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OBJECTIVE--Recently, several drugs have been shown to exert beneficial effects for metabolic syndrome through mild regulation of mitochondrial function. Hence, we explored a strategy of targeting mitochondrial function to improve glucose and lipid metabolism. RESEARCH DESIGN AND METHODS--Mitochondrial membrane potential (ΔΨm) is a marker of mitochondrial function; therefore, we set up a high-throughput screening assay of ΔΨm in L6 myotubes. The effects of a selected lead compound were investigated in vitro and in vivo in relation to metabolic syndrome. RESULTS--A novel small-molecule compound, C1, was identified through this high-throughput screening. C1 depolarized ΔΨm in L6 myotubes without cytotoxicity and led to increased cellular AMP-to-ATP ratio, activation of AMP-activated protein kinase (AMPK), and enhanced glucose uptake. It also stimulated the AMPK pathway in HepG2 cells, leading to decreased lipid content. Intriguingly, C1 inhibited respiration in L6 myotubes but did not affect respiration in isolated muscle mitochondria, suggesting that it may depolarize ΔΨm indirectly by affecting the supply of electron donors. Acute administration of C1 in C57BL/6J mice markedly increased fat oxidation and the phosphorylation of AMPK and acetyl-CoA carboxylase in the liver. In diabetic db/db mice, chronic administration of C1 significantly reduced hyperglycemia, plasma fatty acids, glucose intolerance, and the mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver. CONCLUSIONS--Our results demonstrate a novel small molecule that mildly depolarizes ΔΨm and is able to improve glucose and lipid metabolism to exert beneficial effects for metabolic syndrome. These findings suggest that compounds regulating mitochondrial function may have therapeutic potential for type 2 diabetes. Diabetes 59:256-265, 2010 [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK al catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in a subunits, we screened a chemical library with inactive human a1394 (αl, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK α1394, α1335, α2398, and even heterotrimer α1β1γ1. Based on PT1-docked AMPK α1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 without an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic a subunit and two regulatory subunits, β and γ. It had been reported that the mammalian AMPK a subunit contained an autoinhibitory domain (α1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the a subunit (α1-(313-335)), which includes a predicted α-helix, is responsible for a subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for α1 autoinhibition. An autoinhibitory structural model of human AMPK α1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 Å and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK α1-(1-394) with deletion of residues 313-335 (Δα394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Δα394, V298G, or L328Q mutants, which indicated that these AMPK al mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK a autoinhibitory function. [ABSTRACT FROM AUTHOR]