The AMP kinase α2 isoform antibody, phospho-acetyl CoA carboxylase (ACC) Ser-79 antibody and SAMS peptide were from Upstate Biotechnology. The phospho-AMP kinase Thr-172 and AMP kinase α1 + α2 isoform antibodies were from Cell Signaling Technology. Phospho-Akt Ser-473 antibody and pan-Akt isoform protein antibody were from Cell Signaling Technology (Beverly, MA). Anti−insulin receptor (β-subunit) and anti−insulin receptor substrate-1 (IRS-1) antibodies and anti-phosphotyrosine monoclonal antibodies (clone 4G10) were obtained from Upstate Biotechnology (Lake Placid, NY). All antibodies were polyclonal rabbit antisera, except as otherwise indicated.
ATP was obtained from Amersham Biosciences (Piscataway, NJ). d- glucose and 2-deoxy- d-glucose were obtained from ICN (Costa Mesa, CA). Collagenase, type I, was obtained from Worthington Biochemical (Lakewood, NJ).
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The Acticlean Etox column was from Sterogene Bioseparations (Carlsbad, CA), and the limulus amebocyte lysate pyrogen plus detection kit was from BioWhittaker (Walkersville, MD). The nickel ion-agarose affinity column was from QIAGEN (Valencia, CA). The pTrcHisA vector was obtained from Invitrogen (Carlsbad, CA). Louis, MO) or Fisher Scientific (Pittsburgh, PA). General reagents were of the highest available grade and were obtained from Sigma Chemical (St. AMP kinase is a widely expressed serine kinase responsive to hypoxia and cellular stress that has been strongly implicated in a variety of pleiotropic cellular responses, including the insulin-independent activation of glucose transport in skeletal muscle ( 23). These effects have been associated with increased cellular fat oxidation ( 12) and appear to involve the activation of AMP-activated protein kinase (AMP kinase) in liver and skeletal muscle ( 21, 22).
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Adiponectin has been shown to enhance insulin sensitivity improve plasma clearance of free fatty acids, glucose, and triglycerides and suppress hepatic glucose production ( 11, 12, 16, 20).
The full-length form of adiponectin forms high Mr oligomers in the bloodstream, and some studies have ascribed different signaling properties to various recombinant or processed forms of the protein ( 11, 12, 16– 19).
Coupled with recent evidence that the effects of adiponectin are mediated via AMP kinase activation in liver and skeletal muscle, the findings reported here provide an important mechanistic link in the signaling effects of adiponectin in diverse metabolically responsive tissues.Īdiponectin is highly conserved among mammalian species as a 244-amino acid polypeptide with a stretch of 22 collagen (Gly-X-Y) repeats and a globular domain with an overall homology similar to complement factor C1q ( 5, 15). Inhibition of AMP kinase activation using two pharmacological inhibitors (adenine 9-β- d-arabinofuranoside and compound C) completely abrogated the increase in glucose uptake stimulated by globular adiponectin, indicating that AMP kinase is integrally involved in the adiponectin signal transduction pathway. Cellular treatment with globular adiponectin increased the Thr-172 phosphorylation and catalytic activity of AMP-activated protein kinase and enhanced the Ser-79 phosphorylation of acetyl CoA carboxylase, an enzyme downstream of AMP kinase in adipose cells. Globular adiponectin further enhanced insulin-stimulated glucose uptake at submaximal insulin concentrations and reversed the inhibitory effect of tumor necrosis factor-α on insulin-stimulated glucose uptake. Globular adiponectin increased glucose uptake in adipocytes without stimulating tyrosine phosphorylation of the insulin receptor or insulin receptor substrate-1, and without enhancing phosphorylation of Akt on Ser-473. To characterize the potential effects of adiponectin on glucose uptake into adipose cells, we incubated isolated epididymal rat adipocytes with the globular domain of recombinant adiponectin purified from an E. Adiponectin is an abundant adipocyte-derived plasma protein with anti-atherosclerotic and insulin-sensitizing properties that suppresses hepatic glucose production and enhances glucose uptake into skeletal muscle.