Pancreatic Β-cell Function Research Articles

Perfluorooctane sulfonate-induced oxidative stress contributes to pancreatic β-cell apoptosis by inhibiting cyclic adenosine monophosphate pathway: Prevention by pentoxifylline

Endocrine-disrupting chemical perfluorooctane sulfonate (PFOS) acute exposure stimulates insulin secretion from pancreatic β-cells. However, chronic exposure to PFOS on pancreatic β-cells, its role in insulin secretion, and the underlying mechanisms have not been studied. We used rat insulinoma INS-1 and human 1.1b4 islet cells to investigate the chronic effects of PFOS on glucose-stimulated insulin secretion and toxicity implicated in the downregulation of β-cell functionality. Chronic exposure of INS-1 cells or human pancreatic 1.1b4 β-cells to PFOS stimulated the small G-protein RAC1-guanosine triphosphate-dependent nicotinamide adenine dinucleotide phosphate oxidase (NOX2/gp91phox) subunit expression and activation. Upregulated NOX2/gp91phox activation led to elevated reactive oxygen species (ROS) production with a decrease in the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway in both cell types. Inhibition of cAMP/PKA signaling induces β-cell mitochondrial dysfunction and endoplasmic stress via the loss of PDX1-SERCA2B and glucose-stimulated insulin release. Inhibiting RAC1-NOX2/gp91phox activation or elevating cAMP by pentoxifylline, a Food and Drug Administration-approved phosphodiesterase inhibitor, significantly reduced PFOS-induced ROS production and restored insulin secretory function of pancreatic β-cells. Enhanced secretory function in pentoxifylline-treated cells was associated with increased stability of PDX1-SERCA2B protein levels. Intriguingly, inhibition of cAMP/PKA signaling impaired pentoxifylline-induced insulin secretion caused by the activation of ROS production and mitochondrial dysfunction. Overall, our findings show that PFOS has a new and first-ever direct chronic effect on pancreatic β-cell failure through increased RAC1-NOX2/gp91phox activation and pentoxifylline-induced cAMP/PKA signaling, which inhibits PFOS-mediated mitochondrial dysfunction.

Large yellow tea polysaccharides ameliorate obesity-associated metabolic syndrome by promoting M2 polarization of adipose tissue macrophages.

Obesity-induced metabolic syndrome is strongly associated with infiltrated adipose tissue macrophages (ATMs). Large yellow tea, a traditional functional beverage in China, has been shown to possess anti-obesity effects. However, the effect of large yellow tea polysaccharides (LYPs) against obesity-associated metabolic syndrome and their underlying mechanisms remain unclear and must be extensively investigated. In this study, we investigated the ameliorative effect of LYPs on metabolic syndrome using a high-fat diet (HFD)-induced obese mouse model. Our results indicated that LYPs significantly alleviated weight gain, dyslipidemia, glucose intolerance, and insulin resistance. Moreover, LYPs restored the homeostasis of energy metabolism and pancreatic β-cell function. Notably, LYPs promoted M2 polarization of ATMs by regulating the expression of genes and specific cytokines involved in the assembly and secretion of M2 polarization. The improved metabolic syndrome of LYPs might be associated with the modulation of macrophage polarization. These findings suggest that LYPs might be a novel potential therapeutic agent to prevent or treat HFD-induced metabolic disorders by regulating M2 polarization.

Difference in the early clinical course between children with type 1 diabetes having a single antibody and those having multiple antibodies against pancreatic β-cells.

Islet-cell associated antibodies are predictive and diagnostic markers for type 1 diabetes. We studied the differences in the early clinical course of children with type 1 diabetes with a single antibody and those with multiple antibodies against pancreatic β-cells. Sixty-seven children with type 1 diabetes aged less than 15 years diagnosed between 2010 and 2021 were included in the study and subdivided into two subgroups: children who were single positive for either glutamic acid decarboxylase (GAD) antibodies (n = 16) or insulinoma-associated antigen-2 (IA-2) antibodies (n = 13) and those positive for both antibodies (n = 38) at diagnosis. We compared the patients' clinical characteristics, pancreatic β-cell function, and glycemic control during the 5 years after diagnosis. All clinical characteristics at diagnosis were similar between the two groups. One and two years after diagnosis, children who tested positive for both antibodies showed significantly lower postprandial serum C-peptide (CPR) levels than those who tested positive for either GAD or IA-2 antibodies (p < 0.05). In other periods, there was no significant difference in CPR levels between the two groups. There was a significant improvement in glycosylated hemoglobin (HbA1c) levels after starting insulin treatment in both groups (p < 0.05), but no significant difference in HbA1c levels between the groups. Residual endogenous insulin secretion may be predicted based on the number of positive islet-cell associated antibodies at diagnosis. Although there are differences in serum CPR levels, optimal glycemic control can be achieved by individualized appropriate insulin treatment, even in children with type 1 diabetes.

Islet-on-chip: promotion of islet health and function via encapsulation within a polymerizable fibrillar collagen scaffold.

The protection and interrogation of pancreatic β-cell health and function ex vivo is a fundamental aspect of diabetes research, including mechanistic studies, evaluation of β-cell health modulators, and development and quality control of replacement β-cell populations. However, present-day islet culture formats, including traditional suspension culture as well as many recently developed microfluidic devices, suspend islets in a liquid microenvironment, disrupting mechanochemical signaling normally found in vivo and limiting β-cell viability and function in vitro. Herein, we present a novel three-dimensional (3D) microphysiological system (MPS) to extend islet health and function ex vivo by incorporating a polymerizable collagen scaffold to restore biophysical support and islet-collagen mechanobiological cues. Informed by computational models of gas and molecular transport relevant to β-cell physiology, a MPS configuration was down-selected based on simulated oxygen and nutrient delivery to collagen-encapsulated islets, and 3D-printing was applied as a readily accessible, low-cost rapid prototyping method. Recreating critical aspects of the in vivo microenvironment within the MPS via perfusion and islet-collagen interactions mitigated post-isolation ischemia and apoptosis in mouse islets over a 5-day period. In contrast, islets maintained in traditional suspension formats exhibited progressive hypoxic and apoptotic cores. Finally, dynamic glucose-stimulated insulin secretion measurements were performed on collagen-encapsulated mouse islets in the absence and presence of well-known chemical stressor thapsigargin using the MPS platform and compared to conventional protocols involving commercial perifusion machines. Overall, the MPS described here provides a user-friendly islet culture platform that not only supports long-term β-cell health and function but also enables multiparametric evaluations.

A systematic review of medicinal plants and compounds for restoring pancreatic β-cell mass and function in the management of diabetes mellitus

Pollution Based Study of Heavy Metals in Medicinal plants Aloe vera and Tamarix aphyllaHamid Iqbal, Baharullah Khattak, Sultan Ayaz, Ali Rehman, Muhammad Ishfaq, Muhammad Naseer Abbas, Muhammad Saqib Malik, Abdul Wahab, Imran, Saifullah Mehsud

Soy yoghurts produced with efficient GABA (γ-aminobutyric acid)-producing Lactiplantibacillus plantarum ameliorate hyperglycaemia and re-establish gut microbiota in streptozotocin (STZ)-induced diabetic mice.

Soy yogurt has been gaining popularity as a vegan food produced simply by soymilk fermentation with proper microbial manipulation. It is well known that soy containing rich isoflavones is beneficial for ameliorating hyperglycaemic disorders. Soy fermentation can improve the bioavailability of these precious nutrients. Lactiplantibacillus plantarum is one of the most abundant and frequently isolated species in soymilk manufacturing. Soy yogurts produced with efficient GABA (γ-aminobutyric acid)-producing L. plantarum and the deglycosylating activity of L. plantarum were functionally assessed in a STZ-induced hyperglycaemic mouse model. Hyperglycaemic mice were assigned into groups and treated with daily gavage of either dH2O, soymilk, soy yoghurts produced with high GABA-producing L. plantarum GA30 (LPGA30), low GABA-producing L. plantarum PV30 (LPPV30) or the soy yoghurts fortified with additional 30 mg g-1 GABA counterparts (GA + GABA and PV + GABA groups). Except the dH2O group, all soy yoghurt groups retained body weight with improved glucose homeostasis, glucose tolerance test results and renal tissue integrity, while the soymilk group shows partial benefits. Plasma GABA concentrations in the daily soy yoghurt-supplemented groups (LPGA30 and LPPV30) plateaued at 5 times higher than the average 0.5 μM in dH2O and soymilk groups, and their GABA-fortified soy yoghurt counterparts (GA + GABA and PV + GABA) groups were accountable for the restored plasma insulin levels. Gut microbiome analysis revealed dysbiosis in STZ-induced hyperglycemic mice of the dH2O group with breached out facultative anaerobic Proteobacteria over the normal phyla Firmicutes and Bacteroidetes. Restored gut microbiota with transitionally populated Actinobacteria was demonstrated in the LPGA30 group but not in the LPPV30 group. Soy yoghurts produced with efficient GABA-producing L. plantarum GA30 showed exceptional benefits in modulating gut microbiota with dominant genera of Enterococcus, Lactobacillus and Bifidobacterium, and the presence of some minor beneficial microbial communities including Akkermansia muciniphila, Butyricicoccus pullicaecorum, Corynebacterium spp. and Adlercreutzia spp. Efficient GABA-producing L. plantarum GA30 fermented soymilk to produce soy yoghurts that exhibit profound synergistic protections over rich soy isoflavones to restore pancreatic β-cell functions for insulin production in STZ-induced hyperglycaemic mice. Additionally, the probiotic role of GABA-producing L. plantarum in re-establishing healthy gut microbiota in hyperglycaemic mice implies a possible symbiotic relationship, awaiting further exploration.

Decorin improves human pancreatic β-cell function and regulates ECM expression in vitro

Transplantation of islets of Langerhans is a promising alternative treatment strategy in severe cases of type 1 diabetes mellitus; however, the success rate is limited by the survival rate of the cells post-transplantation. Restoration of the native pancreatic niche during transplantation potentially can help to improve cell viability and function. Here, we assessed for the first time the regulatory role of the small leucine-rich proteoglycan decorin (DCN) in insulin secretion in human β-cells, and its impact on pancreatic extracellular matrix (ECM) protein expression in vitro. In depth analyses utilizing next-generation sequencing as well as Raman microspectroscopy and Raman imaging identified pathways related to glucose metabolism to be upregulated in DCN-treated cells, including oxidative phosphorylation within the mitochondria as well as proteins and lipids of the endoplasmic reticulum. We further showed the effectiveness of DCN in a transplantation setting by treating collagen type 1-encapsulated β-cell-containing pseudo-islets with DCN. Taken together, in this study, we demonstrate the potential of DCN to improve the function of insulin-secreting β-cells while reducing the expression of ECM proteins affiliated with fibrotic capsule formation, making DCN a highly promising therapeutic agent for islet transplantation.

PDX1 is the cornerstone of pancreatic β-cell functions and identity.

Diabetes has been a worldwide healthcare problem for many years. Current methods of treating diabetes are still largely directed at symptoms, aiming to control the manifestations of the pathology. This creates an overall need to find alternative measures that can impact on the causes of the disease, reverse diabetes, or make it more manageable. Understanding the role of key players in the pathogenesis of diabetes and the related β-cell functions is of great importance in combating diabetes. PDX1 is a master regulator in pancreas organogenesis, the maturation and identity preservation of β-cells, and of their role in normal insulin function. Mutations in the PDX1 gene are correlated with many pancreatic dysfunctions, including pancreatic agenesis (homozygous mutation) and MODY4 (heterozygous mutation), while in other types of diabetes, PDX1 expression is reduced. Therefore, alternative approaches to treat diabetes largely depend on knowledge of PDX1 regulation, its interaction with other transcription factors, and its role in obtaining β-cells through differentiation and transdifferentiation protocols. In this article, we review the basic functions of PDX1 and its regulation by genetic and epigenetic factors. Lastly, we summarize different variations of the differentiation protocols used to obtain β-cells from alternative cell sources, using PDX1 alone or in combination with various transcription factors and modified culture conditions. This review shows the unique position of PDX1 as a potential target in the genetic and cellular treatment of diabetes.

Risk Factors for Nonalcoholic Fatty Liver Disease with Different Insulin Resistance in a Nonobese Chinese Population.

The aim of this study is to identify the risk factors of nonobese nonalcoholic fatty liver disease (NAFLD) individuals under different insulin resistance status. This cross-sectional study was conducted at the Medical Center of Beijing Chaoyang Hospital affiliated with Capital Medical University. NAFLD was diagnosed based upon ultrasonographic findings consistent with fatty liver disease. A total of 1257 nonobese adults (625 non-NAFLD and 632 nonobese NAFLD) with body mass index (BMI) 18.5-24.9 kg/m2 were enrolled in the study. And all patients were divided into homeostasis model assessment of insulin resistance (HOMA - IR) > 1 group and HOMA - IR ≤ 1 group. When all the variables were adjusted in both the HOMA - IR > 1 group and HOMA - IR ≤ 1 group, older age (>50 years), higher BMI (23.0-24.9 kg/m2), higher AST (>18 U/L), higher TG (>0.9 mmol/L), higher GLU (>5.25 mmol/L), and higher HbA1C (>5.5%) were associated with higher risks of nonobese NAFLD. In patients with HOMA - IR > 1, lower homeostatic model assessment of β-cell function (HOMA-β) (<47.1%) (OR, 7.460, 95% CI, 3.051-18.238, P < 0.001) was associated with higher risks of nonobese NAFLD. s. Metabolic profiles (i.e., higher BMI, hyperglycemia, hypertriglyceridemia, and higher glycosylated hemoglobin) are risk factors of nonobese NAFLD, regardless of insulin resistance status. Decreased function of pancreatic β-cells may be the risk factor of nonobese NAFLD with insulin resistance, who should pay attention to further development of pancreatic β-cell dysfunction.

Circulating spexin levels are influenced by the glycemic status and correlated with pancreatic β-cell function in Chinese subjects.

Spexin plays a role in regulating glucose metabolism. This study investigated the spexin levels in different glycemic status and its association with insulin secretion in humans. A total of 462 subjects were recruited in this study, including 52 healthy subjects, 106 first-degree relatives (FDRs) of type 2 diabetes mellitus (T2DM), 115 impaired glucose regulation (IGR), 80 newly diagnosed T2DM, and 106 established T2DM. Serum spexin was measured using ELISA. The homeostasis model assessment of insulin resistance (HOMA2-IR) and β-cell function (HOMA2-β), and Stumvoll index estimating first- and second-phase insulin secretion were calculated. Spexin levels were higher in FDRs [235.53pg/ml (185.28, 293.95)] and IGR [239.79pg/ml (191.52, 301.69)], comparable in newly diagnosed T2DM [224.68pg/ml (187.37, 279.74)], and lower in established T2DM [100.11pg/ml (78.50, 137.34)], compared with healthy subjects [200.23pg/ml (160.32, 275.65)]. Spexin levels were negatively correlated with fasting plasma glucose (FPG) (r = - 0.355, P < 0.001), hemoglobin A1C (HbA1c) (r = - 0.379, P < 0.001), and HOMA2-IR (r = - 0.225, P < 0.001), and positively correlated with HOMA2-β (r = 0.245, P < 0.001) after adjusting for age, sex, and BMI. Multivariate linear regression analysis showed that established T2DM and HOMA2-β were independently associated with serum spexin levels. Serum spexin levels represented as a bell-shaped curve along the glycemic continuum and is closely related with insulin secretion in humans.

Vanillic acid potentiates insulin secretion and prevents pancreatic β-cells cytotoxicity under H2O2-induced oxidative stress.

Oxidative stress is known to impair cellular functions and, therefore, plays a significant role in the pathophysiology of various diseases, including diabetes. The persistently elevated glucose levels may cause enhanced mitochondrial reactive oxygen species generation, which in turn can damage the pancreatic β-cells. In this study, we have investigated the effect of vanillic acid on preventing H2O2-induced β-cells death and retaining its insulin secretion potentiating effect in the presence of H2O2. The insulin secretion from the BRIN-BD11 cells was quantified using ELISA-based assays. The viability of the cells was assessed by estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay and DAPI staining. The expression levels of apoptotic and antioxidant proteins were estimated by western blot experiments. Vanillic acid protected pancreatic β-cells viability and function under the H2O2 oxidative stress condition. The Erk1/2 activation appears to play an important role in vanillic acid potentiated insulin secretion and protection of the β-cells in the presence of H2O2. Vanillic acid pretreated cells exhibited enhanced expression of antioxidant enzymes such as catalase and SOD-2 and reduced the expression of proapoptotic markers such as BAX and BAD. In addition, it also enhanced the expression of oxidative stress-sensitive transcription factor Nrf-2 and cell survival protein Akt. The present study shows that vanillic acid potentiates insulin secretion and protects pancreatic β-cells from H2O2-induced oxidative stress.

Exenatide regulates Th17/Treg balance via PI3K/Akt/FoxO1 pathway in db/db mice

BackgroundThe T helper 17 (Th17)/T regulatory (Treg) cell imbalance is involved in the course of obesity and type 2 diabetes mellitus (T2DM). In the current study, the exact role of glucagon-like peptide-1 receptor agonist (GLP-1RA) exenatide on regulating the Th17/Treg balance and the underlying molecular mechanisms are investigated in obese diabetic mice model.MethodsMetabolic parameters were monitored in db/db mice treated with/without exenatide during 8-week study period. The frequencies of Th17 and Treg cells from peripheral blood and pancreas in db/db mice were assessed. The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/Forkhead box O1 (FoxO1) pathway in Th17 and Treg cells from the spleens of male C57BL/6J mice was detected by western blotting. In addition, the expression of glucagon-like peptide-1 receptor (GLP-1R) in peripheral blood mononuclear cells (PBMCs) of male C57BL/6J mice was analyzed.ResultsExenatide treatment improved β-cell function and insulitis in addition to glucose, insulin sensitivity and weight. Increased Th17 and decreased Treg cells in peripheral blood were present as diabetes progressed while exenatide corrected this imbalance. Progressive IL-17 + T cell infiltration of pancreatic islets was alleviated by exenatide intervention. In vitro study showed no significant difference in the level of GLP-1R expression in PBMCs between control and palmitate (PA) groups. In addition, PA could promote Th17 but suppress Treg differentiation along with down-regulating the phosphorylation of PI3K/Akt/FoxO1, which was reversed by exenatide intervention. FoxO1 inhibitor AS1842856 could abrogate all these effects of exenatide against lipid stress.ConclusionsExenatide could restore systemic Th17/Treg balance via regulating FoxO1 pathway with the progression of diabetes in db/db mice. The protection of pancreatic β-cell function may be partially mediated by inhibiting Th17 cell infiltration into pancreatic islets, and the resultant alleviation of islet inflammation.

Glucagon-like peptide-1 therapy for youth with type 2 diabetes.

Glucagon-like peptide-1 therapy for youth with type 2 diabetes.

Effect of chronic exposure to ketohexoses on pancreatic β-cell function in INS-1 rat insulinoma cells.

Glucotoxicity, impaired insulin secretion, suppression of insulin gene expression, and apoptosis, in pancreatic β-cells caused by chronic hyperglycemia is a key component of the pathogenesis of type 2 diabetes. Recently, it has been reported that rare sugar d-allulose has antihyperglycemic and antihyperlipidemic effects in diabetic rats. However, the direct effects of rare sugars including d-allulose on pancreatic β-cell function are unclear. In this study, we investigated whether chronic exposure to ketohexoses causes glucotoxicity, suppression of insulin gene expression, and apoptosis, in INS-1 rat pancreatic insulinoma cells. d-Fructose, d-tagatose, l-allulose, and l-sorbose treatment for 1-week reduced insulin gene expression, whereas d-allulose, d-sorbose, l-fructose, and l-tagatose did not. All ketohexoses were transported into INS-1 cells, but were not metabolized. In addition, the ketohexoses did not induce apoptosis and did not affect glucose metabolism. These results suggest that long-term administration of d-allulose, d-sorbose, l-fructose, and l-tagatose does not affect pancreatic β-cell function.

Insulin Secretion and β-Cell Function Are Preserved in Patients with Treated Hematological Malignancies

INTRODUCTION Cancer survivors are at elevated risk for developing type 2 diabetes mellitus, which decreases quality of life and increases mortality during cancer survivorship. Type 2 diabetes results from a combination of insulin resistance and decreased insulin secretion. Assessing pancreatic β-cell function by measuring insulin secretion dynamics in response to hyperglycemia can provide insight into the pathogenesis of type 2 diabetes. We investigated insulin secretion in patients with treated hematological malignancies and compared them to non-diabetic controls without cancer. We hypothesized that patients with hematological malignancies will have decreased insulin secretion compared to controls without cancer due to β-cell toxicity from previous treatment and chemotherapy. METHODS We evaluated insulin secretion in 16 non-diabetic adult patients with treated hematological malignancies using a hyperglycemic clamp, which assesses pancreatic β-cell function by measuring insulin secretion in response to hyperglycemia (200 mg/dL) maintained by a dextrose infusion. Clamp outcomes included first phase (T0-10 minutes), second phase (T90-120 minutes), and maximal (L-arginine stimulated) insulin response. The insulin sensitivity index (ISI) and disposition index (DI, a measure of the β-cell function to compensate for insulin resistance) were calculated. Controls (n=32) matched for age, sex, race, and body mass index (BMI) were selected 2:1 from a database of 154 hyperglycemic clamps in participants without diabetes or cancer. Demographic characteristics and clamp outcomes were compared using χ2, Wilcoxon rank-sum test, or linear regression. RESULTS Hyperglycemic clamps were performed in 48 individuals without diabetes (16 patients with treated hematological malignancies and 32 matched controls without cancer). Cancer survivors and controls without malignancy were well-matched for age, sex, race (Table 1). Myeloid malignancies were the most common diagnosis (n=11; 69%) with a median time from diagnosis to metabolic testing of 6 months. Treatment included cytotoxic chemotherapy (n=15; 94%), glucocorticoids (n=7; 44%), and radiation (n=1; 6%) with 7 (44%) patients achieving a remission prior to testing. Compared to controls, patients with malignancy had lower fasting glucose levels (93.0 mg/dL [IQR 86.3-96.6] vs 101.5 [IQR 94.9-107.6], p<0.001) and lower fasting insulin levels (11.5 uU/mL [IQR 9.3-15.0] vs 16.3 [IQR 12.3-21.5], p=0.023), suggesting greater insulin sensitivity within the cancer cohort. In univariable analysis, there were no significant differences between first and second phase insulin response between patients with or without treated hematological malignancies (Table 1). Cancer survivors had a higher maximal (L-arginine stimulated) insulin secretion (3606 uU/mL [IQR 3255-4594] vs 2278 [IQR 1900-3555], p=0.026) and a higher disposition index (2521 mg/kg/min [IQR 1986-3360] vs 1300 [IQR 939-1923], p=0.01) than in controls. Cancer survivors demonstrated a trend toward greater insulin sensitivity compared to controls (6.82 mg/kg/min per uU/mL [IQR 5.53-10.83] vs 4.98 [IQR 3.65-7.28], p=0.092). After adjustment for BMI and fasting glucose in linear regression models, both second phase (β=242.5, p=0.005) and maximal insulin secretion (β=1225.6, p=0.019) were greater in cancer survivors then in controls without cancer (Table 2). CONCLUSION Cancer survivors had higher maximal phase insulin secretion and disposition index (a measure of β-cell function adjusted for insulin resistance) compared to non-diabetic controls without cancer. Our data suggests that cancer survivors have preserved β-cell function following treatment of their hematological malignancies, contrary to our hypothesis. Longitudinal follow-up is needed to determine factors that promote diabetes susceptibility in cancer survivors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ferroptosis Signaling in Pancreatic β-Cells: Novel Insights & Therapeutic Targeting.

Metabolic stress impairs pancreatic β-cell survival and function in diabetes. Although the pathophysiology of metabolic stress is complex, aberrant tissue damage and β-cell death are brought on by an imbalance in redox equilibrium due to insufficient levels of endogenous antioxidant expression in β-cells. The vulnerability of β-cells to oxidative damage caused by iron accumulation has been linked to contributory β-cell ferroptotic-like malfunction under diabetogenic settings. Here, we take into account recent findings on how iron metabolism contributes to the deregulation of the redox response in diabetic conditions as well as the ferroptotic-like malfunction in the pancreatic β-cells, which may offer insights for deciphering the pathomechanisms and formulating plans for the treatment or prevention of metabolic stress brought on by β-cell failure.

RF04 | PSUN133 Assessment of the Rnf20 Histone Modifier in Regulating Pancreatic Beta-Cell Identity and Function

Abstract Type 1 and Type 2 Diabetes are characterized by a loss of functional pancreatic islet β-cells. With the prevalence of diabetes increasing worldwide, a complete understanding of the gene regulatory mechanisms governing the development and function of insulin-producing β-cells is requisite for improving diabetes therapies. Islet 1 (Isl1) is a LIM-homeodomain class transcription factor (TF) required for the maturation, proliferation, function, and survival of islet cells. In our previous study, we identified Isl1 interactions with the ring finger ubiquitin ligases Ring Finger 20 (Rnf20) and Rnf40 in mouse β-cell extracts, mouse islets, and human islets. Rnf20 and Rnf40, exist as homodimeric or heterodimeric complexes to deposit a mono-ubiquitin epigenetic mark on histone (H)2B (i.e., H2Bub1) and regulate gene transcription as a precursor to active H3K4 trimethylation (H3K4me3) and H3K79 methylation (H3K79me) marks. We demonstrated that Rnf20 and Rnf40 are required for the expression of several important β-cell genes and glucose-stimulated insulin secretion in vitro. Specifically, loss of Rnf20and Rnf40 in MIN6 β-cells resulted in a significant dysregulation of β-cell Slc2a2/Glut2 and MafA expression, target genes which were occupied by Rnf20 and H2Bub1. Additionally, using proximity ligation assay (PLA) in MIN6 β-cells, we demonstrated a robust increase in interactions between Isl1 and Rnf20 under high glucose conditions. Based on this, we hypothesize that Rnf20 and/or Rnf40 are required to establish and maintain β-cell identity in adult islets. To begin addressing this, we disrupted Rnf20/Rnf20 and/or Rnf20/Rnf40 complexes in β-cells via generation of adult inducible Rnf20 β-cell-specific knockout mice. In the adult inducible model, 2 weeks following tamoxifen induction (i.e., by 8 weeks of age) both male and female mice became hyperglycemic and severely glucose intolerant. These 8-week-old Rnf20-deficient mice also had reduced plasma insulin following a glucose challenge yet normal insulin sensitivity. Islet RNA exhibited dysregulated mRNAs encoding islet hormones, glucose-stimulated insulin secretion markers, and β-cell identity markers, including Slc2a2/Glut2, Ucn3, and Hb9 . Our findings of similar phenotypes between Isl1 and Rnf20 adult knockout models, a glucose sensitive interaction between Isl1 and Rnf20, and their occupancy of the Slc2a2/Glut2 locus support mechanisms of Isl1-Rnf driven β-cell regulation. Presentation: Saturday, June 11, 2022 1:12 p.m. - 1:17 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Loss of tetraspanin-7 expression reduces pancreatic β-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism.

Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate β-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic β-cell function is not yet fully understood. Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. β-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. Tspan7 is expressed in insulin-containing granules of pancreatic β-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- β-cells, but β-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent β-cells, exocytotic Ca2+ sensitivity was reduced in a human β-cell line (EndoC-βH1) following Tspan7 KD. Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in β-cells. Its function is more significant in human β-cells than their rodent counterparts.

Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction

ABSTRACT Macroautophagy/autophagy or mitophagy plays crucial roles in the maintenance of pancreatic β-cell function. PPP3/calcineurin can modulate the activity of TFEB, a master regulator of lysosomal biogenesis and autophagy gene expression, through dephosphorylation. We studied whether PPP3/calcineurin inhibitors can affect the mitophagy of pancreatic β-cells and pancreatic β-cell function employing FK506, an immunosuppressive drug against graft rejection. FK506 suppressed rotenone- or oligomycin+antimycin-A-induced mitophagy measured by Mito-Keima localization in acidic lysosomes or RFP-LC3 puncta colocalized with TOMM20 in INS-1 insulinoma cells. FK506 diminished nuclear translocation of TFEB after treatment with rotenone or oligomycin+antimycin A. Forced TFEB nuclear translocation by a constitutively active TFEB mutant transfection restored impaired mitophagy by FK506, suggesting the role of decreased TFEB nuclear translocation in FK506-mediated mitophagy impairment. Probably due to reduced mitophagy, recovery of mitochondrial potential or quenching of mitochondrial ROS after removal of rotenone or oligomycin+antimycin A was delayed by FK506. Mitochondrial oxygen consumption was reduced by FK506, indicating reduced mitochondrial function by FK506. Likely due to mitochondrial dysfunction, insulin release from INS-1 cells was reduced by FK506 in vitro. FK506 treatment also reduced insulin release and impaired glucose tolerance in vivo, which was associated with decreased mitophagy and mitochondrial COX activity in pancreatic islets. FK506-induced mitochondrial dysfunction and glucose intolerance were ameliorated by an autophagy enhancer activating TFEB. These results suggest that diminished mitophagy and consequent mitochondrial dysfunction of pancreatic β-cells contribute to FK506-induced β-cell dysfunction or glucose intolerance, and autophagy enhancement could be a therapeutic modality against post-transplantation diabetes mellitus caused by PPP3/calcineurin inhibitors.

Novel Loss-of-Function Variant in HNF1a Induces β-Cell Dysfunction through Endoplasmic Reticulum Stress.

Heterozygous variants in the hepatocyte nuclear factor 1a (HNF1a) cause MODY3 (maturity-onset diabetes of the young, type 3). In this study, we found a case of novel HNF1a p.Gln125* (HNF1a-Q125ter) variant clinically. However, the molecular mechanism linking the new HNF1a variant to impaired islet β-cell function remains unclear. Firstly, a similar HNF1a-Q125ter variant in zebrafish (hnf1a+/-) was generated by CRISPR/Cas9. We further crossed hnf1a+/- with several zebrafish reporter lines to investigate pancreatic β-cell function. Next, we introduced HNF1a-Q125ter and HNF1a shRNA plasmids into the Ins-1 cell line and elucidated the molecular mechanism. hnf1a+/- zebrafish significantly decreased the β-cell number, insulin expression, and secretion. Moreover, β cells in hnf1a+/- dilated ER lumen and increased the levels of ER stress markers. Similar ER-stress phenomena were observed in an HNF1a-Q125ter-transfected Ins-1 cell. Follow-up investigations demonstrated that HNF1a-Q125ter induced ER stress through activating the PERK/eIF2a/ATF4 signaling pathway. Our study found a novel loss-of-function HNF1a-Q125ter variant which induced β-cell dysfunction by activating ER stress via the PERK/eIF2a/ATF4 signaling pathway.