Youn-Jung Lee

Protective Role of Autophagy in Palmitate-Induced INS-1 β-Cell Death

Autophagy, a vacuolar degradative pathway, constitutes a stress adaptation that avoids cell death or elicits the alternative cell-death pathway. This study was undertaken to determine whether autophagy is activated in palmitate (PA)-treated beta-cells and, if activated, what the role of autophagy is in the PA-induced beta-cell death. The enhanced formation of autophagosomes and autolysosomes was observed by exposure of INS-1 beta-cells to 400 microm PA in the presence of 25 mm glucose for 12 h. The formation of green fluorescent protein-LC3-labeled structures (green fluorescent protein-LC3 dots), with the conversion from LC3-I to LC3-II, was also distinct in the PA-treated cells. The phospho-mammalian target of rapamycin level, a typical signal pathway that inhibits activation of autophagy, was gradually decreased by PA treatment. Blockage of the mammalian target of rapamycin signaling pathway by treatment with rapamycin augmented the formation of autophagosomes but reduced PA-induced INS-1 cell death. In contrast, reduction of autophagosome formation by knocking down the ATG5, inhibition of fusion between autophagosome and lysosome by treatment with bafilomycin A1, or inhibition of proteolytic degradation by treatment with E64d/pepstatin A, significantly augmented PA-induced INS-1 cell death. These findings showed that the autophagy system could be activated in PA-treated INS-1 beta-cells, and suggested that the induction of autophagy might play an adaptive and protective role in PA-induced cell death.

Glucagon-like peptide-1 inhibits adipose tissue macrophage infiltration and inflammation in an obese mouse model of diabetes

Aims/hypothesisObesity and insulin resistance are associated with low-grade chronic inflammation. Glucagon-like peptide-1 (GLP-1) is known to reduce insulin resistance. We investigated whether GLP-1 has anti-inflammatory effects on adipose tissue, including adipocytes and adipose tissue macrophages (ATM).MethodsWe administered a recombinant adenovirus (rAd) producing GLP-1 (rAd-GLP-1) to an ob/ob mouse model of diabetes. We examined insulin sensitivity, body fat mass, the infiltration of ATM and metabolic profiles. We analysed the mRNA expression of inflammatory cytokines, lipogenic genes, and M1 and M2 macrophage-specific genes in adipose tissue by real-time quantitative PCR. We also examined the activation of nuclear factor κB (NF-κB), extracellular signal-regulated kinase 1/2 and Jun N-terminal kinase (JNK) in vivo and in vitro.ResultsFat mass, adipocyte size and mRNA expression of lipogenic genes were significantly reduced in adipose tissue of rAd-GLP-1-treated ob/ob mice. Macrophage populations (F4/80+ and F4/80+CD11b+CD11c+ cells), as well as the expression and production of IL-6, TNF-α and monocyte chemoattractant protein-1, were significantly reduced in adipose tissue of rAd-GLP-1-treated ob/ob mice. Expression of M1-specific mRNAs was significantly reduced, but that of M2-specific mRNAs was unchanged in rAd-GLP-1-treated ob/ob mice. NF-κB and JNK activation was significantly reduced in adipose tissue of rAd-GLP-1-treated ob/ob mice. Lipopolysaccharide-induced inflammation was reduced by the GLP-1 receptor agonist, exendin-4, in 3T3-L1 adipocytes and ATM.Conclusions/interpretationWe suggest that GLP-1 reduces macrophage infiltration and directly inhibits inflammatory pathways in adipocytes and ATM, possibly contributing to the improvement of insulin sensitivity.

A chemical chaperone 4-PBA ameliorates palmitate-induced inhibition of glucose-stimulated insulin secretion (GSIS).

Free fatty acids (FFAs) are believed to be a stimulus to elicit beta cell dysfunction. The present study was undertaken to determine whether endoplasmic reticulum (ER) stress was involved in palmitate-induced inhibition of glucose-stimulated insulin secretion (GSIS) and whether reduction of ER stress using a chemical chaperone restored the GSIS-inhibition. Treatment of INS-1 cells with 300 microM palmitate for 24h elicited ER stress, showing increased levels of phospho-eIF2alpha, Bip and spliced XBP, and also induced GSIS-inhibition without reduction of cell viability. Replenishment with 4-phenyl butyric acid (4-PBA) as a chemical chaperone reduced the palmitate-induced-ER stress and significantly reversed the palmitate-induced GSIS-inhibition. Furthermore, 4-PBA ameliorated palmitate-induced GSIS-inhibition in primary rat islet cells. These data suggested that ER stress was involved in FFA-induced GSIS-inhibition and that the FFA-induced beta cell dysfunction could be ameliorated by treatment with a chemical chaperone.

Stimulation of Lipogenesis as Well as Fatty Acid Oxidation Protects against Palmitate-Induced INS-1 β-Cell Death

Saturated fatty acids are generally cytotoxic to β-cells. Accumulation of lipid intermediates and subsequent activation of lipid-mediated signals has been suggested to play a role in fatty acid-induced toxicity. To determine the effects of lipid metabolism in fatty acid-induced toxicity, lipid metabolism was modulated by up- and down-regulation of a lipogenic or fatty acid oxidation pathway, and the effects of various modulators on palmitate (PA)-induced INS-1 β-cell death were then evaluated. Treatment with the liver X receptor agonist T0901317 reduced PA-induced INS-1 cell death, regardless of its enhanced lipogenic activity. Furthermore, transient expression of a lipogenic transcription factor sterol regulatory element binding protein-1c (SREBP-1c) was also protective against PA-induced cytotoxicity. In contrast, knockdown of SREBP-1c or glycerol-3-phosphate acyltransferase 1 significantly augmented PA-induced cell death and reduced T0901317-induced protective effects. Conversely, T0901317 increased carnitine PA transferease-1 (CPT-1) expression and augmented PA oxidation. CPT-1 inhibitor etomoxir or CPT-1 knockdown augmented PA-induced cell death and reduced T0901317-induced protective effects, whereas the peroxisome proliferator-activated receptor (PPAR)-α agonist bezafibrate reduced PA-induced toxicity. In particular, T0901317 reduced the levels of PA-induced endoplasmic reticulum (ER) stress markers, including phospho-eukaryotic initiation factor-2α, phospho-C-Jun N terminal kinase, and CCAAT/enhancer-binding protein homologous protein. In contrast, knockdown of SREBP-1c or glycerol-3-phosphate acyltransferase 1 augmented PA-induced ER stress responses. Results of these experiments suggested that stimulation of lipid metabolism, including lipogenesis and fatty acid oxidation, protected β-cells from PA-induced lipotoxicity and that protection through enhanced lipogenesis was likely due to reduced ER stress.

Interleukin‐6 treatment induces beta‐cell apoptosis via STAT‐3‐mediated nitric oxide production

Type 2 diabetes is characterized by progressive beta‐cell failure and apoptosis is probably the main form of beta‐cell death in this disease. It was reported that circulating levels of interleukin‐6 are elevated in type 2 diabetic patients, but whether this is involved in the pathogenesis of type 2 diabetes is still debated. In this study, we examined whether interleukin‐6 can induce beta‐cell damage in vitro and elucidated its mechanisms.

Betacellulin-Induced Beta Cell Proliferation and Regeneration Is Mediated by Activation of ErbB-1 and ErbB-2 Receptors

Background Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation. Methodology/Principal Findings The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes. Conclusions/Significance These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells.

Exendin-4 inhibits glucolipotoxic ER stress in pancreatic β cells via regulation of SREBP1c and C/EBPβ transcription factors.

Prolonged exposure to high glucose (HG) and palmitate (PA) results in increased ER stress and subsequently induces β-cell apoptosis. Exendin-4, a glucagon-like peptide-1 agonist, is known to protect β cells from toxicity induced by cytokines, HG, or fatty acids by reducing ER stress. However, the detailed molecular mechanisms for this protective effect are still not known. In this study, we investigated the role of exendin-4 in the inhibition of glucolipotoxicity-induced ER stress and β-cell apoptosis. Exendin-4 treatment protected INS-1 β cells from apoptosis in response to HG/PA (25 mM glucose+400 μM PA). HG/PA treatment increased cleaved caspase-3 and induced ER stress maker proteins such as PERK (EIF2AK3), ATF6, and phosphorylated forms of PERK, eIF2α, IRE1α (ERN1), and JNK (MAPK8), and these increases were significantly inhibited by exendin-4 treatment. HG/PA treatment of INS-1 cells increased SREBP1 (SREBF1) protein and induced its nuclear translocation and subsequently increased C/EBPβ (CEBPB) protein and its nuclear translocation. Exendin-4 treatment attenuated this increase. Knockdown of SREBP1c reduced the activation of C/EBPβ and also blocked the expression of ER stress markers induced by HG/PA treatment. Our results indicate that exendin-4 inhibits the activation of SREBP1c and C/EBPβ, which, in turn, may reduce glucolipotoxicity-induced ER stress and β-cell apoptosis.

Tumour necrosis factor-α-induced glucose-stimulated insulin secretion inhibition in INS-1 cells is ascribed to a reduction of the glucose-stimulated Ca2+ influx

The present study was undertaken to determine how tumour necrosis factor-alpha (TNF-alpha) elicits the inhibition of glucose-stimulated insulin secretion (GSIS) in rat insulinoma cells (INS)-1 beta-cells. TNF-alpha pretreatment did not change the expression levels of insulin, PDX-1, glucose transporter 2, glucokinase, K(ATP) channels, Ca(2)(+) channels, and exocytotic molecules and, furthermore, did not reduce the glucose-stimulated ATP level. On the other hand, TNF-alpha reduced the glucose-stimulated influx of Ca(2)(+). The TNF-alpha treatment was thought to activate c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inflammatory signals, since TNF-alpha increased phospho-JNK and phospho-p38 and reduced I kappaB levels. Inhibitors of these signaling pathways prevented the TNF-alpha-induced reduction of the Ca(2)(+) influx and GSIS. Overexpression of MEKK3, a possible mediator from the TNF-alpha receptor to the JNK/p38 and NK-kappaB signaling cascade, increased the levels of phospho-JNK, phospho-p38, and NF-kappaB, and reduced the glucose-stimulated Ca(2)(+) influx and GSIS. The reduction of the Ca(2)(+) influx and GSIS in MEKK3-overexpressing INS-1 cells was also prevented by inhibitors of JNK, p38, and NF-kappaB. These data demonstrate that TNF-alpha inhibits GSIS by reducing the glucose-stimulated Ca(2)(+) influx, possibly through the activation of JNK and p38 MAPK and NF-kappaB inflammatory signals. Thus, our findings suggest that the activation of stress and inflammatory signals can contribute to the inhibition of GSIS in the development of diabetes.

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death

The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.

Treatment with glucokinase activator, YH-GKA, increases cell proliferation and decreases glucotoxic apoptosis in INS-1 cells

Glucokinase (GK), an enzyme that phosphorylates glucose to form glucose-6-phosphate, has a role in regulating insulin secretion and proliferation in beta cells. GK activators (GKAs) have been developed as new therapies for type 2 diabetes. In this study, we evaluated the proliferation and anti-apoptotic actions of YH-GKA, a novel and potent GKA, in INS-1 pancreatic β-cells. YH-GKA treatment increased cell numbers at 3 mM glucose via upregulation of insulin receptor substrate-2 and subsequent activation of AKT/protein kinase B phosphorylation. YH-GKA also increased beta-catenin and cyclin D2 mRNA expression and inactivated GSK3β by increasing phosphorylation. These proliferative effects of YH-GKA were attenuated by IRS-2 downregulation. Moreover, YH-GKA reduced annexin-V-stained cells and expression levels of cleaved poly (ADP-ribose) polymerase and caspase-3 induced by glucotoxicity. YH-GKA inhibited apoptotic signaling via induction of ATP content, mitochondrial membrane potential, and citrate synthase activity and was correlated with changes of the mitochondrial function-related genes. YH-GKA also increased interaction between GK and voltage-dependent anion-selective channel protein. Our results suggest that the novel GKA, YH-GKA, promotes beta cell growth and prevents glucotoxic beta cell apoptosis. Therefore, YH-GKA may provide a therapy that compensates for beta cell loss in patients with type 2 diabetes.