GLP-1 regulates exercise endurance and skeletal muscle remodeling via GLP-1R/AMPK pathway.

Exercise-induced physical endurance enhancement and skeletal muscle remodeling can prevent and delay the development of multiple diseases, especially metabolic syndrome. Herein, the study explored the association between glucagon-like peptide-1 (GLP-1) secretion and exercise, and its effect on skeletal muscle remodeling to enhance endurance capacity. We found both acute exercise and short-term endurance training significantly increased the secretion of GLP-1 in mice. Recombinant adeno-associated virus (AAV) encoding Gcg (proglucagon) was used to induce the overexpression of GLP-1 in skeletal muscle of mice. Overexpression of GLP-1 in skeletal muscle enhanced endurance capacity. Meanwhile, glycogen synthesis, glucose uptake, type I fibers proportion, and mitochondrial biogenesis were augmented in GLP-1-AAV skeletal muscle. Furthermore, the in vitro experiment showed that exendin-4 (a GLP-1 receptor agonist) treatment remarkably promoted glucose uptake, type I fibers formation, and mitochondrial respiration. Mechanistically, the knockdown of AMPK could reverse the effects imposed by GLP-1R activation in vitro. Taken together, these results verify that GLP-1 regulates skeletal muscle remodeling to enhance exercise endurance possibly via GLP-1R signaling-mediated phosphorylation of AMPK.

Resveratrol prevents sarcopenic obesity by reversing mitochondrial dysfunction and oxidative stress via the PKA/LKB1/AMPK pathway.

BACKGROUND: The concept of sarcopenic obesity refers to low muscle mass coupled with high adiposity in older adults. Sarcopenic obesity is a new medical challenge that imposes tremendous financial burdens on healthcare authorities worldwide. This study investigated the effects of resveratrol on high-fat diet-induced sarcopenic obesity in aged rats and palmitate acid-induced muscle atrophy in L6 myotubes and explored the underlying mechanisms. RESULTS: In vivo, resveratrol prevented muscle loss and myofiber size decrease, improved grip strength and abolished excessive fat accumulation. In vitro, resveratrol inhibited the palmitate acid-mediated reductions in myosin heavy chain content and myotube diameter. Moreover, resveratrol ameliorated mitochondrial dysfunction and oxidative stress, leading to an improvement in protein metabolism and contributing to the prevention of muscle atrophy. Furthermore, the protective effects of resveratrol on mitochondrial function, oxidative stress and muscle atrophy were abolished by PKA siRNA, LKB1 siRNA and AMPK siRNA transfection in vitro. CONCLUSIONS: Resveratrol prevented high-fat diet-induced muscle atrophy in aged rats by reversing mitochondrial dysfunction and oxidative stress, which was partially mediated by the PKA/LKB1/AMPK pathway. These findings indicate that resveratrol might have potential uses for the prevention and treatment of sarcopenic obesity.

Dihydromyricetin Ameliorates Nonalcoholic Fatty Liver Disease by Improving Mitochondrial Respiratory Capacity and Redox Homeostasis Through Modulation of SIRT3 Signaling.

Aims: Our previous clinical trial indicated that the flavonoid dihydromyricetin (DHM) could improve hepatic steatosis in patients with nonalcoholic fatty liver disease (NAFLD), altough the potential mechanisms of these effects remained elusive. Here, we investigated the hepatoprotective role of DHM on high-fat diet (HFD)-induced NAFLD. Results: DHM supplementation could effectively ameliorate the development of NAFLD by inhibiting hepatic lipid accumulation both in HFD-fed wild-type mice and in palmitic acid-induced hepatocytes. We reveal for the first time that mitochondrial dysfunction characterized by ATP depletion and augmented oxidative stress could be reversed by DHM treatment. Moreover, DHM enhanced the mitochondrial respiratory capacity by increasing the expression and enzymatic activities of mitochondrial complexes and increased mitochondrial reactive oxygen species scavenging by restoring manganese superoxide dismutase (SOD2) activity. Interestingly, the benefits of DHM were abrogated in SIRT3 knockout (SIRT3KO) mice and in hepatocytes transfected with SIRT3 siRNA or treated with an SIRT3-specific inhibitor. We further showed that DHM could increase SIRT3 expression by activating the adenosine monophosphate-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC1α)/estrogen-related receptor-α (ERRα) signaling pathway. Innovation: Our work indicates that SIRT3 plays a critical role in the DHM-mediated beneficial effects that include ameliorating mitochondrial dysfunction and oxidative stress in a nutritional NAFLD model both in vivo and in vitro.Conclusion: Our results suggest that DHM prevents NAFLD by improving mitochondrial respiratory capacity and redox homeostasis in hepatocytes through a SIRT3-dependent mechanism. These results could provide a foundation to identify new DHM-based preventive and therapeutic strategies for NAFLD.

Resveratrol Ameliorates Lipid Droplet Accumulation in Liver Through a SIRT1/ ATF6-Dependent Mechanism.

BACKGROUND/AIMS: Lipid droplets (LDs) are dynamic organelles that store neutral lipids during times of energy excess, and an increased accumulation of LDs in the liver is closely linked to hepatic steatosis. Our previous studies suggested that resveratrol (RSV) supplement could improve hepatic steatosis, but the underlying mechanism, particularly which related to LD accumulation, has not yet been elucidated. METHODS: A high-fat diet (HFD) and palmitic acid were used to induce hepatic steatosis in mouse liver and hepatocytes, respectively. The effects of RSV on LD accumulation were analyzed in vivo and in vitro. The effects of RSV on the expression levels of LD-associated genes (ATF6, Fsp27ß/CIDEC, CREBH, and PLIN1) were measured by qRT-PCR and western blot assays, followed by KD or overexpression of SIRT1 and ATF6 with small interfering RNAs or overexpressed plasmids, respectively. The dual luciferase reporter assay, chromatin immunoprecipitation assay, coimmunoprecipitation, and proximity ligation assay were utilized to clarify the mechanism of transcriptional regulation and possible interaction between SIRT1 and ATF6. RESULTS: There was a significant increase in the accumulation of LDs in liver and hepatocytes during the process of HFD-induced steatosis, respectively, which was significantly inhibited by RSV supplementation. RSV notably activated SIRT1 expression and decreased the expression levels of ATF6, Fsp27ß/CIDEC, CREBH, and PLIN1, which are associated with LD accumulation. Interestingly, the inhibitory effects of RSV on LD accumulation and the associated expression of genes in hepatocytes were abrogated or strengthened with SIRT1 silencing or overexpression, respectively. On the contrary, the benefits of RSV in hepatocytes were eliminated or aggravated when transfected with the overexpressed ATF6 or ATF6 siRNA, respectively. Furthermore, we found that RSV stimulated SIRT1 expression significantly, which was followed by increased deacetylation and inactivation of ATF6, resulting in a positive feedback loop for SIRT1 transcription associated with ATF6 binding to the SIRT1 promoter region. CONCLUSION: Taken together, these findings indicate that RSV supplementation improves hepatic steatosis by ameliorating the accumulation of LDs, and this might be partially mediated by a SIRT1/ATF6-dependent mechanism.

Dietary Factors Modulate Colonic Tumorigenesis Through the Interaction of Gut Microbiota and Host Chloride Channels.

SCOPE: In recent decades, the association among diet, gut microbiota, and the risk of colorectal cancer (CRC) has been established. Gut microbiota and associated metabolites, such as bile acids and butyrate, are now known to play a key role in CRC development. The aim of this study is to identify that the progression to CRC is influenced by cholic acid, sodium butyrate, a high-fat diet, or different dose of dihydromyricetin (DMY) interacted with gut microbiota. METHODS AND RESULTS: An AOM/DSS (azoxymethan/dextran sodium sulfate) model is established to study the gut microbiota compsition before and after tumor formation during colitis-induced tumorigenesis. All above dietary factors profoundly influence the composition of gut microbiota and host colonic tumorigenesis. In addition, mice with DMY-modified initial microbiota display different degrees of chemically induced tumorigenesis. Mechanism analysis reveals that gut microbiota-associated chloride channels participated in colon tumorigenesis. CONCLUSION: Gut microbiota changes occur in the hyperproliferative stage before tumor formation. Gut microbiota and host chloride channels, both of which are regulated by dietary factors, are associated with CRC development.

3,6-Dihydroxyflavone regulates microRNA-34a through DNA methylation.

BACKGROUND: Breast cancer is the common cancer in China. In previous study, we determined that 3,6-dihydroxyflavone (3,6-DHF) increases miR-34a significantly in breast carcinogenesis, but the mechanism remains unclear. METHODS: We used qRT-PCR to analyze miR-34a and ten-eleven translocation (TET)1, TET2, TET3 levels in breast cancer cells. With a cellular breast carcinogenesis model and an experimental model of carcinogenesis in rats, TET1 levels were evaluated by western blot analysis and immunofluorescence. TET1 and 5hmC (5-hydroxymethylcytosine) levels were evaluated by immunofluorescence in nude mouse xenografts of MDA-MB-231 cells. Chromatin immunoprecipitation(ChIP) assayed for TET1 on the TET1 promoter, and dot blot analysis of DNA 5hmC was performed in MDA-MB-231 cells. We evaluated the mechanism of 3,6-DHF on the expression of tumor suppressor miR-34a by transfecting them with DNA methyltransferase (DNMT)1 plasmid and TET1 siRNA in breast cancer cells. Methylation-specific PCR detected methylation of the miR-34a promoter. RESULTS: First, we found that 3,6-DHF promotes the expression of TET1 during carcinogen-induced breast carcinogenesis in MCF10A cells and in rats. 3,6-DHF also increased TET1 and 5hmC levels in MDA-MB-231 cells. Further study indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibited the 3,6-DHF reactivation effect on expression of miR-34a in breast cancer cells. Methylation-specific PCR assays indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibit the effect of 3,6-DHF on the demethylation of the miR-34a promoter. CONCLUSIONS: Our study showed that 3,6-DHF effectively increases TET1 expression by inhibiting DNMT1 and DNA hypermethylation, and consequently up-regulates miR-34a in breast carcinogenesis.

Equol Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice by Inhibiting Endoplasmic Reticulum Stress via Activation of Nrf2 in Endothelial Cells.

The development of atherosclerosis is closely related to excessive endoplasmic reticulum stress (ERs). Equol reportedly protects against cardiovascular disease; however, the underlying mechanism for this protection remains unknown. Herein, the mechanisms contributing to the atheroprotective effect of equol were addressed using apolipoprotein E knockout (apoE-/-) mice fed a high-fat diet (HFD) with or without equol. Equol intervention reduced atherosclerotic lesions in the aorta in HFD-fed apoE-/- mice. Plasma lipid analysis showed that equol intervention reduced triglycerides, total cholesterol and LDL-cholesterol and increased HDL-cholesterol. Additionally, equol administration decreased lipid accumulation in the liver. Simultaneously, equol treatment inhibited cell apoptosis induced by t-BHP and thapsigargin in human umbilical vein endothelial cells (HUVECs). Furthermore, equol treatment attenuated palmitate, t-BHP or thapsigargin-induced upregulation of ER stress markers, including p-PERK, p-eIF2α, GRP78, ATF6 and CHOP proteins expression. The same tendency was also observed in aortic lysates in apoE-/- mice fed with equol plus HFD compared with HFD alone. Moreover, equol treatment dose dependently activated the Nrf2 signaling pathway under oxidative stress. Additionally, elevation of Nrf2 induction was found in aortic lysates in apoE-/- mice fed with a HFD diet containing equol compared with a HFD diet without equol. Importantly, Nrf2 siRNA interference induced CHOP and attenuated the effect of equol to inhibit t-BHP mediated CHOP induction, furthermore, abrogated cell apoptosis induced by t-BHP, suggesting a role for Nrf2 in the protective effect of equol in HUVECs. Collectively, these findings implicate that the improvement of atherosclerosis by equol through attenuation of ER stress is mediated, at least in part, by activating the Nrf2 signaling pathway.

Healthy Subjects Differentially Respond to Dietary Capsaicin Correlating with Specific Gut Enterotypes.

CONTEXT: Previous population studies in evaluating the beneficial effects of capsaicin (CAP) have yielded inconclusive results, and the mechanisms responsible for possible benefit remain unclear. OBJECTIVE: The objective was to assess the effect of dietary CAP on metabolic and immune profiles and its potential associations with gut microbial patterns in healthy adults. DESIGN: In a 6-week controlled feeding trial, subjects were given the weight maintenance diet sequentially contained with 0, 5, 0, and 10 mg/d CAP from chili powder. SETTING AND PARTICIPANTS: The study was conducted in 12 healthy subjects enrolled in Third Military Medical University in Chongqing. MAIN OUTCOME MEASURES: At the end of each period, anthropometric and basal metabolism measures together with blood and fecal samples were collected. Plasma metabolic and inflammatory markers and gut microbial ecology of each subject were subsequently assessed. RESULT: Dietary CAP increased the Firmicutes/Bacteroidetes ratio and Faecalibacterium abundance, accompanied with increased plasma levels of glucagon-like peptide 1 and gastric inhibitory polypeptide and decreased plasma ghrelin level. Further enterotype analysis revealed that these subjects could be clustered into Bacteroides enterotype (E1) and Prevotella enterotype (E2), and the above beneficial effects were mainly obtained in E1 subjects. Moreover, E1 subjects had significantly higher fecal Faecalibacterium abundance and butyrate concentration after CAP interventions than those in E2 subjects. CONCLUSION: Our study showed that gut enterotypes may influence the beneficial effects of dietary CAP, providing new evidence for the personalized nutrition guidance of CAP intervention on health promotion linking with gut microbiota patterns.

Ampelopsin Improves Insulin Resistance by Activating PPARγ and Subsequently Up-Regulating FGF21-AMPK Signaling Pathway.

Ampelopsin (APL), a major bioactive constituent of Ampelopsis grossedentata, exerts a number of biological effects. Here, we explored the anti-diabetic activity of APL and elucidate the underlying mechanism of this action. In palmitate-induced insulin resistance of L6 myotubes, APL treatment markedly up- regulated phosphorylated insulin receptor substrate-1 and protein kinase B, along with a corresponding increase of glucose uptake capacity. APL treatment also increased expressions of fibroblast growth factor (FGF21) and phosphorylated adenosine 5'-monophosphate -activated protein kinase (p-AMPK), however inhibiting AMPK by Compound C or AMPK siRNA, or blockage of FGF21 by FGF21 siRNA, obviously weakened APL -induced increases of FGF21 and p-AMPK as well as glucose uptake capacity in palmitate -pretreated L6 myotubes. Furthermore, APL could activate PPAR γ resulting in increases of glucose uptake capacity and expressions of FGF21 and p-AMPK in palmitate -pretreated L6 myotubes, whereas all those effects were obviously abolished by addition of GW9662, a specific inhibitor of peroxisome proliferator- activated receptor -γ (PPARγ), and PPARγsiRNA. Using molecular modeling and the luciferase reporter assays, we observed that APL could dock with the catalytic domain of PPARγ and dose-dependently up-regulate PPARγ activity. In summary, APL maybe a potential agonist of PPARγ and promotes insulin sensitization by activating PPARγ and subsequently regulating FGF21- AMPK signaling pathway. These results provide new insights into the protective health effects of APL, especially for the treatment of Type 2 diabetes mellitus.

3,6-dihydroxyflavone suppresses the epithelial-mesenchymal transition in breast cancer cells by inhibiting the Notch signaling pathway.

The epithelial to mesenchymal transition (EMT) is a critical developmental program in cancer stem cell (CSC) maintenance and in cancer metastasis. Here, our study found that 3,6-DHF could effectively inhibit EMT in BC cells in vitro and in vivo. 3,6-DHF effectively inhibits the formation and proliferation of BCSCs, and consequently reduces the tumor-initiating capacity of tumor cells in NOD/SCID mice. Optical in vivo imaging of cancer metastasis showed that 3,6-DHF administration suppresses the lung metastasis of BC cells in vivo. Further studies indicated that 3,6-DHF down-regulates Notch1, NICD, Hes-1 and c-Myc, consequently decreasing the formation of the functional transcriptional unit of NICD-CSL-MAML, causing Notch signaling inactivation in BC cells. Over-expression of Notch1 or inhibition of miR-34a significantly reduced the inhibitory effects of 3,6-DHF on EMT, CSCs, as well as cells migration and invasion in BC cells. These data indicated that 3,6-DHF effectively inhibits EMT and CSCs, as well as cells migration and invasion in BC cells, in which miR-34a-mediated Notch1 down-regulation plays a crucial role.

Dihydromyricetin Improves Hypobaric Hypoxia-Induced Memory Impairment via Modulation of SIRT3 Signaling.

Inadequate oxygen availability-for instance at high altitudes-leads to hippocampal neurodegeneration and memory impairment. Although oxidative stress is one factor, the mechanism underlying the effects of hypobaric hypoxia (HH) are unclear, and effective strategies for preventing the resultant damage to the brain are limited. In the present study, we demonstrate that ingesting dihydromyricetin (DM) protects against memory impairment in adult rats subjected to HH for 7 days, equivalent to an altitude of 5000 m above sea level. Moreover, DM treatment stimulated mitochondrial biogenesis and improved mitochondrial morphology and function, suppressed the generation of reactive oxygen species, and reduced lipid peroxidation in the hippocampus. In HT-22 cells exposed to hypoxic conditions, the neuroprotective effects of DM were shown to be exerted via attenuation of oxidative stress through sirtuin 3-induced forkhead box O3 deacetylation.

Fish Oil Supplements Lower Serum Lipids and Glucose in Correlation with a Reduction in Plasma Fibroblast Growth Factor 21 and Prostaglandin E2 in Nonalcoholic Fatty Liver Disease Associated with Hyperlipidemia: A Randomized Clinical Trial.

UNLABELLED: Fish oil has been used effectively in the treatment of cardiovascular disease via triglyceride reduction and inflammation modulation. This study aimed to assess the effects of fish oil on patients with nonalcoholic fatty liver disease (NAFLD) associated with hyperlipidemia. Eighty participants with NAFLD associated with hyperlipidemia were randomly assigned to consume fish oil (n=40, 4 g/d) or corn oil capsules (n=40, 4 g/d) for 3 months in a double-blind, randomized clinical trial. Blood levels of lipids, glucose and insulin, liver enzymes, kidney parameters and cytokines at baseline and the end of the study were measured. Seventy people finished the trial. Plasma concentrations of eicosapentaenoic acid and docosahexaenoic acid significantly increased in the fish oil group after intervention. After adjustment for age, gender and BMI, fish oil significantly decreased fasting serum concentrations of total cholesterol, triglyceride, apolipoprotein B and glucose (by (mean±SD) 0.49±0.43 mmol/L, 0.58±0.89 mmol/L, 0.28±0.33 g/L and 0.76±0.56 mmol/L, respectively, P<0.05), as well as alanine aminotransferase and γ-glutamyl transpeptidase levels (by (median (interquartile)) 9.0(0.5, 21.5) and 7.0(2.2, 20.0) IU/L, respectively, P<0.05), significantly increased serum adiponectin levels (by 1.29±0.62 µg/mL, P<0.001), and reduced serum levels of tumor necrosis factor α, leukotrienes B4, fibroblast growth factor 21 (FGF21), cytokeratin 18 fragment M30 and prostaglandin E2 (by 1.70±1.18 pg/mL, 0.59±0.28 ng/mL, 121±31 pg/mL, 83±60 IU/L and 10.9±2.3 pg/mL, respectively, P<0.001). Corn oil had no effect except for increasing serum creatinine concentrations by 7.7±8.9 µmol/L (P=0.008). The effects of fish oil on lipids, glucose and γ-glutamyl transpeptidase were positively correlated with the reductions of serum FGF21 and prostaglandin E2 concentrations after adjustment for age, gender and BMI (r = 0.275 to 0.360 and 0.261 to 0.375, respectively, P<0.05). In conclusion, our findings suggest that fish oil can benefit metabolic abnormalities associated with NAFLD treatment. TRIAL REGISTRATION: ChiCTR-TRC-12002380.

Dihydromyricetin improves glucose and lipid metabolism and exerts anti-inflammatory effects in nonalcoholic fatty liver disease: A randomized controlled trial.

Ampelopsis grossedentata, a medicinal and edible plant, has been widely used in China for hundreds of years, and dihydromyricetin is the main active ingredient responsible for its various biological actions. We investigated the effects of dihydromyricetin on glucose and lipid metabolism, inflammatory mediators and several biomarkers in nonalcoholic fatty liver disease. In a double-blind clinical trial, sixty adult nonalcoholic fatty liver disease patients were randomly assigned to receive either two dihydromyricetin or two placebo capsules (150 mg) twice daily for three months. The serum levels of alanine, aspartate aminotransferase, γ-glutamyl transpeptidase, glucose, low-density lipoprotein-cholesterol and apolipoprotein B, and the homeostasis model assessment of insulin resistance (HOMA-IR) index were significantly decreased in the dihydromyricetin group compared with the placebo group. In the dihydromyricetin group, the serum levels of tumor necrosis factor-alpha, cytokeratin-18 fragment and fibroblast growth factor 21 were decreased, whereas the levels of serum adiponectin were increased at the end of the study. We conclude that dihydromyricetin supplementation improves glucose and lipid metabolism as well as various biochemical parameters in patients with nonalcoholic fatty liver disease, and the therapeutic effects of dihydromyricetin are likely attributable to improved insulin resistance and decreases in the serum levels of tumor necrosis factor-alpha, cytokeratin-18, and fibroblast growth factor 21.

Resveratrol improves hepatic steatosis by inducing autophagy through the cAMP signaling pathway.

SCOPE: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis. METHODS AND RESULTS: Via Oil red O staining, triglyceride, and ß-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid ß-oxidation in hepatocytes. Based on Western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A), and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis. CONCLUSION: RSV improved hepatic steatosis partially by inducing autophagy via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV's effects on NAFLD treatment.

Dihydromyricetin improves skeletal muscle insulin sensitivity by inducing autophagy via the AMPK-PGC-1α-Sirt3 signaling pathway.

Insulin resistance in skeletal muscle is a key feature in the pathogenesis of type 2 diabetes (T2D) that often manifests early in its development. Pharmaceutical and dietary strategies have targeted insulin resistance to control T2D, and many natural products with excellent pharmacological properties are good candidates for the control or prevention of T2D. Dihydromyricetin (DHM) is a natural flavonol which provides a wide range of health benefits including anti-inflammatory and anti-tumor effects. However, little information is available regarding the effects of DHM on skeletal muscle insulin sensitivity as well as the underlying mechanisms. In the present study, we found that DHM activated insulin signaling and increased glucose uptake in skeletal muscle in vitro and in vivo. The expression of light chain 3, the degradation of sequestosome 1, and the formation of autophagosomes were also upregulated by DHM. DHM-induced insulin sensitivity improvement was significantly abolished in the presence of 3-methyladenine, bafilomycin A1, or Atg5 siRNA in C2C12 myotubes. Furthermore, DHM increased the levels of phosphorylated AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), and Sirt3 in skeletal muscle in vitro and in vivo. Autophagy was inhibited in the presence of Sirt3 siRNA in C2C12 myotubes and in skeletal muscles from Sirt3-/- mice. Additionally, PGC-1α or AMPK siRNA transfection attenuated DHM-induced Sirt3 expression, thereby abrogating DHM-induced autophagy in C2C12 myotubes. In conclusion, DHM improved skeletal muscle insulin sensitivity by partially inducing autophagy via activation of the AMPK-PGC-1α-Sirt3 signaling pathway.

3,6-Dihydroxyflavone Suppresses Breast Carcinogenesis by Epigenetically Regulating miR-34a and miR-21.

Our previous study selected a promising chemopreventive agent 3,6-dihydroxyflavone (3,6-DHF) and found that 3,6-DHF significantly upregulates miR-34a and downregulates miR-21 in breast carcinogenesis, yet the upstream and downstream events of the anticancer mechanism remain unclear. The present study showed that 3,6-DHF cotreatment effectively inhibits carcinogens-induced breast carcinogenic transformation in human breast epithelial MCF10A cells. The data revealed the significant downregulation of miR-34a and upregulation of miR-21 in breast carcinogenesis, which could be mitigated by 3,6-DHF treatment. Methylation-specific PCR detections showed that 3,6-DHF inhibits the hypermethylation of the miR-34a promoter. Further studies indicated that 3,6-DHF is an effective methyltransferase (DNMT)1 inhibitor, docking to the putative cytosine pocket of the protein, and thus decreases the DNMT activity in a dose-dependent manner. Moreover, the ChIP-qPCR analysis for histone modifications showed that 3,6-DHF treatment significantly lowers the H3K9-14ac on the miR-21 promoter. In addition, our study revealed that 3,6-DHF represses the PI3K/Akt/mTOR signaling pathway in breast carcinogenesis in vitro and in vivo. Inhibition of miR-34a or overexpression of miR-21 significantly reduced the effects of 3,6-DHF on Notch-1 and PTEN, and consequently weakened the suppression of 3,6-DHF on PI3K/Akt/mTOR. We concluded that 3,6-DHF upregulates miR-34a via inhibiting DNMT1 and hypermethylation, whereas downregulates miR-21 by modulating histone modification, and consequently suppresses the PI3K/Akt/mTOR signaling pathway in breast carcinogenesis.

Resveratrol improves insulin resistance, glucose and lipid metabolism in patients with non-alcoholic fatty liver disease: a randomized controlled trial.

BACKGROUND: Non-alcoholic fatty liver disease is a major health problem worldwide. Resveratrol is a natural polyphenol found in edible plants that has a variety of biochemical and physiological effects. AIMS: To evaluate the effect of resveratrol on insulin resistance, glucose and lipid metabolism in non-alcoholic fatty liver disease. METHODS: Double-blind, randomized, placebo-controlled trial: 60 subjects with non-alcoholic fatty liver disease were given 2 placebo capsules (placebo group) or 2 150mg resveratrol capsules (resveratrol group) twice daily for three months. Liver ultrasound imaging, anthropometric profile, serum liver enzymes, insulin, glucose, C-peptide, lipid profile, and inflammation-related cytokines were compared pre and post-treatment. RESULTS: Compared with the placebo group, resveratrol significantly decreased aspartate aminotransferase, glucose and low-density lipoprotein cholesterol [-6.00 (-9.00, -3.00) IU/L, -0.64±0.31mmol/L, and -0.41±0.35mmol/L, respectively, P≤0.001] alanine aminotransferase, total cholesterol [-7.00 (-11.0, -2.50) IU/L and -0.67±0.50mmol/L, respectively, P=0.002], and homeostasis model assessment insulin resistance index (-0.60±1.15, P=0.016). In the resveratrol group significant reductions of the levels of tumour necrosis factor-alpha, cytokeratin 18 fragment, and fibroblast growth factor 21 [-0.53±1.30pg/mL, -26.9 (-70.3, 5.12) IU/L and -23.3 (-43.0, 0.31) pg/mL, respectively, P<0.05] and elevation of adiponectin level [1.22 (-0.37, 1.60) ng/mL, P=0.025] were observed. CONCLUSION: Resveratrol supplementation may benefit patients with non-alcoholic fatty liver disease.

Ampelopsin-induced autophagy protects breast cancer cells from apoptosis through Akt-mTOR pathway via endoplasmic reticulum stress.

Our previous study has shown that ampelopsin (AMP), a flavonol mainly found in Ampelopsis grossedentata, could induce cell death in human breast cancer cells via reactive oxygen species generation and endoplasmic reticulum (ER) stress pathway. Here, we examined whether autophagy is activated in AMP-treated breast cancer cells and, if so, sought to find the exact role and underlying molecular profile of autophagy in AMP-induced cell death. Our results showed that AMP treatment activated autophagy in MDA-MB-231 and MCF-7 breast cancer cells, as evidenced by the accumulation of autophagosomes, an increase of microtubule-associated protein 1 light chain 3 beta-2 (LC3B-II) and the conversion of LC3B-I to LC3B-II, the degradation of the selective autophagic target p62/SQSTM1, and the formation of green fluorescent protein (GFP)-LC3 puncta. Blockage of autophagy augmented AMP-induced cell death, suggesting that autophagy has cytoprotective effects. Meanwhile, AMP treatment suppressed Akt-mammalian target of rapamycin (mTOR) pathway as evidenced by dose- and time-dependent decrease of the phosphorylation of Akt, mTOR and ribosomal protein S6 kinase (p70S6K), whereas Akt activator insulin-like growth factor-1 (IGF-1) pretreatment partially restored Akt-mTOR pathway inhibited by AMP and decreased AMP-inuduced autophagy, signifying that AMP activated autophagy via inhibition of the Akt-mTOR pathway. Additionally, blocking ER stress not only reduced autophagy induction, but also alleviated inhibition of the Akt-mTOR pathway induced by AMP, suggesting that activation of ER stress was involved in induction of autophagy and inhibition of the Akt-mTOR pathway. Taken together, these findings indicate that AMP induces protective autophagy in human breast cancer cells through Akt-mTOR pathway via ER stress.

Ampelopsin suppresses breast carcinogenesis by inhibiting the mTOR signalling pathway.

The mammalian target of rapamycin (mTOR), which is a master regulator of cellular catabolism and anabolism, plays an important role in tumourigenesis and progression. In this study, we report the chemopreventive effect of the dietary compound ampelopsin (AMP) on breast carcinogenesis in vivo and in vitro, which acts by inhibiting the mTOR signalling pathway. Our study indicates that AMP treatment effectively suppresses 1-methyl-1-nitrosourea (MNU)-induced breast carcinogenesis in rats and inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P)-induced cellular carcinogenesis. Additionally, AMP inhibits the growth of breast cancer cells in vitro and in vivo. The activity of mTOR kinase was found to be significantly increased in a time-dependent manner during chronic breast carcinogenesis, and this increase can be suppressed by AMP co-treatment. AMP also effectively suppresses mTOR activity in breast cancer MDA-MB-231 cells. We also demonstrated that AMP is an effective mTOR inhibitor that binds to one site on the mTOR target in two ways. Further studies confirmed that AMP inhibits the activation of Akt, suppresses the formation of mTOR complexes (mTORC)1/2 by dissociating regulatory-associated protein of mTOR and rapamycin-insensitive companion of mTOR and, consequently, decreases the activation of the downstream targets of mTOR, including ribosomal p70-S6 kinase, ribosomal protein S6, eukaryotic translation initiation factor 4B and eukaryotic translation initiation factor 4E-binding protein 1. These finding suggest that AMP is a bioactive natural chemopreventive agent against breast carcinogenesis and is an effective mTOR inhibitor that may be developed as a useful chemotherapeutic agent in the treatment of breast cancer.

Genistein inhibits DNA methylation and increases expression of tumor suppressor genes in human breast cancer cells.

It has been previously demonstrated that genistein exhibits anticancer activity against breast cancer. However, the precise mechanisms underlying the anticancer effect of genistein, in particular the epigenetic basis, remain unclear. In this study, we investigated whether genistein could modulate the DNA methylation status and expression of cancer-related genes in breast cancer cells. We treated MCF-7 and MDA-MB-231 human breast cancer cells with genistein in vitro. We found that genistein decreased the levels of global DNA methylation, DNA methyltransferase (DNMT) activity and expression of DNMT1. Yet, the expression of DNMT3A and DNMT3B showed no significant change. Using molecular modeling, we observed that genistein might directly interact with the catalytic domain of DNMT1, thus competitively inhibiting the binding of hemimethylated DNA to the catalytic domain of DNMT1. Furthermore, genistein decreased DNA methylation in the promoter region of multiple tumor suppressor genes (TSGs) such as ataxia telangiectasia mutated (ATM), adenomatous polyposis coli (APC), phosphatase and tensin homolog (PTEN), mammary serpin peptidase inhibitor (SERPINB5), and increased the mRNA expression of these genes. However, we detected no significant changes in the DNA methylation status or mRNA expression of stratifin (SFN). These results suggest that the anticancer effect of genistein on breast cancer may be partly due to its ability to demethylate and reactivate methylation-silenced TSGs through direct interaction with the DNMT1 catalytic domain and inhibition of DNMT1 expression.