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Summary: 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. [ABSTRACT FROM AUTHOR]

Atherosclerosis is associated with alterations in nitric oxide (NO)/ cGMP signaling. In early stages of the disease, inflammatory and possibly other cells produce reactive oxygen species that scavenge vasoprotective NO. In addition to the oxidative stress, expression and activity of enzymes downstream to NO formation may also be affected. Here, we show in the aortas of chronically hypercholesterolemic rabbits (a model of late-stage atherosclerosis), both subunits and specific activity of the NO receptor soluble guanylyl cyclase (sGC) were significantly reduced, whereas overall NO synthase activity was unaffected. These changes were most prominent in the neointimal layer, wherein cGMP-dependent protein kinase I (cGK) levels also were reduced. Additionally, a protein (p38nt) that was constitutively tyrosine-nitrated was detected, and its expression was significantly reduced in atherosclerotic aorta. Phosphorylation of the cGK substrate vasodilator-stimulated phosphoprotein (VASP) at Ser-239, an established biochemical endpoint of NO/cGMP signaling, also was reduced. Thus, late-stage atherosclerosis is associated not only with enhanced NO breakdown but also with altered NO reception and cGMP signaling. Preferential down- regulation in neointima suggests a direct connection of these changes to neointimal proliferation and vascular dysfunction and provides a rationale for future pharmacotherapy using classical and novel sGC activators. [ABSTRACT FROM AUTHOR]

Sterol regulatory element binding protein (SREBP) is a key lipogenic transcription factor that has been implicated in the pathogenesis of dyslipidemia. We previously demonstrated that high glucose and streptozotocin (STZ)-induced type 1 diabetes decrease AMPK activity and cause lipid accumulation both in HepG2 hepatocytes in vitro, and in mouse liver in vivo, associated with hyperlipidemia. These changes were prevented by metformin or a synthetic polyphenol, S17834. We have further investigated the role of S 17834 in regulating SREBP and lipid metabolism. In STZ-induced diabetic mice, the nuclear active form (∼68 kDa) of liver SREBP-1 was increased 5-fold, and the increase was significantly prevented by S17834 (130 mg/kg/day), suggesting that the lipid-lowering effect of S17834 is associated with suppression of SREBP-1. Exposure of HepG2 cells to high glucose in culture resulted in increased expression of the precursor and nuclear active form of SREBP-1, as well as enhanced nuclear translocation of SREBP- 1 as detected by immunofluorescent staining. An increase in the SREBP-1 target enzyme, fatty acid synthase (FAS), was associated with an increase in hepatocellular triglyceride accumulation. In contrast, S17834 (l0 micro;M) or metformin (2 mM) significantly increased AMPK activity and inhibited activation of SREBP and induction of FAS, and thereby lowered hepatocellular triglyceride levels. Moreover, the ability of S17834 to downregulate SREBP-1 and FAS is mimicked by overexpression of the constitutively active AMPK, and largely prevented by a dominant-negative AMPK. These results indicate that polyphenols attenuate hepatic lipid accumulation associated with high glucose via inhibition of de novo fatty acid synthesis via activation of AMPK and suppression of SREBP-1, and point to new approaches using small molecules for treating dyslipidemia associated with diabetes. [ABSTRACT FROM AUTHOR]

Our recent studies show that the synthetic polyphenol, S 17834, stimulates AMP-activated protein kinase (AMPK) more potently than resveratrol or metformin. Stimulation of AMPK by S17834 was shown to attenuate high glucose-induced lipid accumulation in HepG2 cells, as well as to be associated with a reduction in hepatic and serum lipids, and atherosclerosis in type 1 diabetic LDL receptor deficient (LDLR-/-) mice. To determine the effect of S17834 in obesity-induced insulin resistance and type 2 diabetes, LDLR-/- mice were fed a high fat, high sucrose (HFHS) diet for 16 weeks. Compared with normal chow, body weight gain increased ∼3-fold, and insulin resistance index, as determined by the homeostasis model assessment for insulin resistance (HOMAIR), was increased ∼2.5-fold. Hepatic steatosis caused by the HFHS diet was accompanied in the liver by decreased phosphorylation of AMPK and the AMPK downstream effector, acetyl CoA carboxylase (ACC) compared with chow fed mice. The HFHS diet caused a ∼2-fold increase in plasma cholesterol levels as well as a 4-fold increase in oil red O stained atherosclerotic lesions in aortic mot cross sections. S17834 administration (130 mg/kg/day, 16 weeks) significantly protected mice against diet-induced obesity and systemic insulin resistance. Treatment with S 17834 was accompanied by increased AMPK and ACC phosphorylation in the liver and improvement in hepatic steatosis. S17834 also significantly decreased plasma cholesterol levels and attenuated the development of aortic atherosclerotic lesions. These data show that protection afforded by S17834 from the exaggerated hyperlipidemia and vascular disease caused by type 1 diabetes extends to type 2 diabetes in LDLR-/- mice. Improvements in body weight, insulin resistance, hepatic steatosis, hyperlipidemia, and atherosclerosis may be attributed to the ability of the polyphenol to stimulate AMPK as demonstrated here to occur in the mouse liver. Activation of AMPK by polyphenols, like S17834, represents a major new avenue for treatment of hyperlipidemia and vascular disease in both type 1 and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Sirt1 is one of seven mammalian homologs of Sir2, an NAD-dependent deacetylase that functions as a novel metabolic sensor connecting aging and metabolism. We recently discovered that polyphenols stimulate AMPK activity and lower hepatic lipids, contributing to their beneficial effects on accelerated hyperlipidemia and atherosclerosis in diabetes. Here we examined if Sirt1 mediates the effect of polyphenols on AMPK and its lipid metabolic effects. The in vitro Fluor de Lys deacetylation assay showed that Sirt1 catalytic activity was significantly increased by polyphenols including resveratrol and a synthetic polyphenol, S17834, and decreased by Sirt1 inhibitors, nicotinamide and suramin, respectively. The two polyphenolic Sirt1 activators substantially stimulated phosphorylation of AMPK and its downstream effector, acetyl CoA carboxylase, in cultured human HepG2 hepatocytes and HEK293 cells, but not in HeLa cells deficient in LKB1. These responses were largely blocked by inhibition of Sirt1 deacetylation with nicotinamide or splitomicin. Importantly, downregulation of Sirt1 using lentivirus-mediated Sirt1 shRNA completely abolished the basal and polyphenol-induced AMPK activation. Furthermore, studies with a dominant negative Sirt1 (Sirt1H355A) and Sirt1 inhibitors revealed that the effects of polyphenols on hepatocellular AMPK signaling and lipids depend on Sirt1 activity in a HepG2 cell model of high glucose-induced lipid accumulation. Overexpression of wild type Sirt1 also stimulated AMPK, which was inhibited by high glucose, as well as reduced triglyceride levels that were enhanced by high glucose. The effects of wild type Sirtl were completely blocked by a dominant negative AMPK (AMP-Ka1phaK45R). These results implicate Sirt1 stimulation of AMPK as a key regulator of lipid metabolism in bepatocytes. Because of the associated improvements seen in hyperlipidemia and atherosclerosis caused by polyphenols in type 1 and type 2 mouse models, these data suggest that Sirt1 and AMPK may be attractive therapeutic targets for diabetes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]