Molecular mechanisms of insulin resistance in IRS-2-deficient hepatocytes.
To assess the role of insulin receptor (IR) substrate (IRS)-2 in insulin action and resistance in the liver, immortalized neonatal hepatocyte cell lines have been generated from IRS-2(-/-), IRS-2(+/-), and wild-type mice. These cells maintained the expression of the differentiated liver markers albumin and carbamoyl phosphate synthetase, as well as bear a high number of IRs. The lack of IRS-2 did not result in enhanced IRS-1 tyrosine phosphorylation or IRS-1-associated phosphatidylinositol (PI) 3-kinase activity on insulin stimulation. Total insulin-induced PI 3-kinase activity was decreased by 50% in IRS-2(-/-) hepatocytes, but the translocation of PI-3,4,5-trisphosphate to the plasma membrane in these cells was almost completely abolished. Downstream PI 3-kinase, activation of Akt, glycogen synthase kinase (GSK)-3 (alpha and beta isoforms), Foxo1, and atypical protein kinase C were blunted in insulin-stimulated IRS-2(-/-) cells. Reconstitution of IRS-2(-/-) hepatocytes with adenoviral IRS-2 restored activation of these pathways, demonstrating that IRS-2 is essential for functional insulin signaling in hepatocytes. Insulin induced a marked glycogen synthase activity in wild-type and heterozygous primary hepatocytes; interestingly, this response was absent in IRS-2(-/-) cells but was rescued by infection with adenoviral IRS-2. Regarding gluconeogenesis, the induction of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase by dibutyryl cAMP and dexamethasone was observed in primary hepatocytes of all genotypes. However, insulin was not able to suppress gluconeogenic gene expression in primary hepatocytes lacking IRS-2, but when IRS-2 signaling was reconstituted, these cells recovered this response to insulin. Suppression of gluconeogenic gene expression in IRS-2-deficient primary hepatocytes was also restored by infection with dominant negative Delta 256Foxo1.
Valverde
Angela M
AM
Instituto de Bioquímica/Departamento de Bioquímica y Biología Molecular II, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense, Madrid, Spain. valverde@farm.ucm.es
Burks
Deborah J
DJ
Fabregat
Isabel
I
Fisher
Tracey L
TL
Carretero
José
J
White
Morris F
MF
Benito
Manuel
M
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002461:Cell Line, Transformed; D005260:Female; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D005943:Gluconeogenesis / Q000502:physiology; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D006006:Glycogen Synthase / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
D000952:Antigens, Polyomavirus Transforming; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D005952:Glucose-6-Phosphatase; D006006:Glycogen Synthase; D038362:Glycogen Synthase Kinase 3; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D010750:Phosphoproteins; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C467200:Foxo1 protein, mouse; C526298:Irs2 protein, mouse; C060974:phosphatidylinositol 3,4,5-triphosphate