Orexin A stimulates glucose uptake, lipid accumulation and adiponectin secretion from 3T3-L1 adipocytes and isolated primary rat adipocytes

Abstract

Aims/hypothesis Orexin A (OXA) modulates body weight, food intake and energy expenditure. In vitro, OXA increases PPARγ (also known as PPARG) expression and inhibits lipolysis, suggesting direct regulation of lipid metabolism. Here, we characterise the metabolic effects and mechanisms of OXA action in adipocytes. Methods Isolated rat adipocytes and differentiated murine 3T3-L1 adipocytes were exposed to OXA in the presence or absence of phosphoinositide 3-kinase (PI3K) inhibitors. Pparγ expression was silenced using small interfering RNA. Glucose uptake, GLUT4 translocation, phosphatidylinositol (3,4,5)-trisphosphate production, lipogenesis, lipolysis, and adiponectin secretion were measured. Adiponectin plasma levels were determined in rats treated with OXA for 4 weeks.Results OXA PI3K-dependently stimulated active glucose uptake by translocating the glucose transporter GLUT4 from cytoplasm into the plasma membrane. OXA increased cellular triacylglycerol content via PI3K. Cellular triacylglycerol accumulation resulted from increased lipogenesis as well as from a decrease of lipolysis. Adiponectin levels in chow-and high-fat diet-fed rats treated chronically with OXA were increased. OXA stimulated adiponectin expression and secretion in adipocytes. Both pharmacological blockade of peroxisome proliferator-activated receptor γ (PPARγ) activity or silencing Pparγ expression prevented OXA from stimulating triacylglycerol accumulation and adiponectin production. Conclusions/interpretation Our study demonstrates that OXA stimulates glucose uptake in adipocytes and that the evolved energy is stored as lipids. OXA increases lipogenesis, inhibits lipolysis and stimulates the secretion of adiponectin. These effects are conferred via PI3K and PPARγ2. Overall, OXA's effects on lipids and adiponectin secretion resemble that of insulin sensitisers, suggesting a potential relevance of this peptide in metabolic disorders.Electronic supplementary material The online version of this article

Glucagon increases circulating fibroblast growth factor 21 independently of endogenous insulin levels: a novel mechanism of glucagon-stimulated lipolysis?

“…Isolation of rat adipocytes and pre-adipocytes Adipocytes were isolated according to the method of Rodbell [26] with some modifications, as described [27]. Adipocytes (10 6 cells/ml) were incubated in Krebs-Ringer-bicarbonate-HEPES (KRBH) (5 mmol/l glucose, 3% wt/vol.…”

“…Lipolysis and FGF-21 assay Lipolysis was determined by measuring the concentration of released glycerol in incubation medium using 'free glycerol reagent' [27]. The results were normalised to cell number and are expressed in mmol/10 6 cells.…”

“…Differentiation of 3T3-L1 fibroblasts into adipocytes, and secretion assays 3T3-L1 fibroblasts (American Type Culture Collection, LGC Standards, Wesel, Germany) were differentiated to mature adipocytes [27,28]. Criteria for differentiation were: ≥1,000-fold increase of Fabp4 relative to Gapdh and lipid droplet formation in ≥95% cells (data not shown).…”

“…Western blot Proteins were isolated from 3T3-L1 adipocytes, as described and resolved on SDS-PAGE [27]. Membranes were incubated with anti-FGF21 antibody (ESM Table 2), washed and exposed to a secondary antibody (ESM Table 2).…”

“…Signals were detected by enhanced chemiluminescence (GE Healthcare Europe, Freiburg, Germany) and visualised using x-ray film. Membranes were stripped and incubated with anti-β-actin antibody, as the loading control [27].…”