title: Development of a new model system to dissect isoform specific Akt signalling in adipocytes.
abstract: Protein kinase B (Akt) kinases are critical signal transducers mediating insulin action. Genetic studies revealed that Akt1 and Akt2 signalling differentially contribute to sustain lipid and glucose homoeostasis; however Akt isoform-specific effectors remain elusive due to the lack of a suitable model system to mechanistically interrogate Akt isoform-specific signalling. To overcome those technical limitations we developed a novel model system that provides acute and specific control of signalling by Akt isoforms. We generated mutants of Akt1 and Akt2 resistant to the allosteric Akt inhibitor MK-2206. We then developed adipocyte cell lines, in which endogenous Akt1 or Akt2 has been replaced by their corresponding drug-resistant Akt mutant. Treatment of those cells with MK-2206 allowed for acute and specific control of either Akt1 or Akt2 function. Our data showed that Akt1(W80A) and Akt2(W80A) mutants are resistant to MK-2206, dynamically regulated by insulin and able to signal to Akt downstream effectors. Analyses of insulin action in this cellular system showed that Akt1 and Akt2 are both able to mediate insulin regulation of the transcription factor forkhead box O1 (FoxO1) and the glucose transporter 4 (GLUT4), revealing a redundant role for these Akt kinases in the control of glucose transport into fat cells. In contrast, Akt1 signalling is uniquely required for adipogenesis, by controlling the mitotic clonal expansion (MCE) of pre-adipocytes that precedes white adipose cell differentiation. Our data provide new insights into the role of Akt kinases in glucose transport and adipogenesis and support our model system as a valuable tool for the biochemical characterization of signalling by specific Akt isoforms.
authors: Biochemistry Department, Weill Cornell Medical College, 1300 York Avenue, Room W-320, New York, NY 10065, U.S.A.
authors: Esi
authors: E
authors: Kajno
authors: Biochemistry Department, Weill Cornell Medical College, 1300 York Avenue, Room W-320, New York, NY 10065, U.S.A.
authors: Timothy E
authors: TE
authors: McGraw
authors: Biochemistry Department, Weill Cornell Medical College, 1300 York Avenue, Room W-320, New York, NY 10065, U.S.A. evg2005@med.cornell.edu evamgonzalez09@gmail.com.
authors: Eva
authors: E
authors: Gonzalez
mesh_terms: D041721:3T3-L1 Cells; D052438:Adipocytes, White / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D050156:Adipogenesis / Q000187:drug effects; D000494:Allosteric Regulation / Q000187:drug effects; D019943:Amino Acid Substitution; D000818:Animals; D004351:Drug Resistance; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D007333:Insulin Resistance; D051379:Mice; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D051275:Glucose Transporter Type 4; D006575:Heterocyclic Compounds, 3-Ring; C548887:MK 2206; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C495693:Slc2a4 protein, mouse; C514862:Akt1 protein, mouse; C494922:Akt2 protein, mouse; D051057:Proto-Oncogene Proteins c-akt
keywords: Akt isoform signalling; MK-2206; adipocyte; adipogenesis; glucose transporter 4 (GLUT4); insulin action