Pancreatic cancer-derived exosomal microRNA-19a induces β-cell dysfunction by targeting ADCY1 and EPAC2

New-onset diabetes mellitus has a rough correlation with pancreatic cancer (PaC), but the underlying mechanism remains unclear. This study aimed to explore the exosomal microRNAs and their potential role in PaC-induced β-cell dysfunction. The pancreatic β cells were treated with isolated exosomes from PaC cell lines, SW1990 and BxPC-3, before measuring the glucose-stimulated insulin secretion (GSIS), validating that SW1990 and BxPC-3 might disrupt GSIS of both β cell line MIN6 and primary mouse pancreatic islets. The difference in expression profiles between exosomes and exosome-free medium of PaC cell lines was further defined, revealing that miR-19a secreted by PaC cells might be an important signaling molecule in this process. Furthermore, adenylyl cyclase 1 (Adcy1) and exchange protein directly activated by cAMP 2 (Epac2) were verified as the direct targets of exogenous miR-19a, which was involved in insulin secretion. These results indicated that exosomes might be an important mediator in the pathogenesis of PaC-DM, and miR-19a might be the effector molecule. The findings shed light on the pathogenesis of PaC-DM.     

Correlation between TGF-β2/3 promoter DNA methylation and Smad signaling during palatal fusion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant that is strongly associated with a number of human diseases and birth defects, including cleft palate. Transforming growth factor (TGF) plays a significant role during mammalian palatogenesis. However, the epigenetic mechanism of transforming growth factors in the process of TCDD-induced cleft palate is unclear. The purpose of this research was to investigate the relationship and potential mechanism between TGF-β2/3 promoter DNA methylation and Smad signaling during TCDD-induced cleft palate. Pregnant C57BL/6N mice were exposed to 64 µg/kg TCDD on gestational day 10 (GD10) to establish the cleft palate model and palatal tissues of embryos were collected on GD13, GD14, and GD15 for subsequent experiments. TGF-β2/3 mRNA expression, TGF-β2/3 promoter methylation, and Smad signaling molecules expression were assessed in the palate of the two groups. The results showed that the incidence of cleft palate was 94.7% in the TCDD-treated group whereas no cleft palate was found in the control group. TCDD-treated group altered specific CpG sites of TGF-β2/3 promoter methylation. Compared to the control group, the proliferation of mouse embryonic palate mesenchymal stromal cells (MEPM), the expressions of TGF-β2/3, p-Smad2, and Smad4 were all reduced, while the expression of Smad7 was significantly increased in the atAR group. Smad signaling was downregulated by TCDD. Therefore, we suggest that TGF-β2/3 promoter methylation and Smad signaling may be involved in TCDD-induced cleft palate formation in fetal mice.     

Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

The adipocyte-derived hormone leptin increases trafficking of K_ATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca²⁺ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.     

Intra-islet glucagon confers β-cell glucose competence for first-phase insulin secretion and favors GLP-1R stimulation by exogenous glucagon

We report that intra-islet glucagon secreted from α-cells signals through β-cell glucagon and GLP-1 receptors (GcgR and GLP-1R), thereby conferring to rat islets their competence to exhibit first-phase glucose-stimulated insulin secretion (GSIS). Thus, in islets not treated with exogenous glucagon or GLP-1, first-phase GSIS is abolished by a GcgR antagonist (LY2786890) or a GLP-1R antagonist (Ex[9–39]). Mechanistically, glucose competence in response to intra-islet glucagon is conditional on β-cell cAMP signaling because it is blocked by the cAMP antagonist prodrug Rp-8-Br-cAMPS-pAB. In its role as a paracrine hormone, intra-islet glucagon binds with high affinity to the GcgR, while also exerting a “spillover” effect to bind with low affinity to the GLP-1R. This produces a right shift of the concentration-response relationship for the potentiation of GSIS by exogenous glucagon. Thus, 0.3 nM glucagon fails to potentiate GSIS, as expected if similar concentrations of intra-islet glucagon already occupy the GcgR. However, 10 to 30 nM glucagon effectively engages the β-cell GLP-1R to potentiate GSIS, an action blocked by Ex[9–39] but not LY2786890. Finally, we report that the action of intra-islet glucagon to support insulin secretion requires a step-wise increase of glucose concentration to trigger first-phase GSIS. It is not measurable when GSIS is stimulated by a gradient of increasing glucose concentrations, as occurs during an oral glucose tolerance test in vivo. Collectively, such findings are understandable if defective intra-islet glucagon action contributes to the characteristic loss of first-phase GSIS in an intravenous glucose tolerance test, that is, diagnostic of type 2 diabetes in the clinical setting.     

Glucose-6-phosphatase catalytic subunit 2 negatively regulates glucose oxidation and insulin secretion in pancreatic β-cells

Elevated fasting blood glucose (FBG) is associated with increased risks of developing type 2 diabetes (T2D) and cardiovascular-associated mortality. G6PC2 is predominantly expressed in islets, encodes a glucose-6-phosphatase catalytic subunit that converts glucose-6-phosphate (G6P) to glucose, and has been linked with variations in FBG in genome-wide association studies. Deletion of G6pc2 in mice has been shown to lower FBG without affecting fasting plasma insulin levels in vivo. At 5 mM glucose, pancreatic islets from G6pc2 knockout (KO) mice exhibit no glucose cycling, increased glycolytic flux, and enhanced glucose-stimulated insulin secretion (GSIS). However, the broader effects of G6pc2 KO on β-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in βTC3 cells, a murine pancreatic β-cell line, to examine the role of G6pc2 in regulating glycolytic and mitochondrial fluxes. We found that deletion of G6pc2 led to ∼60% increases in glycolytic and citric acid cycle (CAC) fluxes at both 5 and 11 mM glucose concentrations. Furthermore, intracellular insulin content and GSIS were enhanced by approximately two-fold, along with increased cytosolic redox potential and reductive carboxylation flux. Normalization of fluxes relative to net glucose uptake revealed upregulation in two NADPH-producing pathways in the CAC. These results demonstrate that G6pc2 regulates GSIS by modulating not only glycolysis but also, independently, citric acid cycle activity in β-cells. Overall, our findings implicate G6PC2 as a potential therapeutic target for enhancing insulin secretion and lowering FBG, which could benefit individuals with prediabetes, T2D, and obesity.     

Pancreatic β-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in β-cell death leading to reduced β-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic β-cell death. In Pdx1 haploinsufficient mice, Bax ablation in β-cells increased β-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates β-cell apoptosis in Pdx1-deficient diabetes.     

TFPI2 suppresses the interaction of TGF-β2 pathway regulators to promote endothelial–mesenchymal transition in diabetic nephropathy

Endothelial–mesenchymal transition (EndMT) is an important source of myofibroblasts, but also contributes to the progression of diabetic nephropathy (DN). By several differential gene expression analyses from the Gene Expression Omnibus (GEO) database, the tissue factor pathway inhibitor 2 (TFPI2) gene, known as a tumor suppressor, was shown to be dysregulated in DN; however, the potential role and regulatory mechanism of TFPI2 in DN are unclear. Here, we found abnormal upregulation of TFPI2 in the renal cortex of diabetic mice, accompanied by impaired renal function. We also injected a single dose of adeno-associated virus (AAV)2 carrying shRNA targeting TFPI2 intravenously into these mice and found that knockdown of TFPI2 improved renal function and reduced renal fibrosis and cell apoptosis in experimental DN. Furthermore, hyperglycemia-induced EndMT was inhibited in the absence of TFPI2, as evidenced by increased expression of endothelial markers (VE-cadherin and CD31) and decreased expression of mesenchymal markers (α-SMA, desmin, and FSP-1). To further explore the mechanism in vitro, human renal glomerular endothelial cells (hRGECs) were incubated in the presence of high glucose or transforming growth factor beta (TGF-β)2. TFPI2 deficiency inhibited high glucose-induced cell apoptosis and TGF-β2-induced EndMT in hRGECs, while overexpression of TFPI2 had the opposite effects. Importantly, TGF-β2 is a crucial driver of EndMT, and we found that TFPI2 promoted TGF-β2/Smad signaling activation by interferring the interaction of TGF-β pathway regulators (SMURF2 with SMAD7). Our results show that TFPI2 regulates EndMT and the TGF-β2 signaling pathway and is a potential promoter of DN pathogenesis.     

Latent TGF-β1 protects against diabetic kidney disease via Arkadia/Smad7 signaling

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.     

Chronic alcohol consumption potentiates the development of diabetes through pancreatic β-cell dysfunction

Chronic ethanol consumption is well established as a major risk factor for type-2 diabetes(T2D), which is evidenced by impaired glucose metabolism and insulin resistance. However, the relationships between alcoholconsumption and the development of T2 D remain controversial. In particular, the direct effects of ethanol consumption on proliferation of pancreatic β-cell and the exact mechanisms associated with ethanolmediated β-cell dysfunction and apoptosis remain elusive. Although alcoholism and alcohol consumption are prevalent and represent crucial public health problems worldwide, many people believe that low-tomoderate ethanol consumption may protect against T2 D and cardiovascular diseases. However, the J- or U-shaped curves obtained from cross-sectional and large prospective studies have not fully explained the relationship between alcohol consumption and T2 D. This review provides evidence for the harmful effects of chronic ethanol consumption on the progressive development of T2 D, particularly with respect to pancreatic β-cell mass and function in association with insulin synthesis and secretion. This review also discusses a conceptual framework for how ethanolproduced peroxynitrite contributes to pancreatic β-cell dysfunction and metabolic syndrome.     

Wild-type IDH1 inhibits the tumor growth through degrading HIF-α in renal cell carcinoma

The purpose of our study was to explore the effect and intrinsic mechanism of wild-type IDH1 and its substrate α-KG on renal cell carcinoma (RCC). IDH1 was observed lower expression in RCC cell lines. Phenotype experiment was carried out in the wild-type IDH1 and mutant IDH1^R132H plasmid treated cell line. The results showed that the wild-type IDH1 could significantly inhibit the proliferation, migration and promote the apoptosis of RCC cell lines, which were consistent with the IDH1''s substrate α-KG. The mutant IDH1^R132H was found to lose this biological function of IDH1. Moreover, we verified the proliferation inhibition of IDH1 in vivo. In addition, we verified the correlation between IDH1 and hypoxia signal-related proteins in vitro and in vivo, specifically, IDH1 overexpression could significantly reduce the expression of HIF-1α and HIF-2α proteins and its downstream proteins (VEGF, TGF-α). Furthermore, we preliminarily verified the possibility of α-KG in the RCC''s treatment by injecting α-KG into the xenograft model. α-KG significantly reduced tumor size and weight in tumor-bearing mice. This study provided a new therapeutic target and small molecule for the study of the treatment and mechanism of RCC.     

PINK1 deficiency in β-cells increases basal insulin secretion and improves glucose tolerance in mice

The Parkinson''s disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis. Given the link between glucose metabolism, mitochondrial function and insulin secretion in β-cells, and the reported association of PD with type 2 diabetes, we investigated the response of PINK1-deficient β-cells to glucose stimuli to determine whether loss of PINK1 affected their function. We find that loss of PINK1 significantly impairs the ability of mouse pancreatic β-cells (MIN6 cells) and primary intact islets to take up glucose. This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions. Finally, we investigated the effect of PINK1 deficiency in vivo and find that PINK1 knockout mice have improved glucose tolerance. For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.     

High temperature promotes amyloid β-protein production and γ-secretase complex formation via Hsp90

Alzheimer''s disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aβ production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aβ production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aβ production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.     

Inhibitory evaluation of Curculigo latifolia on α-glucosidase,DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC₅₀ 2.72 ± 0.32) as compared with the fruit extracts (IC₅₀ 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.     

The complexity of TGFβ/activin signaling in regeneration

The role of transforming growth factor β TGFβ/activin signaling in wound repair and regeneration is highly conserved in the animal kingdom. Various studies have shown that TGF-β/activin signaling can either promote or inhibit different aspects of the regeneration process (i.e., proliferation, differentiation, and re-epithelialization). It has been demonstrated in several biological systems that some of the different cellular responses promoted by TGFβ/activin signaling depend on the activation of Smad-dependent or Smad-independent signal transduction pathways. In the context of regeneration and wound healing, it has been shown that the type of R-Smad stimulated determines the different effects that can be obtained. However, neither the possible roles of Smad-independent pathways nor the interaction of the TGFβ/activin pathway with other complex signaling networks involved in the regenerative process has been studied extensively. Here, we review the important aspects concerning the TGFβ/activin signaling pathway in the regeneration process. We discuss data regarding the role of TGF-β/activin in the most common animal regenerative models to demonstrate how this signaling promotes or inhibits regeneration, depending on the cellular context.