Michel Wierzbicki

AMPK Phosphorylates and Inhibits SREBP Activity to Attenuate Hepatic Steatosis and Atherosclerosis in Diet-Induced Insulin-Resistant Mice

AMPK has emerged as a critical mechanism for salutary effects of polyphenols on lipid metabolic disorders in type 1 and type 2 diabetes. Here we demonstrate that AMPK interacts with and directly phosphorylates sterol regulatory element binding proteins (SREBP-1c and -2). Ser372 phosphorylation of SREBP-1c by AMPK is necessary for inhibition of proteolytic processing and transcriptional activity of SREBP-1c in response to polyphenols and metformin. AMPK stimulates Ser372 phosphorylation, suppresses SREBP-1c cleavage and nuclear translocation, and represses SREBP-1c target gene expression in hepatocytes exposed to high glucose, leading to reduced lipogenesis and lipid accumulation. Hepatic activation of AMPK by the synthetic polyphenol S17834 protects against hepatic steatosis, hyperlipidemia, and accelerated atherosclerosis in diet-induced insulin-resistant LDL receptor-deficient mice in part through phosphorylation of SREBP-1c Ser372 and suppression of SREBP-1c- and -2-dependent lipogenesis. AMPK-dependent phosphorylation of SREBP may offer therapeutic strategies to combat insulin resistance, dyslipidemia, and atherosclerosis.

SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser428, and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKβ, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.

Polyphenols Stimulate AMP-Activated Protein Kinase, Lower Lipids, and Inhibit Accelerated Atherosclerosis in Diabetic LDL Receptor–Deficient Mice

Because polyphenols may have beneficial effects on dyslipidemia, which accelerates atherosclerosis in diabetes, we examined the effect of polyphenols on hepatocellular AMP-activated protein kinase (AMPK) activity and lipid levels, as well as hyperlipidemia and atherogenesis in type 1 diabetic LDL receptor–deficient mice (DMLDLR−/−). In HepG2 hepatocytes, polyphenols, including resveratrol (a major polyphenol in red wine), apigenin, and S17834 (a synthetic polyphenol), increased phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC), and they increased activity of AMPK with 200 times the potency of metformin. The polyphenols also prevented the lipid accumulation that occurred in HepG2 cells exposed to high glucose, and their ability to do so was mimicked and abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Furthermore, treatment of DMLDLR−/− mice with S17834 prevented the decrease in AMPK and ACC phosphorylation and the lipid accumulation in the liver, and it also inhibited hyperlipidemia and the acceleration of aortic lesion development. These studies 1) reveal that inactivation of hepatic AMPK is a key event in the pathogenesis of hyperlipidemia in diabetes, 2) point to a novel mechanism of action of polyphenols to lower lipids by activating AMPK, and 3) emphasize a new therapeutic avenue to benefit hyperlipidemia and atherosclerosis specifically in diabetes via activating AMPK.

Nox4 mediates the expression of plasminogen activator inhibitor-1 via p38 MAPK pathway in cultured human endothelial cells

INTRODUCTIONnPlasminogen Activator Inhibitor-1 (PAI-1) is the most potent endogenous inhibitor of fibrinolysis which is implicated in the pathogenesis of myocardial infarction and metabolic syndrome. The formation of reactive oxygen species (ROS) plays an important role in the pathology of vascular disorders and has been shown to increase PAI-1 expression by endothelial cells. Growing evidence indicates that NADPH oxidase and in particular the constitutively active Nox4-p22(phox) complexes are major sources of ROS in endothelial cells. The aim of the present study was to characterize the role of NADPH oxidase and in particular Nox4 in the regulation of PAI-1 expression in cultured Human Umbilical Venous Endothelial Cells (HUVECs).nnnMETHODS AND RESULTSnN-acetylcysteine (NAC, scavenger of ROS), diphenylene iodonium chloride (DPI, inhibitor of flavoproteins), M40403 (superoxyde dismutase mimic) and S17834 (inhibitor of NADPH oxidase) inhibited PAI-1 release and promoter activity in HUVECs. Specific knock down of Nox4 mRNA by siRNA caused a decrease in ROS production and NADPH oxidase activity. Moreover, Nox4 silencing decreased PAI-1 expression, release and activity as well as p38 MAPK pathways and NFkappaB activation. These signalling pathways are also involved in PAI-1 release.nnnCONCLUSIONSnThe NADPH oxidase inhibitors DPI and S 17834 as well as Nox4 silencing decreased PAI-1 synthesis in human cultured endothelial cells demonstrating the involvement of the constitutively active Nox4-containing NADPH oxidase in ROS-mediated PAI-1 transcription via p38 MAPK pathways. NADPH oxidase targeting with inhibitors such as S17834 could be an interesting strategy to decrease both oxidative stress and PAI-1 synthesis.