The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the ␣ subunit of AMPK in a dose-and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviralmediated expression of a constitutively active AMPK␣ or a kinase-inactive AMPK␣, respectively. An insulinresistant state was induced by exposing cells to 30 mM glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3␣/␤. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPK␣. The kinase-inactive AMPK␣ increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPK␣ negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance. AMP-activated protein kinase (AMPK)1 is a phylogenetically conserved intracellular energy sensor that has been implicated in the regulation of glucose and lipid homeostasis (1-4). AMPK is activated by physiological stimuli, such as exercise, muscle contraction, and hormones including adiponectin and leptin, as well as by pathological stresses, glucose deprivation, hypoxia, oxidative stress, and osmotic shock (2, 5). AMPK serine/threonine protein kinase is a heterotrimeric complex consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥) (5). Regulation of AMPK activity is complex; it involves allosteric activation by AMP, which increases during states of stress where ATP is depleted, and phosphorylation via the presumptive upstream activator AMPK kinase (6 -9), which may also be allosterically activated by AMP (5). Moreover, phosphorylation of Thr-172 within the activation loop of the catalytic domain of the ␣ subunit is necessary for AMPK activity because sitedirected mutagenesis of Thr-172 to Ala completely abolishes AMPK activity (10, 11). Once activated, AMPK phosphorylates its downstream substrates to reduce ATP-consuming anabolic pathways, including ...

Abstract-The purpose of this study was to determine whether superoxide anion is produced endogenously in the rat aortic adventitia and whether sufficient superoxide anion is produced to interfere with the response of the rat aorta to nitric oxide. Relaxation was measured in rings of the rat thoracic aorta, which were oriented so that the adventitial or luminal surface could be preferentially exposed to nitric oxide or sodium nitroprusside. To accomplish this, the rings were mounted (1) with the adventitia facing outward, (2) with the adventitia facing inward after inverting, or (3) with the adventitia facing outward after inverting twice (to control for the inverting procedure). The relaxation to nitric oxide, but not to sodium nitroprusside, was less in rings with the adventitia facing outward compared with those in which it faced inward. In contrast, the response to nitric oxide via either surface was similar when extracellular superoxide anion was scavenged with superoxide dismutase. Incubation of rings with nitro blue tetrazolium (NBT) resulted in blue formazan staining of the adventitia, and lucigenin chemiluminescence was significantly greater when detected from the adventitial compared with the intimal aspect of the artery. The reduction of NBT in intact aortic rings was 30Ϯ2 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1 and was significantly decreased by superoxide dismutase to 19Ϯ2 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1 and by a synthetic superoxide dismutase mimic, Euk-8, to 11Ϯ2 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1. The NADPH oxidase inhibitor, diphenyleneiodonium, decreased NBT reduction to 9Ϯ1 pmol ⅐ min Ϫ1 ⅐ mg Ϫ1, whereas inhibitors of xanthine oxidase, mitochondrial oxidases, and nitric oxide synthase were ineffective. Immunohistochemical staining indicated the localization of NADPH oxidase proteins gp91 phox , p22 phox , p47 phox , and p67 phox almost exclusively in the adventitia of the rat aorta with no substantial staining in the media. These results indicate that NADPH oxidase located in the adventitia of rat thoracic aorta generates sufficient extracellular superoxide anion to constitute a barrier capable of inactivating nitric oxide. This study suggests that adventitial superoxide anion can play a role in the pathophysiology of the arterial wall. (Circ Res. 1998;82:810-818.)

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