobility shift assays were conducted. Results: Among the detected and considerably changed genes in microarray, FGF21 was the most consistently downregulated in TRH-deficient mice islets. FGF21 was strongly co-expressed with insulin in mouse islets, and TRH stimulated endogenous Fgf21 mRNA expression in the islet cell line βHC9. The E-box site in the FGF21 promoter was responsible for TRH-induced stimulation via the extracellular signal-regulated kinase (ERK)1/2 signaling pathway. The transcription factor upstream stimulatory factor 1 (USF1) could specifically bind to the E-box site. Overexpression of USF1 increased FGF21 promoter activity. Conclusion: FGF21 was transcriptionally upregulated by TRH through the ERK1/2 and USF1 pathways in pancreatic β cells.

3D-printed polyurethane immunoisolation bags with controlled pore architecture for macroencapsulation of islet clusters encapsulated in alginate gel.

Diabetes mellitus is a fast-growing chronic metabolic condition caused by insulin deficiency or resistance, leading to lifelong insulin use. It has become one of the world's most difficult non-communicable diseases. The goal of this study was to view the effectiveness of the combined method of macro- and microencapsulation for islet transplantation. The process of 3D printing is used to make macroencapsulation bags with regulated diffusion properties thanks to the emerging small pored channels. The ink used to manufacture 3D-printed bags with controlled specifications was polyurethane solution(13% w/v). Swelling experiments revealed that there was very little swelling and that the membrane maintained its structural stability. Alginate beads (made from 5% w/v solution) were used to microencapsulate islet cell clusters. Direct contact assay was used to confirm in vitro cytocompatibility. The insulin release from the encapsulated rabbit islets was confirmed using a glucose challenge assay. When challenged with 20mM glucose on day 7, the encapsulated islet cells released insulin at a rate of 9.72 ± 0.65mU/L, which was identical to the RIN-5F islet cell line control, confirming the functioning of the encapsulated islets. After 21days of culture, the islets were shown to be viable utilizing a live-dead assay. As a result, our work demonstrates that 3D printing for macroencapsulating cells, as well as microencapsulation with alginates, is a viable scale-up technology with great potential in the field of pancreatic islet transplantation.

Jiangtang Sanhuang tablet inhibits endoplasmic reticulum stress and autophagy in diabetic mouse islet cells

To investigate effects of Jiangtang Sanhuang tablet (JTSHT) for regulating blood glucose and alleviating islet cell damage in db/db mice and its protective effects against endoplasmic reticulum stress (ERS) and autophagy induced by glycolipid toxicity. Forty db/db mice were randomized into 4 groups for daily intragastric administration of saline, JTSHT of 2.64 and 1.32 g/kg, and metformin at 0.225g/kg for 8 weeks, using 10 C57BL/6J mice as the normal control. After the treatments, the metabolic indexes of the mice were measured, and morphological changes of the islet cells were observed. A mouse islet cell line (MIN6) was exposed to high glucose (22 mmol/L glucose) and 0.1 mmol/L palmitic acid, followed by treatment with the sera from JTSHT- or saline- treated SD rats, alone or in combination with SP600125, and the changes in cell apoptosis, ERS and autophagy were evaluated using flow cytometry, RT-qPCR and Western blotting. In db/db mice, treatment with JTSHT significantly improved glucose and lipid metabolism (P < 0.05) and suppressed progressive weight gain (P < 0.05) without significant effect on drinking water volume (P > 0.05). JTSHT was also found to promote repair of islet cell injuries. In the cell experiments, high glucose exposure significantly increased apoptosis rate of MIN6 cells (P < 0.05), which was obviously lowered by treatment with JTSHT-treated rat serum (P < 0.05). Western blotting showed that JTSHT significantly reduced the level of ERS and autophagy caused by glycolipid toxicity in MIN6 cells (P < 0.05). Interference with ERS using SP600125 significantly attenuated the protective effect of JTSHT against MIN6 cell injury, apoptosis and autophagy induced by glycolipid toxicity (P < 0.05). JTSHT has protective effects against glycolipid toxicity in MIN6 cells possibly by inhibiting ERS and autophagy.

SORLA mediates endocytic uptake of proIAPP and protects against islet amyloid deposition

ObjectiveSorting-related receptor with type A repeats (SORLA) is a neuronal sorting receptor that prevents accumulation of amyloid-beta peptides, the main constituent of senile plaques in Alzheimer disease. Recent transcriptomic studies show that SORLA transcripts are also found in beta cells of pancreatic islets, yet the role of SORLA in islets is unknown. Based on its protective role in reducing the amyloid burden in the brain, we hypothesized that SORLA has a similar function in the pancreas via regulation of amyloid formation from islet amyloid polypeptide (IAPP). MethodsWe generated human IAPP transgenic mice lacking SORLA (hIAPP:SORLA KO) to assess the consequences of receptor deficiency for islet histopathology and function in vivo. Using both primary islet cells and cell lines, we further investigated the molecular mechanisms whereby SORLA controls the cellular metabolism and accumulation of IAPP. ResultsLoss of SORLA activity in hIAPP:SORLA KO resulted in a significant increase in islet amyloid deposits and associated islet cell death compared to hIAPP:SORLA WT animals. Aggravated islet amyloid deposition was observed in mice fed a normal chow diet, not requiring high-fat diet feeding typically needed to induce islet amyloidosis in mouse models. In vitro studies showed that SORLA binds to and mediates the endocytic uptake of proIAPP, but not mature IAPP, delivering the propeptide to an endolysosomal fate. ConclusionsSORLA functions as a proIAPP-specific clearance receptor, protecting against islet amyloid deposition and associated cell death caused by IAPP.

Phosphoproteome reveals molecular mechanisms of aberrant rhythm in neurotransmitter-mediated islet hormone secretion in diabetic mice.

BackgroundAcetylcholine (ACh) and norepinephrine (NE) are representative neurotransmitters of parasympathetic and sympathetic nerves, respectively, that antagonize each other to coregulate internal body functions. This also includes the control of different kinds of hormone secretion from pancreatic islets. However, the molecular mechanisms have not been fully elucidated, and whether innervation in islets is abnormal in diabetes mellitus also remains unclear.Methods and resultsImmunofluorescence colocalization and islet perfusion were performed and the results demonstrated that ACh/NE and their receptors were highly expressed in islet and rapidly regulated different hormones secretion. Phosphorylation is considered an important posttranslational modification in islet innervation and it was identified by quantitative proteomic and phosphoproteomic analyses in this study. The phosphorylated islet proteins were found involved in many biological and pathological processes, such as synaptic signalling transduction, calcium channel opening and insulin signalling pathway. Then, the kinases were predicted by motif analysis and further screened and verified by kinase‐specific siRNAs in different islet cell lines (αTC1‐6, Min6 and TGP52). After functional verification, Ksr2 and Pkacb were considered the key kinases of ACh and NE in insulin secretion, and Cadps, Mlxipl and Pdcd4 were the substrates of these kinases measured by immunofluorescence co‐staining. Then, the decreased expression of receptors, kinases and substrates of ACh and NE were found in diabetic mice and the aberrant rhythm in insulin secretion could be improved by combined interventions on key receptors (M3 (pilocarpine) or α2a (guanfacine)) and kinases (Ksr2 or Pkacb).ConclusionsAbnormal innervation was closely associated with the degree of islet dysfunction in diabetic mice and the aberrant rhythm in insulin secretion could be ameliorated significantly after intervention with key receptors and kinases in the early stage of diabetes mellitus, which may provide a promising therapeutic strategy for diabetes mellitus in the future.

Human and Rodent Cell Lines as Models of Functional Melatonin-Responsive Pancreatic Islet Cells.

Cell culture of different pancreatic islet cell lines, like the murine α-cell line αTC1.9, the rat β-cell lines INS-1 and INS-1832/13, and the human δ-cell line QGP-1, can serve as valuable cell models for the analysis of melatonin-dependent modulation of hormone secretion. The paper summarizes in detail the requirements of culture for each cell line and includes batch protocols to stimulate hormone secretion and to treat cells with several melatonin concentrations as previously published. We here describe the processing of collected cell pellets or cell culture supernatants as well as different methods to analyze cell experiments after melatonin treatment on the basis of our own experience. Finally, we outlined for each cell line under which conditions the melatonin treatment should be performed to gain reproducible results.

Food-derived cyanidin-3-O-glucoside alleviates oxidative stress: evidence from the islet cell line and diabetic db/db mice.

Type 2 diabetes mellitus is a disease associated with an oxidative milieu that often leads to adverse health outcomes. Multiple anthocyanins have been reported to possess outstanding antioxidant activity, however, their effects on hyperglycemia-related oxidative stress remain elusive. In the present study, cyanidin-3-O-glucoside (C3G), a typical anthocyanin with various widely accepted health benefits, was applied to alleviate oxidative stress in pancreas islets under the conditions of hyperglycemia. Firstly, significantly decreased mitochondrial membrane potential (MMP) and antioxidant enzymes, as well as increased reactive oxygen species (ROS) and O2- levels, were detected after exposure to a series of concentrations of high glucose (HG) and palmitic acid (PA), which manifested oxidative stress triggered by mitochondrial damage. To evaluate the antioxidant effect of C3G in vitro, the islet cell line NIT-1 was used, and results proved that C3G could effectively relieve cellular oxidative stress induced by HG and PA. Furthermore, we found that the antioxidant effect of C3G was achieved by activating mitophagy via the PINK1-PARKIN signaling pathway. More importantly, an autophagy inhibitor chloroquine (CQ) was added to verify our findings at the protein level, and we observed the co-localization of mitochondria and lysosomes, which may form autophagolysosomes to clean damaged mitochondria. Immediately afterwards, more studies were conducted on pancreatic islets of diabetic db/db mice to verify the antioxidant effect of C3G discovered in islet cells. Along with the decline in fasting blood glucose, the oxidative stress in pancreas islets was successfully alleviated in diabetic db/db mice after supplementation with C3G. This was demonstrated by increased levels of ROS, and the impaired activities of anti-oxidative enzymes superoxide dismutase (SOD) and catalase (CAT) were partly reversed by C3G intervention. Our study has provided evidence for the alleviation effect of C3G against oxidative stress in pancreas islets, which may provide enlightenment for improving the health situation of diabetic patients in the future.

The RNA-binding protein, HuD regulates proglucagon biosynthesis in pancreatic α cells

Glucagon is a peptide hormone generated by pancreatic α cells. It is the counterpart of insulin and plays an essential role in the regulation of blood glucose level. Therefore, a tight regulation of glucagon levels is pivotal to maintain homeostasis of blood glucose. However, little is known about the mechanisms regulating glucagon biosynthesis. In this study, we demonstrate that the RNA-binding protein HuD regulates glucagon expression in pancreatic α cells. HuD was found in α cells from mouse pancreatic islet and mouse glucagonoma αTC1 cell line. Ribonucleoprotein immunoprecipitation analysis, followed by RT-qPCR showed the association of HuD with glucagon mRNA. Knockdown of HuD resulted in a reduction in both proglucagon expression and cellular glucagon level by decreasing its de novo synthesis. Reporter analysis using the EGFP reporter containing 3′ untranslated region (3′UTR) of glucagon mRNA showed that HuD regulates proglucagon expression via its 3′UTR. In addition, the relative level of glucagon in the islets and plasma was lower in HuD knockout (KO) mice compared to age-matched control mice. Taken together, these results suggest that HuD is a novel factor regulating the biosynthesis of proglucagon in pancreatic α cells.

The Secretome of Mesenchymal Stem Cells Prevents Islet Beta Cell Apoptosis via an IL-10-Dependent Mechanism

Background:Type 1 Diabetes Mellitus (T1DM) is partly driven by autoimmune destruction of the pancreatic beta cell, facilitated by the release of inflammatory cytokines, including IFN-γ, TNF-α and IL-1β by cells of the innate immune system. Mesenchymal Stem Cells (MSCs) have been used to counteract autoimmunity in a range of therapeutic settings due to their secretion of trophic and immunomodulatory factors that ameliorate disease independently of the cells themselves.Objective:The aim of this study was to assess the effect of the secretome of human bone-marrow derived MSCs on cytokine-driven beta cell apoptosis.Methods:All experiments were conducted in two insulin-secreting islet cell lines (BRIN-BD11 and βTC1.6) with selected experiments confirmed in primary islets. MSC secretome was generated by conditioning serum-free media (MSC-CM) for 24 hours on sub-confluent MSC populations. The media was then removed and filtered in readiness for use.Results:Exposure to IFN-γ, TNF-α and IL-1β induced apoptosis in cell lines and primary islets. The addition of MSC-CM to cell lines and primary islets partially reversed cytokine-driven apoptosis. MSC-CM also restored glucose-stimulated insulin secretion in cytokine-treated cell lines, which was linked to improved cell viability following from cytokine challenge. Characterization of MSC-CM revealed significant concentrations of IL-4, IL-10, PIGF and VEGF. Of these, IL-10 alone prevented cytokine-driven apoptosis. Furthermore, the inhibition of IL-10 through the addition of a blocking antibody reversed the anti-apoptotic effects of MSC-CM.Conclusion:Overall, the protective effects of MSC-CM on islet beta cell survival appear to be largely IL-10-dependent.

Adrenomedullin has a cytoprotective role against endoplasmic reticulum stress for pancreatic β-cells in autocrine and paracrine manners.

Aims/IntroductionPancreatic β‐cells are sensitive to endoplasmic reticulum (ER) stress, which has a major role in the context of β‐cell death. Adrenomedullin (ADM) has been shown to exert a cytoprotective effect under various pathophysiological conditions. Several studies have suggested that thiazolidinediones have protective effects on β‐cells. During the course to elucidate the molecular mechanisms by which pioglitazone prevents β‐cell death, ADM emerged as a candidate. Here, we studied the regulation of ADM expression, including the effects of pioglitazone, and its role in pancreatic islets.Materials and MethodsWe analyzed ADM expression in islet cell lines treated with pioglitazone. The effects of ER stress on ADM and ADM receptor expressions were investigated by analyzing thapsigargin‐treated MIN6 cells and islets isolated from Wfs1−/− and db/db mice. To study the anti‐apoptotic effect of ADM, ER stress‐exposed MIN6 cells were treated with ADM peptides or transfected with ADM expression plasmid.ResultsPioglitazone increased the production and secretion of ADM in islets through peroxisome‐proliferator activated receptor‐γ‐dependent mechanisms. Thapsigargin treatment increased expressions of both ADM and ADM receptor, composed of Ramp2, Ramp3 and Crlr, in MIN6 cells. ADM and ADM receptor expressions were also increased in isolated islets from Wfs1−/− and db/db mice. ADM peptides and ADM overexpression protected MIN6 cells from thapsigargin‐induced apoptosis.ConclusionsER stress stimulates ADM production and secretion in islets. ADM signaling might protect β‐cells from ER stress‐induced apoptosis, and might be one of the self‐protective mechanisms. β‐Cell protection by pioglitazone is partly through induction of ADM. ADM‐based therapy could be a novel strategy for treating diabetes.

Stress-Associated Endoplasmic Reticulum Protein 1 Protected High Glucose-Induced Islet β Cells from Apoptosis by Attenuating Endoplasmic Reticulum Stress

The incidence of type II diabetes caused by islet cell injury is increasing in recent years. Endoplasmic reticulum stress is one of the crucial causes of islet β cell damage, and stress-associated endoplasmic reticulum protein 1 (SERP1) could inhibit the occurrence and development of endoplasmic reticulum stress. But whether SERP1 could inhibit the damage of islet β cell caused by endoplasmic reticulum stress is unclear. In this study, we detected the levels of SERP1 and endoplasmic reticulum stress related proteins (p-PERK, p-Eif2 α, ATF-4 and CHOP) by western blotting. Next the lentivirus was used to construct the islet cell line which was stable overexpressed SERP1. Then the expression of endoplasmic reticulum stress related proteins and inflammatory factors was determined with western blotting. At last the apoptosis rates of islet β cells were detected by flow cytometry. We found that high glucose medium promoted the expression of p-PERK, p-Eif2 α, ATF-4 and CHOP while downregulated the levels of SERP1 in isletβ cells. Moreover, overexpression of SERP1 induced the downregulation of levels of p-PERK, p-Eif2 α, ATF-4, CHOP, TNF-α , IL-1β and IL-6 and alleviated the apoptosis of islet cells. At last, the overexpression of CHOP rescued the apoptosis rates and the expression of TNF-α, IL-1β and IL-6. These results indicated SERP1 relieved the inflammation response and apoptosis of islet β cells by inhibiting the expression of CHOP and alleviating the endoplasmic reticulum stress induced damage.

LncRNA53106 regulates CXCL10 and affects the apoptosis of islet β cells

Objective To investigate the regulatory mechanism of long non-coding RNA (lncRNA) 53106 in the apoptosis model of MIN6 cells stimulated by cytokines. Methods The stimulation model of cytokines 10 ng/ml interleukin-1β, 50 ng/ml tumor necrosis factor-α, 50 ng/ml interferon-γ in MIN6 islet cell lines were established. The apoptosis rate was measured by flow cytometry and the expression levels of lncRNA53106 and (C-X-C motif) ligand (CXCL)10 were detected by realtime quantitative PCR (qPCR). LncRNA53106 smart silencer and CXCL10 siRNA were constructed, and lncRNA53106 and CXCL10 were knocked down respectively. Then inflammatory cytokines were combined to stimulate, and their roles in the apoptosis of min6 cells were detected by flow cytometry, qPCR, and Western blotting. Results In the apoptosis model of MIN6 cells stimulated by cytokines, the apoptosis rate of cytokines group was significantly increased and reached statistical significance. The apoptosis rate of the knocked down lncRNA53106 group was significantly lower than that of the control group (P<0.05). The expression of CXCL10 was also decreased in the knockdown group by qPCR and Western blotting, the expressions of the apoptosis-related factors Bax and Caspase3 mRNA were decreased. The apoptosis rate in the knocked down CXCL10 group was significantly lower than that in the control group (P<0.05), and the expression of lncRNA53106 was slightly increased, but the difference was not significant (P=0.61). Conclusion LncRNA53106 may promote the expression of apoptosis factor by upregulation of CXCL10, and promote the apoptosis of β cells of the pancreas, which may lead to the occurrence of type 1 diabetes. Key words: IncRNA53106; CXCL10; Islet β cell apoptosis; Diabetes mellitus, type 1

Enhanced Secretion of Functional Insulin with DNA-Functionalized Gold Nanoparticles in Cells.

We have previously shown the use of gold nanoparticles (AuNPs) functionalized with DNA (AuNP-DNA) to increase insulin mRNA translation in a cell-free system. In this study, we translate the concept into a whole cell system to demonstrate functionality despite the additional complexity of intracellular delivery and mRNA translation inside living cells. We selected an insulin-secreting pancreatic islet cell line, RIN-5F, as our model and designed a DNA oligomer (insDNA) that is complementary to the 3'-untranslated region of insulin mRNA for conjugation to AuNPs (AuNP-insDNA). AuNP-insDNA was stable in the extracellular environment of RIN-5F cells for up to 24 h, without eliciting any cell toxicity. Upon cellular entry, AuNP-insDNA was able to sustain enhanced insulin secretion from 6 to 12 h post-incubation, peaking at 10 h with an enhancement factor of 1.69-fold. This enhancement was not observed when insDNA was removed or replaced with poly thymine or poly adenine DNAs. The enhanced insulin secreted was 100% functional and capable of binding to its insulin receptor. The outcome of this study demonstrated the feasibility of AuNP-DNA to enhance the synthesis of proteins in whole cells and could serve as a new direction of invoking a patient's own beta cells to increase insulin secretion for treatment of diabetes.

Research progress on the donor cell sources of pancreatic islet transplantation for treatment of diabetes mellitus

To summarize the research progress on the source and selection of donor cells in the field of islet replacement therapy for diabetes mellitus. Domestic and abroad literature concerning islet replacement therapy for diabetes mellitus, as well as donor source and donor selection was reviewed and analyzed thoroughly. The shortage of donor supply is still a major obstacle for the widely clinical application of pancreatic islet transplantation (PIT). Currently, in addition to the progress on the allogeneic/autologous donor islet supply, some remarkable achievements have been also attained in the application of xenogeneic islet (from pig donor), as well as islet like cells derived from stem cells and islet cell line, potentially enlarging the source of implantable cells. Adequate and suitable donor cell supply is an essential prerequisite for widely clinical application of PIT therapy for type 1 diabetes mellitus (T1DM). Further perfection of organ donation system, together with development of immune-tolerance induction, gene and bioengineering technology etc. will possibly solve the problem of donor cell shortage and provide a basis for clinical application of cellular replacement therapy for T1DM.

Islet β cell: An endocrine cell secreting miRNAs

The pancreas is an important endocrine organ. Pancreatic beta (β) cells secrete insulin to regulate the metabolism of glucose and maintain the stability of blood glucose. MicroRNAs (miRNAs), small (20-25 nt) non-coding RNA molecules, function in target gene silencing through post-transcriptional patterns, and multiple miRNAs regulate insulin secretion, insulin signaling pathways in pancreatic β cells and islet activity. In recent years, it has been found that many types of cells can secrete regulatory miRNA, but whether islet cells can secrete miRNA has not been clarified yet. In this study, we detected miRNA expressions in cell culture medium of primary mouse islet cells and islet cell line MIN6. And we found that islet cells can selectively and actively secrete miRNAs, mainly through the way of exosome package, and that miRNA secretion profiling patterns vary according to different insulin secretion stimulating conditions (high Glucose, high Kcl, high Arginine and high Free fat acid (FFA)). Additionally, we found that these miRNAs secreted by islet cells can be transported and have gene-regulating functions on recipient tissue cells, such as liver and muscles. In conclusion, we find another secretory function of the islet β cells: not only insulin, but also miRNAs.

Artemether Does Not Turn α Cells into β Cells

Pancreatic α cells retain considerable plasticity and can, under the right circumstances, transdifferentiate into functionally mature β cells. In search of a targetable mechanistic basis, a recent paper suggested that the widely used anti-malaria drug artemether suppresses the α cell transcription factor Arx to promote transdifferentiation into β cells. However, key initial experiments in this paper were carried out in islet cell lines, and most subsequent validation experiments implied transdifferentiation without direct demonstration of α to β cell conversion. Indeed, we find no evidence that artemether promotes transdifferentiation of primary α cells into β cells. Moreover, artemether reduces Ins2 expression in primary β cells >100-fold, suppresses glucose uptake, and abrogates β cell calcium responses and insulin secretion in response to glucose. Our observations suggest that artemether induces general islet endocrine cell dedifferentiation and call into question the utility of artemisinins to promote α to β cell transdifferentiation in treating diabetes.

Alpha TC1 and Beta-TC-6 genomic profiling uncovers both shared and distinct transcriptional regulatory features with their primary islet counterparts

Alpha TC1 (αTC1) and Beta-TC-6 (βTC6) mouse islet cell lines are cellular models of islet (dys)function and type 2 diabetes (T2D). However, genomic characteristics of these cells, and their similarities to primary islet alpha and beta cells, are undefined. Here, we report the epigenomic (ATAC-seq) and transcriptomic (RNA-seq) landscapes of αTC1 and βTC6 cells. Each cell type exhibits hallmarks of its primary islet cell counterpart including cell-specific expression of beta (e.g., Pdx1) and alpha (e.g., Arx) cell transcription factors (TFs), and enrichment of binding motifs for these TFs in αTC1/βTC6 cis-regulatory elements. αTC1/βTC6 transcriptomes overlap significantly with the transcriptomes of primary mouse/human alpha and beta cells. Our data further indicate that ATAC-seq detects cell-specific regulatory elements for cell types comprising ≥ 20% of a mixed cell population. We identified αTC1/βTC6 cis-regulatory elements orthologous to those containing type 2 diabetes (T2D)-associated SNPs in human islets for 33 loci, suggesting these cells’ utility to dissect T2D molecular genetics in these regions. Together, these maps provide important insights into the conserved regulatory architecture between αTC1/βTC6 and primary islet cells that can be leveraged in functional (epi)genomic approaches to dissect the genetic and molecular factors controlling islet cell identity and function.

Protective role of berberine and Coptischinensis extract on T2MD rats and associated islet Rin‑5f cells.

The aim of the present study was to compare the different effects of berberine (Ber) and Coptischinensis extract (CCE) on a rat model of type 2 diabetes mellitus (T2DM), and the islet Rin‑5f cell line was used to examine the differences between Ber and CCE and the underlying mechanisms. CCE was extracted and purified prior to analysis. Male Sprague‑Dawley rats were provided with a high‑fat diet to induce insulin resistance prior to injecting with streptozotocinto establish the T2DM model, the T2DM rats were treated with Ber and CCE, and blood samples and pancreatic tissues were obtained and compared to examine T2DM metabolic syndromes among the groups of rats, which included healthy rats, model rats, and model rats treated with Ber and CCE at different doses between 0 and 8weeks. The protective effects of Ber and CCE on the Rin‑5f islet cell line were also evaluated. The effects on Rin‑5f cell proliferation and cell cycle, glucose‑stimulated insulin release test (GSIS), the anti‑apoptotic effects caused by fat induction, and protein expression levels of poly ADP‑ribose polymerase (PARP‑1) were evaluated. The results showed that the content of the prepared CCE was 96.07% for five alkaloids. When it was used for treatment of the T2DM rats, compared with Ber, metformin and rosiglitazone, the fasting blood glucose, glucosylated serum protein (GSP) and glucose infusion rate indicesin the fasting rats were ameliorated, compared with those in the T2MD rats, with no significant differences between treatment with Ber or CCE and metformin or rosiglitazone. The indices of mean optical density and fasting β‑cell function index (FBCI) were different following treatment with Ber and CCE, compared with those in the model rats, which may have stimulated the pancreatic secretion of insulin. When Ber and CCE were used to examine the protective effects on Rin‑5F cells, it was found that the Rin‑5f cell GSIS, cell cycle, lipotoxic islet cell proliferation and protein expression of PARP‑1 were altered and improved, which may have protected pancreatic islet β‑cells by improving islet β‑cell proliferation and the protein expression of PARP‑1. CCE and Ber exerted similar effects when used for the treatment of T2MD rats, and may have stimulated the pancreatic secretion of insulin through the protective effect on islet β‑cells via improving islet β‑cell proliferation and the protein expression of PARP‑1.

Stress-impaired transcription factor expression and insulin secretion in transplanted human islets.

Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity.