Impaired Akt phosphorylation in insulin-resistant human muscle is accompanied by selective and heterogeneous downstream defects.

AIMS/HYPOTHESIS: Muscle insulin resistance, one of the earliest defects associated with type 2 diabetes, involves changes in the phosphoinositide 3-kinase/Akt network. The relative contribution of obesity vs insulin resistance to perturbations in this pathway is poorly understood. METHODS: We used phosphospecific antibodies against targets in the Akt signalling network to study insulin action in muscle from lean, overweight/obese and type 2 diabetic individuals before and during a hyperinsulinaemic-euglycaemic clamp. RESULTS: Insulin-stimulated Akt phosphorylation at Thr309 and Ser474 was highly correlated with whole-body insulin sensitivity. In contrast, impaired phosphorylation of Akt substrate of 160 kDa (AS160; also known as TBC1D4) was associated with adiposity, but not insulin sensitivity. Neither insulin sensitivity nor obesity was associated with defective insulin-dependent phosphorylation of forkhead box O (FOXO) transcription factor. In view of the resultant basal hyperinsulinaemia, we predicted that this selective response within the Akt pathway might lead to hyperactivation of those processes that were spared. Indeed, the expression of genes targeted by FOXO was downregulated in insulin-resistant individuals. CONCLUSIONS/INTERPRETATION: These results highlight non-linearity in Akt signalling and suggest that: (1) the pathway from Akt to glucose transport is complex; and (2) pathways, particularly FOXO, that are not insulin-resistant, are likely to be hyperactivated in response to hyperinsulinaemia. This facet of Akt signalling may contribute to multiple features of the metabolic syndrome.

Conservation of signaling pathways of xenobiotic-sensing orphan nuclear receptors, chicken xenobiotic receptor, constitutive androstane receptor, and pregnane X receptor, from birds to humans.

Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor are orphan nuclear receptors that have recently been discovered to regulate drug- and steroid-mediated induction of hepatic cytochromes P450 (CYP). This induction is part of an adaptive response involving numerous genes to exposure to drugs and chemicals and has major clinical and toxicological implications. Here we report experiments in the chicken hepatoma cell line LMH that suggest evolutionary conservation of the signaling pathways triggered by pregnane X receptor, constitutive androstane receptor, and chicken xenobiotic receptor. Thus, the phenobarbital-inducible enhancer units of the mouse Cyp2b10, rat CYP2B2, and human CYP2B6 genes were activated in reporter gene assays by the same compounds that activate the chicken CYP2H1 phenobarbital-inducible enhancer units. Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor all bound to the CYP2H1 phenobarbital-inducible enhancer units in gel-shift experiments. In CV-1 cell transactivation assays, mammalian pregnane X receptors activate the chicken phenobarbital-inducible enhancer units to the same extent as does chicken xenobiotic receptor, each receptor maintaining its species-specific ligand spectrum. To assess the reported role of protein phosphorylation in drug-mediated induction, we treated LMH cells with okadaic acid and observed increased mRNA of delta-aminolevulinate synthase and CYP2H1 whereas expression of CYP3A37 was decreased. The effects of okadaic acid and other modifiers of protein phosphorylation in LMH cells are comparable to those seen on CYP2Bs and CYP3As in mammalian primary hepatocyte cultures. These results indicate that closely related nuclear receptors, transcription factors, and signaling pathways are mediating the transcriptional activation of multiple genes by xenobiotics in chicken, rodents, and man.