Jihyun Song

Coupling mitochondrial dysfunction to endoplasmic reticulum stress response: a molecular mechanism leading to hepatic insulin resistance.

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered critical components in the development of insulin resistance and Type 2 diabetes. However, understanding the molecular mechanisms underlying these individual disorders and how they are linked has been challenging. Here, we provide evidence that elevated levels of cytosolic free Ca(2+) due to mitochondrial dysfunction and concomitant activation of p38 mitogen activated protein kinase (MAPK) induce ER stress response in human liver sk-HepI cells. Blocking Ca(2+) release from mitochondria or ER using ruthenium red or ryanodine ameliorated the increase in expression of gluconeogenic enzymes due to mitochondrial dysfunction. Disturbance in mitochondrial function results in the activation of p38 MAPK and related transcription factors that are directly responsible for increased phosphoenolpyruvate carboxykinase (PEPCK) expression. In addition, abnormal activation of c-Jun N-terminal kinase (JNK) influences the PEPCK expression by affecting insulin signaling and Forkhead box O (Foxo) 1 activity. Alleviation of ER stress response using a chemical chaperone reduces p38 MAPK activation, as well as PEPCK overexpression, indicating that ER stress response strengthens mitochondrial stress-induced abnormalities. Our results demonstrate that mitochondrial dysfunction is directly linked to the ER stress response, and together, cause aberrant insulin signaling and an abnormal increase of hepatic gluconeogenesis.

Therapeutic Potential of Peroxisome Proliferators–Activated Receptor-α/γ Dual Agonist With Alleviation of Endoplasmic Reticulum Stress for the Treatment of Diabetes

OBJECTIVE—Peroxisome proliferator–activated receptor (PPAR) α/γ dual agonists have the potential to be used as therapeutic agents for the treatment of type 2 diabetes. This study evaluated the function of macelignan, a natural compound isolated from Myristica fragrans, as a dual agonist for PPARα/γ and investigated its antidiabetes effects in animal models. RESEARCH DESIGN AND METHODS—GAL4/PPAR chimera transactivation was performed and the expression of PPARα/γ target genes was monitored to examine the ability of macelignan to activate PPARα/γ. Additionally, macelignan was administrated to obese diabetic (db/db) mice to investigate antidiabetes effects and elucidate its molecular mechanisms. RESULTS—Macelignan reduced serum glucose, insulin, triglycerides, free fatty acid levels, and triglycerides levels in the skeletal muscle and liver of db/db mice. Furthermore, macelignan significantly improved glucose and insulin tolerance in these mice, and without altering food intake, their body weights were slightly reduced while weights of troglitazone-treated mice increased. Macelignan increased adiponectin expression in adipose tissue and serum, whereas the expression and serum levels of tumor necrosis factor-α and interleukin-6 decreased. Macelignan downregulated inflammatory gene expression in the liver and increased AMP-activated protein kinase activation in the skeletal muscle of db/db mice. Strikingly, macelignan reduced endoplasmic reticulum (ER) stress and c-Jun NH2-terminal kinase activation in the liver and adipose tissue of db/db mice and subsequently increased insulin signaling. CONCLUSIONS—Macelignan enhanced insulin sensitivity and improved lipid metabolic disorders by activating PPARα/γ and attenuating ER stress, suggesting that it has potential as an antidiabetes agent for the treatment of type 2 diabetes.

Mitochondrial dysfunction induces aberrant insulin signalling and glucose utilisation in murine C2C12 myotube cells

Aims/hypothesisMitochondrial dysfunction is considered a critical component in the development of diabetes. The aim of this study was to elucidate the molecular mechanisms involved in the development of insulin resistance and diabetes through investigation of mitochondrial retrograde signalling.Materials and methodsMitochondrial function of C2C12 myotube cells was impaired by genetic (ethidium bromide) and metabolic (oligomycin) stress, and changes in target molecules related to insulin signalling were analysed.ResultsConcomitant with reductions in mitochondrial membrane potential (ΔΨm) and ATP synthesis, production of IRS1 and solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4, formerly known as GLUT4) were reduced. Moreover, serine phosphorylation of IRS1 increased, resulting in decreased tyrosine phosphorylation. This indicates that mitochondrial dysfunction decreases insulin-stimulated SLC2A4 translocation and glucose uptake. Mitochondrial stress activated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) signalling in a Ca2+-dependent manner, and removal of free Ca2+ by BAPTA-AM, as well as inhibition of JNK and p38 MAPK, abrogated mitochondrial stress-induced reductions in IRS1 and SLC2A4 production. Mitochondrial dysfunction after oligomycin treatment significantly increased levels of activating transcription factor 3 (ATF3), which represses Irs1 promoter activity. Removal of the 5′ flanking region of Irs1 demonstrated that the promoter region within 191 bases from the transcription site may be involved in the transcriptional repression of Irs1 by mitochondrial stress.Conclusions/interpretationWe show distinct mitochondrial retrograde signalling, where Irs1 is downregulated through ATF3 in a Ca2+-, JNK- and p38 MAPK-dependent manner, and IRS1 is inactivated. Therefore, mitochondrial dysfunction causes aberrant insulin signalling and abnormal utilisation of glucose, as observed in many insulin resistance states.

Consumption of barley β‐glucan ameliorates fatty liver and insulin resistance in mice fed a high‐fat diet

Consumption of a diet high in barley beta-glucan (BG) has been shown to prevent insulin resistance. To investigate the mechanism for the effects of barley BG, three groups of male 7-wk-old C57BL/6J mice were fed high-fat diets containing 0, 2, or 4% of barley BG for 12 wk. The 2% BG and 4% BG groups had significantly lower body weights compared with the 0% BG group. The 4% BG group demonstrated improved glucose tolerance and lower levels of insulin-resistance index and glucose-dependent insulinotropic polypeptide. Consumption of the BG diet decreased hepatic lipid content. Mice on the BG diet also demonstrated decreased fatty acid synthase and increased cholesterol 7alpha-hydroxylase gene expression levels. The BG diet promoted hepatic insulin signaling by decreasing serine phosphorylation of insulin receptor substrate 1 and activating Akt, and it decreased mRNA levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. In summary, consumption of BG reduced weight gain, decreased hepatic lipid accumulation, and improved insulin sensitivity in mice fed a high-fat diet. Insulin signaling enhanced due to the expression changes of glucose and lipid metabolism genes by BG consumption. Consumption of barley BG could be an effective strategy for preventing obesity, insulin resistance, and the metabolic syndrome.

Effects of three different conjugated linoleic acid preparations on insulin signalling, fat oxidation and mitochondrial function in rats fed a high-fat diet.

To investigate the effects of three different conjugated linoleic acid (CLA) preparations containing different ratios of CLA isomers on insulin signalling, fatty acid oxidation and mitochondrial function, Sprague-Dawley rats were fed a high-fat diet either unsupplemented or supplemented with one of three CLA preparations at 1 % of the diet for 8 weeks. The first CLA preparation contained approximately 30 % cis-9, trans-11 (c9, t11)-CLA isomer and 40 % trans-10, cis-12 (t10, c12)-CLA isomer (CLA-mix). The other two preparations were an 80:20 mix (c9, t11-CLA-mix) or a 10:90 mix of two CLA isomers (t10, c12-CLA-mix). Insulin resistance was decreased in all three supplemented groups based on the results of homeostasis model assessment and the revised quantitative insulin-sensitivity check index. The phosphorylation of insulin receptor substrate-1 on serine decreased in the livers of all three supplemented groups, while subsequent Akt phosphorylation increased only in the t10, c12-CLA-mix group. Both the c9, t11-CLA-mix and the t10, c12-CLA-mix increased the expression of hepatic adiponectin receptors R1 and 2, which are thought to enhance insulin sensitivity and fat oxidation. The c9, t11-CLA-mix increased protein and mRNA levels of PPAR alpha, acyl-CoA oxidase and uncoupling protein, which are involved in fatty acid oxidation and energy dissipation. The c9, t11-CLA-mix enhanced mitochondrial function and protection against oxidative stress by increasing the activities of cytochrome c oxidase, manganese-superoxide dismutase, glutathione peroxidase, and glutathione reductase and the level of GSH. In conclusion, all three CLA preparations reduced insulin resistance. Among them, the c9, t11-CLA-mix was the most effective based on the parameters reflecting insulin resistance and fat oxidation, and mitochondrial antioxidative enzyme activity in the liver.

Chronic ethanol consumption-induced pancreatic β-cell dysfunction and apoptosis through glucokinase nitration and its down-regulation.

Chronic ethanol consumption is known as an independent risk factor for type 2 diabetes, which is characterized by impaired glucose homeostasis and insulin resistance; however, there is a great deal of controversy concerning the relationships between alcohol consumption and the development of type 2 diabetes. We investigated the effects of chronic ethanol consumption on pancreatic β-cell dysfunction and whether generated peroxynitrite participates in the impaired glucose homeostasis. Here we show that chronic ethanol feeding decreases the ability of pancreatic β-cells to mediate insulin secretion and ATP production in coordination with the decrease of glucokinase, Glut2, and insulin expression. Specific blockade of ATF3 using siRNA or C-terminally deleted ATF3(ΔC) attenuated ethanol-induced pancreatic β-cell apoptosis or dysfunction and restored the down-regulation of glucokinase (GCK), insulin, and pancreatic duodenal homeobox-1 induced by ethanol. GCK inactivation and down-regulation were predominantly mediated by ethanol metabolism-generated peroxynitrite, which were suppressed by the peroxynitrite scavengers Nγ-monomethyl-l-arginine, uric acid, and deferoxamine but not by the S-nitrosylation inhibitor DTT, indicating that tyrosine nitration is the predominant modification associated with GCK down-regulation and inactivation rather than S-nitrosylation of cysteine. Tyrosine nitration of GCK prevented its association with pBad, and GCK translocation into the mitochondria results in subsequent proteasomal degradation of GCK following ubiquitination. This study identified a novel and efficient pathway by which chronic ethanol consumption may induce GCK down-regulation and inactivation by inducing tyrosine nitration of GCK, resulting in pancreatic β-cell apoptosis and dysfunction. Peroxynitrite-induced ATF3 may also serve as a potent upstream regulator of GCK down-regulation and β-cell apoptosis.

Mitochondrial dysfunction: Glucokinase downregulation lowers interaction of glucokinase with mitochondria, resulting in apoptosis of pancreatic β-cells

Mitochondrial dysfunction has been considered a critical component in the development of diabetes. In pancreatic beta-cells especially, mitochondrial dysfunction impairs insulin secretion and the eventual apoptosis of beta-cells. The aim of this study was to elucidate the molecular mechanism underlying these events. Metabolic stress induced by antimycin or oligomycin was used to impair mitochondrial function in MIN6N8 cells, a mouse pancreatic beta-cells, and the effects of glucokinase (GCK) and mitochondria were investigated. Concurrent with reduction in mitochondrial membrane potential (DeltaPsim) and cellular ATP content, impaired mitochondrial function reduced GCK expression and resulted in decreased insulin secretion and beta-cell apoptosis. Specifically, lowered GCK expression led to decreased interactions between GCK and mitochondria, which increased Bax binding to mitochondria and cytochrome C release into cytoplasm. However, these events were blocked by treatment with the antioxidant, N-acetyl-cysteine (NAC), as well as GCK overexpression. Moreover, examination of the GCK promoter in antimycin-treated cells demonstrated that the promoter region within -287 bases from transcription site is involved in the transcriptional repression of GCK by mitochondrial stress, whose region contains a putative binding site for pancreatic duodenal homeobox-1 (PDX-1). Mitochondrial stress reduced PDX-1 expression, and increased ATF3 expression dependent on reactive oxygen species (ROS). Collectively, these data demonstrate that mitochondrial dysfunction by metabolic stress reduces GCK expression through PDX-1 downregulation via production of ROS, which then decreases the association of GCK with mitochondria, resulting in pancreatic beta-cell apoptosis and reduction of insulin secretion.

Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic β-cells

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic beta-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic beta-cells, two Nnat isotypes (alpha and beta) were expressed using adenoviruses in murine MIN6N8 pancreatic beta-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatalpha and Nnatbeta were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatbeta to Nnatalpha ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatbeta was inclined to form aggresome-like structures, and we found that Nnatbeta aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatbeta sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatbeta increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatbeta under high glucose conditions decreased the expression of genes important for beta-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal beta-cell function, as well as in the pathogenesis of type 2 diabetes.

AdipoR2 is transcriptionally regulated by ER stress‐inducible ATF3 in HepG2 human hepatocyte cells

Adiponectin acts as an insulin‐sensitizing adipokine that protects against obesity‐linked metabolic disease, which is generally associated with endoplasmic reticulum (ER) stress. The physiological effects of adiponectin on energy metabolism in the liver are mediated by its receptors. We found that the hepatic expression of adiponectin receptoru20032 (AdipoR2) was lower, but the expression of markers of the ER stress pathway, 78 kDa glucose‐regulated protein (GRP78) and activating transcription factoru20033 (ATF3), was higher in the liver of ob/ob mice compared with control mice. To investigate the regulation of AdipoR2 by ER stress, we added thapsigargin, an ER stress inducer, to a human hepatocyte cell line, HepG2. Addition of the ER stress inducer increased the levels of GRP78 and ATF3, and decreased that of AdipoR2, whereas addition of a chemical chaperone, 4‐phenyl butyric acid (PBA), could reverse them. Up‐ or down‐regulation of ATF3 modulated the AdipoR2 protein levels and AdipoR2 promoter activities. Reporter gene assays using a series of 5′‐deleted AdipoR2 promoter constructs revealed the location of the repressor element responding to ER stress and ATF3. In addition, using electrophoretic mobility shift and chromatin immunoprecipitation assays, we identified a region between nucleotides −94 and −86 of the AdipoR2 promoter that functions as a putative ATF3‐binding site inu2003vitro and inu2003vivo. Thus, our findings suggest that the ER stress‐induced decrease in both protein and RNA of AdipoR2 results from a concomitant increase in expression of ATF3, which may play a role in the development of obesity‐induced insulin resistance and related ER stress in hepatocytes.

Effect of genistein on insulin resistance, renal lipid metabolism, and antioxidative activities in ovariectomized rats.

OBJECTIVESnPostmenopausal women develop obesity, insulin resistance, and potentially renal dysfunction because of decreased serum estrogen levels. We investigated the effects of genistein, an estrogen-like compound thought to exert antioxidative effects, on insulin resistance, renal lipid accumulation, and oxidative stress in ovariectomized rats.nnnMETHODSnThree weeks after an ovariectomy or a sham surgery, rats were put on a high-fat diet containing 0% or 0.1% genistein for 4 wk. We examined the following treatment groups: sham surgery + high-fat diet (sham), ovariectomy + high-fat diet (OVX), and ovariectomy + high-fat diet with 0.1% genistein (OVX + G).nnnRESULTSnThe OVX + G group had increased serum estradiol levels and renal expression of estrogen receptors-alpha and -beta compared with the OVX group. OVX + G rats showed decreases in serum insulin levels and the insulin resistance index. OVX + G rats also exhibited decreased renal triacylglycerol and cholesterol levels, which may have been the result of decreased sterol response element binding protein-1 and -2 expressions, and increased adenosine triphosphate-binding cassette transporter-1 and adiponectin receptor expression. The observed increases in renal lipid levels and serum and renal transforming growth factor-beta in OVX rats may be associated with the increased expression of extracellular matrix proteins, such as fibronectin, and the decreased activity of metalloproteinase-2, an extracellular matrix-degrading enzyme. Ovariectomy also induced oxidative stress by the reduction of antioxidative enzymes, whereas genistein reversed these detrimental ovariectomy-induced effects.nnnCONCLUSIONnGenistein may help to maintain normal kidney function through the alleviation of many ovariectomy-induced risk factors for renal damage, including an increased insulin resistance index, renal oxidative stress, lipid accumulation, and extracellular matrix protein expression.