cyanidin-3-o-beta-glucopyranoside and Fatty-Liver

Apoptosis is an early event of steatohepatitis in non-alcoholic fatty liver disease (NAFLD), and an increase in oxidative stress induced by hyperglycemia has been linked to an acceleration of apoptosis in hepatocytes. Cyanidin-3-O-β-glucoside (C3G), a classic anthocyanin, has been reported to reduce oxidative stress and attenuate non-alcoholic steatohepatitis in mice. In this study, we evaluated the toxicity of high glucose in primary hepatocytes of mice fed with a high fat diet and amelioration of this toxicity by C3G. Incubation of hepatocytes with 35mM glucose for 12h resulted in a significant decrease in cell viability and increase in apoptotic cell death. Furthermore, hyperglycemia-induced mitochondrial depolarization was accompanied by the release of cytochrome c and altered expression of Bax and Bcl-2, suggesting a mitochondria-mediated apoptotic mode of cell death. Pre-incubation with 50μM C3G induced changes associated with better cell survival and function, including a reduction in reactive species generation, improvement of mitochondrial membrane potential, inactivation of caspase-3 and -9, and down-regulation of the pro-apoptotic Bax protein. We further investigated the role of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the anti-apoptotic action of C3G, and our results showed that C3G could activate Akt. Additionally, C3G inactivated c-Jun N-terminal protein kinase (JNK), but not extracellular signal-regulated kinase or p38 MAPK, in glucose-stressed cells. Interestingly, JNK inhibitor enhanced the protective effect of C3G on cell survival. Our results suggest that anthocyanin C3G may exhibit hepatoprotective potential against NAFLD by promoting functional integrity and survival of hepatocytes.

Topics: Animals; Anthocyanins; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cytochromes c; Diet, High-Fat; Dietary Fats; Fatty Liver; Gene Expression Regulation; Glucose; Glucosides; Hepatocytes; JNK Mitogen-Activated Protein Kinases; Male; Membrane Potential, Mitochondrial; Mice; Mitochondria; Non-alcoholic Fatty Liver Disease; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinase; Primary Cell Culture; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction

Obesity is a major risk factor for the development of type 2 diabetes, and both conditions are now recognized to possess significant inflammatory components underlying their pathophysiologies. Here, we hypothesized that cyanidin 3-glucoside (C3G), a typical anthocyanin reported to possess potent anti-inflammatory properties, would ameliorate obesity-associated inflammation and metabolic disorders, such as insulin resistance and hepatic steatosis in mouse models of diabesity. Male C57BL/6J obese mice fed a high-fat diet for 12 weeks and genetically diabetic db/db mice at an age of 6 weeks received dietary C3G supplementation (0.2%) for 5 weeks. We found that dietary C3G lowered fasting glucose levels and markedly improved the insulin sensitivity in both high-fat diet fed and db/db mice as compared with unsupplemented controls. White adipose tissue messenger RNA levels and serum concentrations of inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1) were reduced by C3G, as did macrophage infiltration in adipose tissue. Concomitantly, hepatic triglyceride content and steatosis were alleviated by C3G. Moreover, C3G treatment decreased c-Jun N-terminal kinase activation and promoted phosphorylation and nuclear exclusion of forkhead box O1 after refeeding. These findings clearly indicate that C3G has significant potency in antidiabetic effects by modulating the c-Jun N-terminal kinase/forkhead box O1 signaling pathway and the related inflammatory adipocytokines.

Topics: Adipokines; Adipose Tissue, White; Animals; Anthocyanins; Chemokine CCL2; Diet, High-Fat; Dietary Fats; Fatty Liver; Forkhead Box Protein O1; Forkhead Transcription Factors; Glucosides; Hypoglycemic Agents; Insulin Resistance; Interleukin-6; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Signal Transduction; Triglycerides; Tumor Necrosis Factor-alpha

Enhanced oxidative stress due to high glucose contributes to pathological changes in diabetes-related liver complications. Reducing oxidative stress may alleviate these pathogenic processes. Anthocyanin, a natural antioxidant, has been reported to reduce intracellular reactive oxygen species (ROS) levels but the mechanism of this reduction is not fully understood. The glutathione (GSH) antioxidant system is critical for counteracting oxidative stress-induced intracellular injury. In this study, we evaluated the mechanism of the anthocyanin-mediated regulation of GSH synthesis and reduction in intracellular ROS levels. We observed that treatment of human HepG2 cells with the anthocyanin C3G significantly reduced ROS levels induced by high glucose. C3G incubation increased glutamate-cysteine ligase expression, which in turn mediated the reduction in ROS levels. However, the upregulation of glutamate-cysteine ligase catalytic subunit (Gclc) expression by C3G occurred independent of the Nrf1/2 transcription factors. Notably, the cAMP-response element binding protein (CREB) was identified as the target transcription factor involved in the C3G-mediated upregulation of Gclc expression. C3G increased phosphorylation of CREB through protein kinase A (PKA) activation, which induced a CREB-mediated upregulation of Gclc transcription. In vivo, treatment with C3G increased the GSH synthesis in the liver of diabetic db/db mice through PKA-CREB-dependent induction of Gclc expression. Finally, oxidative stress determined by lipid peroxidation, neutrophil infiltration, and hepatic steatosis was attenuated in C3G-treated db/db mice. Our results demonstrate that the anthocyanin C3G has an effect of activating GSH synthesis through a novel antioxidant defense mechanism against excessive ROS production, contributing to the prevention of hyperglycemia-induced hepatic oxidative damage.

Topics: Animals; Anthocyanins; Antioxidants; Apoptosis; Cell Survival; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cytoprotection; Enzyme Activation; fas Receptor; Fatty Liver; Gene Expression; Glucosides; Glutamate-Cysteine Ligase; Glutathione; Hep G2 Cells; Hepatocytes; Humans; Hyperglycemia; Liver; Male; MAP Kinase Kinase 4; Mice; Mice, Obese; Oxidative Stress; Protein Binding; Reactive Oxygen Species; Signal Transduction; Thiobarbituric Acid Reactive Substances; Up-Regulation