ABSTRACT
mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.
Subject(s)
ATP Citrate (pro-S)-Lyase/metabolism , Adipocytes, Brown/metabolism , Lipogenesis/physiology , Mechanistic Target of Rapamycin Complex 2/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Acetate-CoA Ligase/metabolism , Animals , Carrier Proteins , Epigenesis, Genetic , Fatty Acid Synthases , Gene Editing , Gene Expression , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , HEK293 Cells , Histones/metabolism , Humans , Lipogenesis/genetics , Mice , Mice, Inbred C57BL , PPAR gamma/metabolism , Phosphorylation , Proteomics , Response ElementsABSTRACT
Previous studies from our laboratory demonstrated the involvement of COX-2 in the stimulation of steroid production by LPS in murine adrenocortical Y1 cells, as well as in the adrenal cortex of male Wistar rats. In this paper we analyzed signaling pathways involved in the induction of this key regulatory enzyme in adrenocortical cells and demonstrated that LPS triggers an increase in COX-2 mRNA levels by mechanisms involving the stimulation of reactive oxygen species (ROS) generation and the activation of p38 MAPK and Akt, in addition to the previously demonstrated increase in NFκB activity. In this sense we showed that: (1) inhibition of p38 MAPK or PI3K/Akt (pharmacological or molecular) prevented the increase in COX-2 protein levels by LPS, (2) LPS induced p38 MAPK and Akt phosphorylation, (3) antioxidant treatment blocked the effect of LPS on p38 MAPK phosphorylation and in COX-2 protein levels, (4) PI3K inhibition with LY294002 prevented p38 MAPK phosphorylation and, (5) the activity of an NFκB reporter was decreased by p38 MAPK or PI3K inhibition. These results suggest that activation of both p38 MAPK and PI3K/Akt pathways promote the stimulation of NFκB activity and that PI3K/Akt activity might regulate both p38 MAPK and NFκB signaling pathways. In summary, in this study we showed that in adrenal cells, LPS induces COX-2 expression by activating p38 MAPK and PI3K/Akt signaling pathways and that both pathways converge in the modulation of NFκB transcriptional activity.
Subject(s)
Adrenal Cortex/drug effects , Cyclooxygenase 2/genetics , Lipopolysaccharides/pharmacology , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , p38 Mitogen-Activated Protein Kinases/genetics , Adrenal Cortex/cytology , Adrenal Cortex/metabolism , Animals , Antioxidants/pharmacology , Cell Line, Tumor , Chromones/pharmacology , Cyclooxygenase 2/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Male , Mice , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Primary Cell Culture , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Rats , Reactive Oxygen Species/metabolism , Signal Transduction , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Stimulation of adrenal steroidogenesis is involved in the HPA response to exogenous noxa. Although inflammatory cytokines can mediate the LPS-triggered activation of the HPA, direct effects of LPS on glucocorticoid release have been described. Present studies were undertaken to characterize the molecular mechanisms underlying the effect of LPS on steroid secretion in isolated rodent adrenal cells, assessing the participation of NFκB and COX-2 activities in this response. Our results show that LPS treatment stimulates steroidogenesis in murine and rat adrenocortical cells, and that Y1 cells express the binding-transducing complex TLR-4/CD14/MD-2, as demonstrated by RT-PCR. NFκB activity and COX-2 protein levels are increased in this cell line by LPS treatment, and pharmacologic and molecular manipulation of the NFκB pathway significantly affected both COX-2 protein levels and steroid production. Finally, pharmacological inhibition of COX-2 activity significantly impairs steroid production. Thus, our results strongly suggest that the mechanism involved in the stimulation of steroidogenesis by LPS in rodent adrenal cells involves the activation of the NFκB signaling pathway and the induction of COX-2.
Subject(s)
Adrenal Glands/cytology , Cyclooxygenase 2/metabolism , Enzyme Activation/drug effects , Lipopolysaccharides/pharmacology , NF-kappa B/metabolism , Adrenal Glands/enzymology , Adrenal Glands/metabolism , Animals , Cell Culture Techniques , Cell Line, Tumor , Corticosterone/biosynthesis , Heterocyclic Compounds, 3-Ring/pharmacology , I-kappa B Kinase/antagonists & inhibitors , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Lymphocyte Antigen 96/genetics , Lymphocyte Antigen 96/metabolism , Mice , Phosphoproteins/metabolism , Progesterone/biosynthesis , Pyridines/pharmacology , Rats , Signal Transduction/drug effects , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Transcription, Genetic/drug effectsABSTRACT
BACKGROUND: Increased activity of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in enhanced adrenocorticotropin (ACTH) and serum glucocorticoid levels, has been described in patients with diabetes mellitus and in animal models of this disease; however, altered steroid production by adrenocortical cells could result from local changes triggered by increased reactive oxygen species (ROS), induced in turn by chronic hyperglycaemia. Experiments were designed (1) to analyse the effects of incubating murine adrenocortical cells in hyperglycaemic media on the generation of oxidative stress, on steroid synthesis and on its modulation by the activity of haeme oxygenase (HO); and (2) to evaluate the effect of antioxidant treatment on these parameters. METHODS: Y1 cells were incubated for 7 days with either normal or high glucose (HG, 30 mmol/L) concentrations, with or without antioxidant treatment. Parameters of oxidative stress and expression levels of haeme oxygenase-1 (HO-1), nitrite levels, L-arginine uptake and progesterone production were determined. RESULTS: HG augmented ROS and lipoperoxide production, decreasing glutathione (GSH) levels and increasing antioxidant enzymes and HO-1 expression. Basal progesterone production was reduced, while a higher response to ACTH was observed in HG-treated cells. The increase in HO-1 expression and the effects on basal steroid production were abolished by antioxidant treatment. Inhibition of HO activity increased basal and ACTH-stimulated steroid release. Similar results were obtained by HO-1 gene silencing while the opposite effect was observed in Y1 cells overexpressing HO-1. CONCLUSIONS: HG induces oxidative stress and affects steroid production in adrenal cells; the involvement of HO activity in the modulation of steroidogenesis in Y1 cells is postulated.
