Pharmacological activation of AMPK prevents Drp1-mediated mitochondrial fission and alleviates endoplasmic reticulum stress-associated endothelial dysfunction.

BACKGROUND AND PURPOSE: This study aims to investigate whether and how pharmacological activation of AMP-activated protein kinase (AMPK) improves endothelial function by suppressing mitochondrial ROS-associated endoplasmic reticulum stress (ER stress) in the endothelium. Experimental approach Palmitate stimulation induced mitochondrial fission and ER stress-associated endothelial dysfunction. The effects of AMPK activators salicylate and AICA riboside (AICAR) on mitochondrial ROS production, Drp1 phosphorylation, mitochondrial fission, ER stress, thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation, inflammation, cell apoptosis and endothelium-dependent vasodilation were observed. Key results "Silencing" of TXNIP by RNA interference inhibited NLRP3 inflammasome activation in response to ER stress, indicating that TXNIP was a key link between ER stress and NLRP3 inflammasome activation. AMPK activators salicylate and AICAR prevented ROS-induced mitochondrial fission by enhancing dynamin-related protein 1 (Drp1) phosphorylation (Ser 637) and thereby attenuated IRE-1α and PERK phosphorylation, but their actions were blocked by knockdown of AMPK. Salicylate and AICAR reduced TXNIP induction and inhibited NLRP3 inflammasome activation by reducing NLRP3 and caspase-1 expression, leading to a reduction in IL-1ß secretion. As a result, salicylate and AICAR inhibited inflammation and reduced cell apoptosis. Meanwhile, salicylate and AICAR enhanced eNOS phosphorylation and restored the loss of endothelium-dependent vasodilation in the rat aorta. Immunohistochemistry staining showed that AMPK activation inhibited ER stress and NLRP3 inflammasome activation in the vascular endothelium. CONCLUSION AND IMPLICATIONS: Pharmacological activation of AMPK regulated mitochondrial morphology and ameliorated endothelial dysfunction by suppression of mitochondrial ROS-associated ER stress and subsequent TXNIP/NLRP3 inflammasome activation. These findings suggested that regulation of Drp1 phosphorylation by AMPK activation contributed to suppression of ER stress and thus presented a potential therapeutic strategy for AMPK activation in the regulation of endothelium homeostasis.

Rare ginsenosides ameliorate lipid overload-induced myocardial insulin resistance via modulating metabolic flexibility.

BACKGROUND: Rare ginsenosides are found in ginseng and notoginseng, two medicinal plants widely used in China for treatment of cardiovascular diseases and type 2 diabetes. However, their pharmacological studies regarding myocardial fuel metabolism and insulin signaling are not clear. PURPOSE: To explore the effect of a rare ginsenoside-standardized extract (RGSE), derived from steamed notoginseng, on cardiac fuel metabolism and insulin signaling. STUDY DESIGN: We used palmitic acid (PA) to treat H9c2 cells in vitro and high fat diet (HFD) to mice to induce insulin resistance in vivo. METHODS: In vitro, differentiated H9c2 cells were pretreated with RGSE, metformin, mildronate or dichloroacetate (DCA) and stimulated with PA. In vivo, mice were fed with HFD and received RGSE, metformin or DCA for 6 weeks. Protein expression was determined by Western blotting. Mitochondrial membrane potential (Δψm), glucose uptake and reactive oxygen species (ROS) production were measured by fluorescence labeling. Other assessments including oxygen consumption rate (OCR) were also performed. RESULTS: RGSE prevented PA-induced decrease in pyruvate dehydrogenase (PDH) activity and increase in carnitine palmitoyltransferase 1 (CPT1) expression, and ameliorated insulin-mediated glucose uptake and utilization in H9c2 cells. Metformin and mildronate exhibited similar effects. In vivo, RGSE counteracted HFD-induced increase in myocardial expression of p-PDH and CPT1 and ameliorated cardiac insulin signaling. Metformin and DCA also showed beneficial effects. Further study showed that RGSE decreased OCR and mitochondrial complex I activity in PA-treated H9c2 cells, reduced ROS production and relieved mitochondrial oxidative stress, thus decreased serine phosphorylation in IRS-1. CONCLUSION: RGSE ameliorated myocardial insulin sensitivity under conditions of lipid overload, which was tightly associated with the decrease in mitochondrial oxidative stress via modulating glucose and fatty acid oxidation.

Quercetin oppositely regulates insulin-mediated glucose disposal in skeletal muscle under normal and inflammatory conditions: The dual roles of AMPK activation.

SCOPE: Quercetin is a dietary flavonoid whose role in the regulation of the activity of insulin remains controversial. Our study aimed to investigate how quercetin and its major metabolite quercetin-3-glucuronide (Q-3-G) regulate insulin-mediated glucose disposal in skeletal muscle under normal and inflammatory conditions. METHODS AND RESULTS: Under normal conditions, quercetin impaired glucose and insulin tolerance and attenuated insulin-mediated phosphorylation of Akt substrate of 160 kDa (AS160) and TBC1D1 without affecting Akt activity in male Institute of Cancer Research (ICR) mice. However, under inflammatory conditions, quercetin exhibited an opposite effect in these animals. In C2C12 cells, quercetin also decreased insulin-stimulated AS160 and TBC1D1 phosphorylation and glucose uptake in the absence of an inflammatory insult, whereas it improved the action of insulin under inflammatory conditions. Knockdown of adenosine 5'-monophosphate-activated protein kinase α (AMPKα) blocked the differential effects of quercetin under both conditions. Unlike quercetin, Q-3-G had no influence on insulin-induced phosphorylation of AS160 and TBC1D1 and glucose uptake in C2C12 myotubes under normal conditions. Q-3-G displayed a similar regulation with quercetin in glucose disposal under inflammatory conditions. CONCLUSION: Quercetin suppressed insulin-mediated glucose disposal in skeletal muscle tissue/cells under normal conditions while it ameliorated impaired glucose uptake under inflammatory conditions with activation of AMPK. In contrast, Q-3-G ameliorated insulin resistance in skeletal cells under inflammatory conditions without affecting glucose disposal under normal conditions.