AMP-activated protein kinase (AMPK) is a key sensor and regulator of intracellular and whole-body energy metabolism. We have identified a thienopyridone family of AMPK activators. A-769662 directly stimulated partially purified rat liver AMPK (EC50 = 0.8 microM) and inhibited fatty acid synthesis in primary rat hepatocytes (IC50 = 3.2 microM). Short-term treatment of normal Sprague Dawley rats with A-769662 decreased liver malonyl CoA levels and the respiratory exchange ratio, VCO2/VO2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreased hepatic expression of PEPCK, G6Pase, and FAS, lowered plasma glucose by 40%, reduced body weight gain and significantly decreased both plasma and liver triglyceride levels. These results demonstrate that small molecule-mediated activation of AMPK in vivo is feasible and represents a promising approach for the treatment of type 2 diabetes and the metabolic syndrome.

In wound healing, myofibroblast transdifferentiation (MFT) is a metaplastic change in phenotype producing profibrotic effector cells that secrete and remodel the extracellular matrix. Unlike pathways that induce MFT, the molecular mechanisms that negatively regulate MFT are poorly understood. Here, we report that AMP-activated protein kinase (AMPK) blocks MFT in response to transforming growth factor-␤ (TGF␤). Pharmacological activation of AMPK inhibited TGF␤-induced secretion of extracellular matrix proteins collagen types I and IV and fibronectin. AMPK activation also prevented induction of the myofibroblast phenotype markers ␣-smooth muscle actin and the ED-A fibronectin splice variant. AMPK activators did not prevent MFT in cells transduced with an adenovirus expressing dominant negative, kinase-dead AMPK␣2. Moreover, AMPK activators did not inhibit MFT induction in AMPK ␣1,2 ؊/؊ fibroblasts, demonstrating a requirement for AMPK ␣ expression. Adenoviral transduction of constitutively active AMPK ␣2 was sufficient to prevent TGF␤-induced collagen I, ␣-smooth muscle actin, and ED-A fibronectin. AMPK did not reduce TGF␤-stimulated Smad3 COOH-terminal phosphorylation and nuclear translocation, which are necessary for MFT. However, AMPK activation inhibited TGF␤-induced transcription driven by Smad3-binding cis-elements. Consistent with a role for AMPK in transcriptional regulation, nuclear translocation of AMPK␣2 correlated with the appearance of active AMPK␣ in the nucleus. Collectively, these results demonstrate that AMPK inhibits TGF␤-induced transcription downstream of Smad3 COOH-terminal phosphorylation and nuclear translocation. Furthermore, activation of AMPK is sufficient to negatively regulate MFT in vitro.Myofibroblast transdifferentiation (MFT) 2 is a fundamental cellular program activated in embryonic development, tumorstroma interactions, wound repair, and fibrosis in the lung, heart, liver, and kidney (1-4). Myofibroblasts are mesenchymal cells that display phenotypic markers of both muscle and nonmuscle cells. At sites of tissue injury, MFT produces profibrotic effector cells that assemble a fibrotic lesion (1, 3, 4). MFT is induced in an autocrine or paracrine fashion by cytokines, growth factors, and mechanical forces such as shear stress (3, 5).The temporal sequence of MFT in wound healing is not entirely clear, but it involves early de novo expression of the ED-A fibronectin (ED-A FN) splice variant and formation of a proto-myofibroblast (1, 6). The transition of proto-myofibroblasts into myofibroblasts is marked by abundant de novo expression of ␣-smooth muscle actin (␣SMA) (7). Stress fibers rich in ␣SMA develop and link to focal adhesions at the plasma membrane (1, 2). Finally, the mature myofibroblasts secrete a fibrillar collagen-and fibronectin-rich extracellular matrix that is remodeled into a fibrotic lesion (3,8).AMP-activated protein kinase (AMPK) was initially characterized as a protein activated by nutrient and bioenergetic stress that raises intracellular AMP and lowers ATP (9 -11...

A novel and innovative high-throughput screening assay was developed to identify both activators and inhibitors of AMP-activated protein kinase (AMPK) using microarrayed compound screening (µARCS) technology. Test compounds were arrayed at a density of 8640 on a polystyrene sheet, and the enzyme and peptide substrate were introduced into the assay by incorporating them into an agarose gel followed by placement of the gels onto the compound sheet. Adenosine triphosphate (ATP) was delivered via a membrane, and the phosphorylated biotinylated substrate was captured onto a streptavidin affinity membrane (SAM™). For detection, the SAM™ was removed, washed, and imaged on a phosphor screen overnight. A library of more than 700,000 compounds was screened using this format to identify novel activators and inhibi-

Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome.

A critical early event in the inflammatory cascade is the induced expression of cell adhesion molecules on the lumenal surface of vascular endothelial cells. These adhesion molecules include E-selectin, ICAM-1, and VCAM-1, which serve to recruit circulating leukocytes to the site of the inflammation. These adhesive interactions allow the leukocytes to firmly adhere to and cross the vascular endothelium and migrate to the site of tissue injury. Pharmaceutical agents which would prevent the induced expression of one or more of the cell adhesion molecules on the endothelium might be expected to provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A thieno[2,3-d]pyrimidine, A-155918, was identified from a whole-cell high-throughput assay for compounds which inhibited the tumor necrosis factor-alpha (TNFalpha)-induced expression of E-selectin, ICAM-1, or VCAM-1 on human vascular endothelial cells. Traditional medicinal chemistry methods were applied to this low-micromolar inhibitor, resulting in the 2,4-disubstituted thieno[2,3-c]pyridine A-205804, a potent and selective lead inhibitor of E-selectin and ICAM-1 expression (IC(50) = 20 and 25 nM, respectively). The relative position of the nitrogen atom in the thienopyridine isomer was shown to be critical for activity, as was a small amide 2-substituent.