Fatty acids suppress autophagic turnover in β-cells

Recent studies have shown that autophagy is essential for proper β-cell function and survival. However, it is yet unclear under what pathogenic conditions autophagy is inhibited in β-cells. Here, we report that long term exposure to fatty acids and glucose block autophagic flux in β-cells, contributing to their toxic effect. INS1 cells expressing GFP-LC3 (an autophagosome marker) were treated with 0.4 mm palmitate, 0.4 mm oleate, and various concentrations of glucose for 22 h. Kinetics of the effect of fatty acids on autophagy showed a biphasic response. During the second phase of autophagy, the size of autophagosomes and the content of autophagosome substrates (GFP-LC3, p62) and endogenous LC3 was increased. During the same phase, fatty acids suppressed autophagic degradation of long lived protein in both INS1 cells and islets. In INS1 cells, palmitate induced a 3-fold decrease in the number and the acidity of Acidic Vesicular Organelles. This decrease was associated with a suppression of hydrolase activity, suppression of endocytosis, and suppression of oxidative phosphorylation. The combination of fatty acids with glucose synergistically suppressed autophagic turnover, concomitantly suppressing insulin secretion. Rapamycin treatment resulted in partial reversal of the inhibition of autophagic flux, the inhibition of insulin secretion, and the increase in cell death. Our results indicate that excess nutrient could impair autophagy in the long term, hence contributing to nutrient-induced β-cell dysfunction. This may provide a novel mechanism that connects diet-induced obesity and diabetes.     

Palmitate activates autophagy in INS-1E β-cells and in isolated rat and human pancreatic islets

We have investigated the in vitro effects of increased levels of glucose and free fatty acids on autophagy activation in pancreatic beta cells. INS-1E cells and isolated rat and human pancreatic islets were incubated for various times (from 2 to 24 h) at different concentrations of glucose and/or palmitic acid. Then, cell survival was evaluated and autophagy activation was explored by using various biochemical and morphological techniques. In INS-1E cells as well as in rat and human islets, 0.5 and 1.0 mM palmitate markedly increased autophagic vacuole formation, whereas high glucose was ineffective alone and caused little additional change when combined with palmitate. Furthermore, LC3-II immunofluorescence co-localized with that of cathepsin D, a lysosomal marker, showing that the autophagic flux was not hampered in PA-treated cells. These effects were maintained up to 18-24 h incubation and were associated with a significant decline of cell survival correlated with both palmitate concentration and incubation time. Ultrastructural analysis showed that autophagy activation, as evidenced by the occurrence of many autophagic vacuoles in the cytoplasm of beta cells, was associated with a diffuse and remarkable swelling of the endoplasmic reticulum. Our results indicate that among the metabolic alterations typically associated with type 2 diabetes, high free fatty acids levels could play a role in the activation of autophagy in beta cells, through a mechanism that might involve the induction of endoplasmic reticulum stress.     

Inhibition of SCD1 impairs palmitate-derived autophagy at the step of autophagosome-lysosome fusion in pancreatic β-cells

Autophagy is indispensable for the proper architecture and flawless functioning of pancreatic β-cells. A growing body of evidence indicates reciprocal communication between autophagic pathways, apoptosis, and intracellular lipids. The way in which elevated levels of free saturated or unsaturated FAs contribute to progressive β-cell failure remains incompletely understood. Stearoyl-CoA desaturase (SCD)1, a key regulatory enzyme in biosynthesis of MUFAs, was shown to play an important role in regulation of β-cell function. Here, we investigated whether SCD1 activity is engaged in palmitate-induced pancreatic β-cell autophagy. We found augmented apoptosis and diminished autophagy upon cotreatment of INS-1E cells with palmitate and an SCD1 inhibitor. Furthermore, we found that additional treatment of the cells with monensin, an inhibitor of autophagy at the step of fusion, exacerbates palmitate-induced apoptosis. Accordingly, diminished SCD1 activity affected the accumulation, composition, and saturation status of cellular membrane phospholipids and neutral lipids. Such an effect was accompanied by aberrant endoplasmic reticulum stress, mitochondrial injury, and decreases in insulin secretion and cell proliferation. Our data reveal a novel mechanism by which the inhibition of SCD1 activity affects autophagosome-lysosome fusion because of perturbations in cellular membrane integrity, thus leading to an aberrant stress response and β-cell failure.     

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...     

ATF3 represses PDX-1 expression in pancreatic β-cells

The downregulation of PDX-1 expression plays an important role in development of type 2 diabetes. However, the negative regulator of PDX-1 expression is not well known. In this study, we analyzed the mouse PDX-1 promoter to characterize the effects of ATF3 on PDX-1 expression in pancreatic β-cells. Both thapsigargin treatment, an inducer of ER stress, and ATF3 expression decreased PDX-1 expression in pancreatic β-cells, MIN6N8. Furthermore, they also repressed the activity of −4.5 Kb promoter of mouse PDX-1 gene. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of 0.9 Kb PDX-1 promoter, whereas it did not affect the activity of 0.7 Kb PDX-1 promoter, suggesting that ATF3 responsive element is located between the −903 and −702. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds directly to the promoter region spanning from −759 to −738. Moreover, mutation of the putative ATF/CRE site between −752 and −745 abrogated ATF3-mediated transrepression of the PDX-1 promoter. PDX-1 was decreased in MIN6N8 cells treated with high glucose or high palmitate, whereas ATF3 was increased, indicating that ATF3 plays a role in hyperglycemia or hyperlipidemia-mediated downregulation of PDX-1 expression. Collectively, these results demonstrate that ATF3 represses PDX-1 expression via binding to an ATF3-responsive element in its promoter, which plays an important role in suppression of pancreatic β-cells function.     

The role of autophagy in pancreatic β-cell and diabetes

Pancreatic β-cells play a key role in glucose homeostasis in mammals. Although large-scale protein synthesis and degradation occur in pancreatic β-cells, the mechanism underlying dynamic protein turnover in β-cells remains largely unknown. We found low-level constitutive autophagy in β-cells of C57BL/6 mice fed a standard diet; however, autophagy was markedly upregulated in mice fed a high-fat diet. β-cells of diabetic db/db mice contained large numbers of autophagosomes, compared with non-diabetic db/misty controls. The functional importance of autophagy was analyzed using β-cell-specific Atg7 knockout mice. Autophagy-deficient mice showed degeneration of β-cells and impaired glucose tolerance with reduced insulin secretion. While a high-fat diet stimulated β-cell autophagy in control mice, it induced a profound deterioration of glucose intolerance in β-cell autophagy-deficient mutants, partly because of the lack of a compensatory increase in β-cell mass. These results suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of β-cells. Autophagy also serves as a crucial element of stress responses to protect β-cells under insulin resistant states. Impairment of autophagic machinery could thus predispose individuals to type 2 diabetes.     

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.     

Mechanism for antioxidative effects of thiazolidinediones in pancreatic β-cells

Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily. TZDs are known to increase insulin sensitivity and also to have an antioxidative effect. In this study, we tested whether TZDs protect pancreatic β-cells from oxidative stress, and we investigated the mechanism involved in this process. To generate oxidative stress in pancreatic β-cells (INS-1 and βTC3) or isolated islets, glucose oxidase was added to the media. The extracellular and intracellular reactive oxygen species (ROS) were measured to directly determine the antioxidant effect of TZDs. The phosphorylation of JNK/MAPK after oxidative stress was detected by Western blot analysis, and glucose-stimulated insulin secretion and cell viability were also measured. TZDs significantly reduced the ROS levels that were increased by glucose oxidase, and they effectively prevented β-cell dysfunction. The antioxidative effect of TZDs was abolished in the presence of a PPARγ antagonist, GW9662. Real-time PCR was used to investigate the expression levels of antioxidant genes. The expression of catalase, an antioxidant enzyme, was increased by TZDs in pancreatic β-cells, and the knockdown of catalase significantly inhibited the antioxidant effect of TZDs. These results suggest that TZDs effectively protect pancreatic β-cells from oxidative stress, and this effect is dependent largely on PPARγ. In addition, the expression of catalase is increased by TZDs, and catalase, at least in part, mediates the antioxidant effect of TZDs in pancreatic β-cells.     

Ultrastructural changes in β-cells of pancreatic islets in α-amanitin-poisoned Mice

In mice poisoned by alpha-amanitin nuclear changes typical of this toxin were observed in beta-cells of pancreatic islets. The lesions became progressively more severe and at 48 h after toxin injection some cells were necrotic. The damage to these cells could have implications in the changes in glycogen metabolism which occur after alpha-aminitin poisoning.     

RyR channels and glucose-regulated pancreatic β-cells

Ryanodine receptor channel model is introduced to a dynamical model of pancreatic beta-cells to discuss the effects of RyR channels and glucose concentration on membrane potential. The results show Ca(2+) concentration changes responding to enhance of glucose concentration is more quickly than that of activating RyR channels, and both methods can induce bursting action potential and increase free cytosolic Ca(2+) concentration. An interesting finding is that moderate stimulation to RyR channels will result in a kind of "complex bursting", which is more effective in enhancing average Ca(2+) concentration and insulin section.     

Calcium signaling in pancreatic β-cells in health and in Type 2 diabetes

Changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic β-cell. Secretion is controlled by the finely tuned balance between Ca²⁺ influx (mainly through voltage-dependent Ca²⁺ channels, but also through voltage-independent Ca²⁺ channels like store-operated channels) and efflux pathways. Changes in [Ca²⁺]_c directly affect [Ca²⁺] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca²⁺ influx and efflux pathways, they mutually influence free [Ca²⁺] in the others. In this article, we review the mechanisms of control of [Ca²⁺] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca²⁺]_ER), acidic stores and mitochondrial matrix ([Ca²⁺]_mito), focusing chiefly on the most important physiological stimulus of β-cells, glucose. We also briefly review some alterations of β-cell Ca²⁺ homeostasis in Type 2 diabetes.     

Nodal induces apoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 β-cells

We demonstrated previously that the activation of ALK7 (activin receptor-like kinase-7), a member of the type I receptor serine/threonine kinases of the TGF-β superfamily, resulted in increased apoptosis and reduced proliferation through suppression of Akt signaling and the activation of Smad2-dependent signaling pathway in pancreatic β-cells. Here, we show that Nodal activates ALK7 signaling and regulates β-cell apoptosis. We detected Nodal expression in the clonal β-cell lines and rodent islet β-cells. Induction of β-cell apoptosis by treatment with high glucose, palmitate, or cytokines significantly increased Nodal expression in clonal INS-1 β-cells and isolated rat islets. The stimuli induced upregulation of Nodal expression levels were associated with elevation of ALK7 protein and enhanced phosphorylated Smad3 protein. Nodal treatment or overexpression of Nodal dose- or time-dependently increased active caspase-3 levels in INS-1 cells. Nodal-induced apoptosis was associated with decreased Akt phosphorylation and reduced expression level of X-linked inhibitor of apoptosis (XIAP). Remarkably, overexpression of XIAP or constitutively active Akt, or ablation of Smad2/3 activity partially blocked Nodal-induced apoptosis. Furthermore, siRNA-mediated ALK7 knockdown significantly attenuated Nodal-induced apoptosis of INS-1 cells. We suggest that Nodal-induced apoptosis in β-cells is mediated through ALK7 signaling involving the activation of Smad2/3-caspase-3 and the suppression of Akt and XIAP pathways and that Nodal may exert its biological effects on the modulation of β-cell survival and β-cell mass in an autocrine fashion.     

Systems analysis of GLP-1 receptor signaling in pancreatic β-cells

Glucagon-like peptide-1 (GLP-1) elevates intracellular concentration of cAMP ([cAMP]) and facilitates glucose-dependent insulin secretion in pancreatic β-cells. There has been much evidence to suggest that multiple key players such as the GLP-1 receptor, G(s) protein, adenylate cyclase (AC), phosphodiesterase (PDE), and intracellular Ca(2+) concentration ([Ca(2+)]) are involved in the regulation of [cAMP]. However, because of complex interactions among these signaling factors, the kinetics of the reaction cascade as well as the activities of ACs and PDEs have not been determined in pancreatic β-cells. We have constructed a minimal mathematical model of GLP-1 receptor signal transduction based on experimental findings obtained mostly in β-cells and insulinoma cell lines. By fitting this theoretical reaction scheme to key experimental records of the GLP-1 response, the parameters determining individual reaction steps were estimated. The model reconstructed satisfactorily the dynamic changes in [cAMP] and predicted the activities of cAMP effectors, protein kinase A (PKA), and cAMP-regulated guanine nucleotide exchange factor [cAMP-GEF or exchange protein directly activated by cAMP (Epac)] during GLP-1 stimulation. The simulations also predicted the presence of two sequential desensitization steps of the GLP1 receptor that occur with fast and very slow reaction rates. The cross talk between glucose- and GLP-1-dependent signal cascades for cAMP synthesis was well reconstructed by integrating the direct regulation of AC and PDE by [Ca(2+)]. To examine robustness of the signaling system in controlling [cAMP], magnitudes of AC and PDE activities were compared in the presence or absence of GLP-1 and/or the PDE inhibitor IBMX.(1).     

Electrophysiology of pancreatic β-cells in intact mouse islets of Langerhans

When exposed to intermediate glucose concentrations (6–16 mol/l), pancreatic β-cells in intact islets generate bursts of action potentials (superimposed on depolarised plateaux) separated by repolarised electrically silent intervals. First described more than 40 years ago, these oscillations have continued to intrigue β-cell electrophysiologists. To date, most studies of β-cell ion channels have been performed on isolated cells maintained in tissue culture (that do not burst). Here we will review the electrophysiological properties of β-cells in intact, freshly isolated, mouse pancreatic islets. We will consider the role of ATP-regulated K⁺-channels (K_ATP-channels), small-conductance Ca²⁺-activated K⁺-channels and voltage-gated Ca²⁺-channels in the generation of the bursts. Our data indicate that K_ATP-channels not only constitute the glucose-regulated resting conductance in the β-cell but also provide a variable K⁺-conductance that influence the duration of the bursts of action potentials and the silent intervals. We show that inactivation of the voltage-gated Ca²⁺-current is negligible at voltages corresponding to the plateau potential and consequently unlikely to play a major role in the termination of the burst. Finally, we propose a model for glucose-induced β-cell electrical activity based on observations made in intact pancreatic islets.     

Voltage-dependent metabolic regulation of Kv2.1 channels in pancreatic β-cells

Voltage-gated potassium channels (Kv channels) play a crucial role in formation of action potentials in response to glucose stimulation in pancreatic β-ells. We previously reported that the Kv channel is regulated by glucose metabolism, particularly by MgATP. We examined whether the regulation of Kv channels is voltage-dependent and mechanistically related with phosphorylation of the channels. In rat pancreatic β-cells, suppression of glucose metabolism with low glucose concentrations of 2.8 mM or less or by metabolic inhibitors decreased the Kv2.1-channel activity at positive membrane potentials, while increased it at potentials negative to −10 mV, suggesting that modulation of Kv channels by glucose metabolism is voltage-dependent. Similarly, in HEK293 cells expressing the recombinant Kv2.1 channels, 0 mM but not 10 mM MgATP modulated the channel activity in a manner similar to that in β-cells. Both steady-state activation and inactivation kinetics of the channel were shifted toward the negative potential in association with the voltage-dependent modulation of the channels by cytosolic dialysis of alkaline phosphatase in β-cells. The modulation of Kv-channel current-voltage relations were also observed during and after glucose-stimulated electrical excitation. These results suggest that the cellular metabolism including MgATP production and/or channel phosphorylation/dephosphorylation underlie the physiological modulation of Kv2.1 channels during glucose-induced insulin secretion.