Acacetin antagonized lipotoxicity in pancreatic β-cells via ameliorating oxidative stress and endoplasmic reticulum stress

Molecular Biology Reports - During the pathogenesis and progression of diabetes, lipotoxicity is a major threat to the function and survival of pancreatic β-cells. To battle against the...     

N-Acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic β-cells

Pancreatic β-cells secrete insulin in response to various stimuli to control blood glucose levels. This insulin release is the result of a complex interplay between signaling, membrane potential and intracellular calcium levels. Various nutritional and hormonal factors are involved in regulating this process. N-Acyl taurines are a group of fatty acids which are amidated (or conjugated) to taurine and little is known about their physiological functions. In this study, treatment of pancreatic β-cell lines (HIT-T15) and rat islet cell lines (INS-1) with N-acyl taurines (N-arachidonoyl taurine and N-oleoyl taurine), induced a high frequency of calcium oscillations in these cells. Treatment with N-arachidonoyl taurine and N-oleoyl taurine also resulted in a significant increase in insulin secretion from pancreatic β-cell lines as determined by insulin release assay and immunofluorescence (p < 0.05). Our data also show that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in insulin secretion in response to N-arachidonoyl taurine and N-oleoyl taurine treatment. However our data also suggest that receptors other than TRPV1 are involved in the insulin secretion response to treatment with N-oleoyl taurine.     

NAD kinase regulates the size of the NADPH pool and insulin secretion in pancreatic β-cells

NADPH is an important component of the antioxidant defense system and a proposed mediator in glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. An increase in the NADPH/NADP(+) ratio has been reported to occur within minutes following the rise in glucose concentration in β-cells. However, 30 min following the increase in glucose, the total NADPH pool also increases through a mechanism not yet characterized. NAD kinase (NADK) catalyzes the de novo formation of NADP(+) by phosphorylation of NAD(+). NAD kinases have been shown to be essential for redox regulation, oxidative stress defense, and survival in bacteria and yeast. However, studies on NADK in eukaryotic cells are scarce, and the function of this enzyme has not been described in β-cells. We employed INS-1 832/13 cells, an insulin-secreting rat β-cell line, and isolated rodent islets to investigate the role of NADK in β-cell metabolic pathways. Adenoviral-mediated overexpression of NADK resulted in a two- to threefold increase in the total NADPH pool and NADPH/NADP(+) ratio, suggesting that NADP(+) formed by the NADK-catalyzed reaction is rapidly reduced to NADPH via cytosolic reductases. This increase in the NADPH pool was accompanied by an increase in GSIS in NADK-overexpressing cells. Furthermore, NADK overexpression protected β-cells against oxidative damage by the redox cycling agent menadione and reversed menadione-mediated inhibition of GSIS. Knockdown of NADK via shRNA exerted the opposite effect on all these parameters. These data suggest that NADK kinase regulates intracellular redox and affects insulin secretion and oxidative defense in the β-cell.     

Nuclear SREBP-1a causes loss of pancreatic β-cells and impaired insulin secretion

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic β-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, ΒΕΤΑ2, MafA, and IRS-2 were suppressed, partially explaining the loss and dysfunction of β-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous β-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts β-cell mass and function.     

Imaging glucose-regulated insulin secretion and gene expression in single islet β-cells

The mechanisms by which changes in glucose concentration regulate gene expression and insulin secretion in pancreatic islet β-cells are only partly understood. Here we describe the development of new technologies for examining these processes at the level of single living β-cells. We also present recent findings, made using these and other techniques, which implicate a role for adenosine 5′-monophosphate-activated protein kinase in glucose signaling in these cells.     

Involvement of RhoA/ROCK in insulin secretion of pancreatic β-cells in 3D culture

Cell-cell contacts and interactions between pancreatic β-cells and/or other cell populations within islets are essential for cell survival, insulin secretion, and functional synchronization. Three-dimensional (3D) culture systems supply the ideal microenvironment for islet-like cluster formation and functional maintenance. However, the underlying mechanisms remain unclear. In this study, mouse insulinoma 6 (MIN6) cells were cultured in a rotating 3D culture system to form islet-like aggregates. Glucose-stimulated insulin secretion (GSIS) and the RhoA/ROCK pathway were investigated. In the 3D-cultured MIN6 cells, more endocrine-specific genes were up-regulated, and GSIS was increased to a greater extent than in cells grown in monolayers. RhoA/ROCK inactivation led to F-actin remodeling in the MIN6 cell aggregates and greater insulin exocytosis. The gap junction protein, connexin 36 (Cx36), was up-regulated in MIN6 cell aggregates and RhoA/ROCK-inactivated monolayer cells. GSIS dramatically decreased when Cx36 was knocked down by short interfering RNA and could not be reversed by RhoA/ROCK inactivation. Thus, the RhoA/ROCK signaling pathway is involved in insulin release through the up-regulation of Cx36 expression in 3D-cultured MIN6 cells.     

p24 family type 1 transmembrane proteins are required for insulin biosynthesis and secretion in pancreatic β-cells

The p24 protein family have multiple functions in protein transport in the early secretory pathway. In this study we examined the role of p24 proteins in insulin transport. Several members were detected in insulinoma cell lines and rat islets and expression levels positively correlated with insulin abundance, particularly for p24δ1 and p24β1. Knocking down p24δ1 in insulinoma cell lines, which also resulted in the concomitant knock-down of other family members, impaired glucose-stimulated insulin secretion, decreased total cellular insulin content and reduced proinsulin biosynthesis. There was no effect on overall protein biosynthesis or ER stress. These results suggest that p24δ1 and possibly other p24 family proteins are required for normal insulin biosynthesis and subsequent secretion in pancreatic β-cells.     

PCSK9 is expressed in pancreatic δ-cells and does not alter insulin secretion

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immunohistochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancreatic δ-cells, with no expression in α- and β-cells. PCSK9 seems not to be secreted by mouse isolated islets maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated islets, mainly in β-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin secretion in mouse islets. Finally, invivo glucose tolerance was similar in Pcsk9^+/+ and Pcsk9^−/− mice under basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that PCSK9 does not alter insulin secretion.     

The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells

Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.     

Thiopental-induced insulin secretion via activation of IP3-sensitive calcium stores in rat pancreatic β-cells

While glucose-stimulated insulin secretion depends on Ca(2+) influx through voltage-gated Ca(2+) channels in the cell membrane of the pancreatic β-cell, there is also ample evidence for an important role of intracellular Ca(2+) stores in insulin secretion, particularly in relation to drug stimuli. We report here that thiopental, a common anesthetic agent, triggers insulin secretion from the intact pancreas and primary cultured rat pancreatic β-cells. We investigated the underlying mechanisms by measurements of whole cell K(+) and Ca(2+) currents, membrane potential, cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and membrane capacitance. Thiopental-induced insulin secretion was first detected by enzyme-linked immunoassay, then further assessed by membrane capacitance measurement, which revealed kinetics distinct from glucose-induced insulin secretion. The thiopental-induced secretion was independent of cell membrane depolarization and closure of ATP-sensitive potassium (K(ATP)) channels. However, accompanied by the insulin secretion stimulated by thiopental, we recorded a significant intracellular [Ca(2+)] increase that was not from Ca(2+) influx across the cell membrane, but from intracellular Ca(2+) stores. The thiopental-induced [Ca(2+)](i) rise in β-cells was sensitive to thapsigargin, a blocker of the endoplasmic reticulum Ca(2+) pump, as well as to heparin (0.1 mg/ml) and 2-aminoethoxydiphenyl borate (2-APB; 100 μM), drugs that inhibit inositol 1,4,5-trisphosphate (IP(3)) binding to the IP(3) receptor, and to U-73122, a phospholipase C inhibitor, but insensitive to ryanodine. Thapsigargin also diminished thiopental-induced insulin secretion. Thus, we conclude that thiopental-induced insulin secretion is mediated by activation of the intracellular IP(3)-sensitive Ca(2+) store.     

M19 modulates skeletal muscle differentiation and insulin secretion in pancreatic β-cells through modulation of respiratory chain activity

Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.     

The effect of ATP-sensitive K+ channels on the electrical burst activity and insulin secretion in pancreatic β-cells

In recent years, the electrical burst activity of the insulin releasing pancreatic β-cells has attracted many experimentalists and theoreticians, largely because of its functional importance, but also because of the nonlinear nature of the burst activity. The ATP-sensitive K⁺ channels are believed to play an important role in electrical activity and insulin release. In this paper, we show by computer simulation how ATP and antidiabetic drugs can lengthen the plateau fraction of bursting and how these chemicals can increase the intracellular Ca²⁺ level in the pancreatic β-cell.     

PACAP stimulates insulin secretion by PAC1 receptor and ion channels in β-cells

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays a crucial role in the endocrine system. The present study aimed to investigate the effect of PACAP38 on insulin secretion and the underlying mechanism in rat pancreatic β-cells. The insulin secretion results showed that PACAP38 stimulated insulin secretion in a glucose- and dose-dependent manner. The insulinotropic effect was mediated by PAC₁ receptor, but not by VPAC₁ and VPAC₂ receptors. Inhibition of adenylyl cyclase and protein kinase A suppressed PACAP38-augmented insulin secretion. Glucose-regulated insulin secretion is dependent on a series of electrophysiological activities. Current-clamp technology suggested that PACAP38 prolonged action potential duration. Voltage-clamp recordings revealed that PACAP38 blocked voltage-dependent potassium currents, and this effect was reversed by inhibition of PAC₁ receptor, adenylyl cyclase, or protein kinase A. Activation of Ca²⁺ channels by PACAP38 was also observed, which could be antagonized by the PAC₁ receptor antagonist. In addition, calcium-imaging analysis indicated that PACAP38 increased intracellular Ca²⁺ concentration, which was decreased by PAC₁ receptor antagonist. These findings demonstrate that PACAP38 stimulates glucose-induced insulin secretion mainly by acting on PAC₁ receptor, inhibiting voltage-dependent potassium channels, activating Ca²⁺ channels and increasing intracellular Ca²⁺ concentration. Further, PACAP blocks voltage-dependent potassium currents via the adenylyl cyclase/protein kinase A signaling pathway.     

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.     

SNAP-251–180 enhances insulin secretion by blocking Kv2.1 channels in rat pancreatic islet β-cells

Voltage-gated outward K⁺ currents from pancreatic islet β-cells are known to repolarize the action potential during a glucose stimulus, and consequently to modulate Ca²⁺ entry and insulin secretion. The voltage gated K⁺ (Kv) channel, Kv2.1, which is expressed in rat islet β-cells, mediates over 60% of the Kv outward K⁺ currents. A novel peptidyl inhibitor of Kv2.1/Kv2.2 channels, guangxitoxin (GxTX)-1, has been shown to enhance glucose-stimulated insulin secretion. Here, we show that SNAP-25_1–180 (S180), an N-terminal SNAP-25 domain, but not SNAP-25_1–206 (S206), inhibits Kv current and enhances glucose-dependent insulin secretion from rat pancreatic islet β-cells, and furthermore, this enhancement was induced by the blockade of the Kv2.1 current. This study indicates that the Kv2.1 channel is a potential target for novel therapeutic agent design for the treatment of type 2 diabetes. This target may possess advantages over currently-used therapies, which modulate insulin secretion in a glucose-independent manner.     

GABA coordinates with insulin in regulating secretory function in pancreatic INS-1 β-cells

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABA(A)Rs). We and others recently reported that islet β-cells also express GABA(A)Rs and that activation of GABA(A)Rs increases insulin release. Here we investigate the effects of insulin on the GABA-GABA(A)R system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 μM) suppressed the GABA-induced current (I(GABA)) by 43%. Zinc-free insulin also suppressed I(GABA) to the same extent of inhibition by regular insulin. The inhibition of I(GABA) occurs within 30 seconds after application of insulin. The insulin-induced inhibition of I(GABA) persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABA(A)Rs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 μM, p<0.01) and insulin (1 μM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABA(A)Rs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning β-cell secretion.