Hif 1α

Fasting blood was used to determine glucose, insulin, glycohemoglobin, lipids (non-esterified fatty acids [NEFA (Wako Chemicals GmbH, Neuss, DE)], total cholesterol [TC], low-density lipoprotein cholesterol [LDL], high density lipoprotein cholesterol [HDL] and triglycerides [TG]). Also, circulating levels of GH, IGF-1, cortisol and PRL (DRG International, Inc., US); leptin, TNF-α, VEGF (Invitrogen, CA, US), adiponectin (R&D Systems, MN, US), RANK-L (BioVendor, CR, US), HIF1α (RayBiotech, GA, US) and estradiol (Life Technologies, CA, US) were measured using commercial ELISA kits. All the information regarding each assay can be accessed at the company website.

Fasting serum insulin and plasma leptin and ghrelin concentrations, was assessed (SPI bio, Germany). Lipid profile including total cholesterol, triglyceride, high density lipid (HDL), low density lipid (LDL) and very low-density lipid (VLDL) were measured in serum (Grenier Diagnostic, Germany). Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K, Bioassay Laboratory Technology, China), insulin (Ray Biotech, USA), phospho-Akt (Ray Biotech, USA), Resistin-Like Molecule (RELM, Ray Biotech, USA), phosphorylated-signal transducer and activator of transcription-3 (p-tyr-STAT-3, Ray Biotech, USA), uric acid (Abcam Lab, USA), serum glutamic-oxaloacetic transaminase (SGOT, Erba Lab, India), serum glutamic-pyruvic transaminase (SGPT, Erba Lab, India), hypoxia-inducible factor 1-alpha (HIF-1α, Ray Biotech, USA), vascular endothelial growth factor (VEGF, Krishgen Biosystems, India) and tumor necrosis factor (TNF-α, Krishgen Biosystems, India) were measured in liver sample.

The cell lysate was used for estimating glycogen concentration, enzyme activities (Hexokinase, HK; Aldehyde dehydrogenase, ALDH; Ketohexokinase, KHK; Phosphofructokinase, PFK) using respective ELISA kits (Sincere, China). The phosphatidylinositol-4,5-bisphosphate 3-kinase (P13K), signal transducer and activator of transcription 3 (STAT-3), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor 1-alpha (HIF-1α, Ray Biotech, USA), were estimated in cell lysate. In supernatant tumor necrosis factor (TNF-α, Krishgen Biosystems, India) and vascular endothelial growth factor (VEGF, Krishgen Biosystems, India), were estimated.