A-769662 activates AMPK β₁-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle

Abstract

A-769662 activates AMPK ␤1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle.

Prior AICAR Stimulation Increases Insulin Sensitivity in Mouse Skeletal Muscle in an AMPK-Dependent Manner

“…Five different AMPK trimer complexes have been detected in mouse skeletal muscle: a2b2g3, a2b1g1, a2b2g1, a1b1g1, and a1b2g1 (19). a2b2g3-AMPK activity was measured on g3-AMPK immunoprecipitates (IPs) from 300 mg of muscle lysate using AMPK-g3 antibody, G-protein-coupled agarose beads (Millipore) and IP buffer (50 mmol/L NaCl, 1% Triton X-100, 50 mmol/L sodium fluoride, 5 mmol/L sodium-pyrophosphate, 20 mmol/L Trisbase, pH 7.5, 500 mmol/L PMSF, 2 mmol/L dithiothreitol, 4 mg/mL leupeptin, 50 mg/mL soybean trypsin inhibitor, 6 mmol/L benzamidine, and 250 mmol/L sucrose).…”

“…a2b2g3-AMPK activity was measured on g3-AMPK immunoprecipitates (IPs) from 300 mg of muscle lysate using AMPK-g3 antibody, G-protein-coupled agarose beads (Millipore) and IP buffer (50 mmol/L NaCl, 1% Triton X-100, 50 mmol/L sodium fluoride, 5 mmol/L sodium-pyrophosphate, 20 mmol/L Trisbase, pH 7.5, 500 mmol/L PMSF, 2 mmol/L dithiothreitol, 4 mg/mL leupeptin, 50 mg/mL soybean trypsin inhibitor, 6 mmol/L benzamidine, and 250 mmol/L sucrose). Samples were treated as previously described (19,36). In short, after overnight end-over-end rotation at 4°C, IPs were centrifuged for 1 min at 2,000g and washed once in IP buffer, once in 63 assay buffer (240 mmol/L HEPES, 480 mmol/L NaCl, pH 7.0), and twice in 33 assay buffer (1:1).…”

“…Furthermore, prior AICAR stimulation failed to increase insulin sensitivity in mouse soleus muscle in which the a2b2g3 complex represents ,2% of all AMPK trimer complexes (19). In both human vastus lateralis muscle (18) and mouse EDL muscle (19), the AMPK a2b2g3 trimer complex accounts for one-fifth of all AMPK complexes (36). Of interest, AMPK-g3 protein level is markedly decreased in skeletal muscle from trained humans (50), although insulin sensitivity in general is increased.…”

“…Of the 12 heterotrimeric combinations, only 3 and 5 combinations have been found in the skeletal muscle of human and mouse, respectively (18,19). AMPK is activated in response to various stimuli that increase cellular energy stress (e.g., metformin, hypoxia, hyperosmolarity, muscle contraction, and exercise) (20).…”

Assay Development and High-Throughput Screening of Small Molecular c-Abl Kinase Activators

“…2 efforts have been made to discover and develop aMpk-specific small-molecule nonnucleoside activators of aMpk, [3][4][5] and the newly discovered thienopyridone a769662 validates the approach of aMpk activation as a way to ameliorate diabetes symptoms by inhibiting lipogenesis 6 and increasing glucose uptake 7 in cellular and animal models. c-abl, a member of the src family of nonreceptor tyrosine kinases ubiquitously expressed in mammalian cells, is found throughout the cell, including the nucleus, cytoplasm, mitochondria, endoplasmic reticulum, and plasma membrane.…”