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

Aim: To investigate the pathogenesis of age related macular degeneration (ARM) with respect to lipid accumulation within Bruch's membrane (BrM) in a knockout model with low density lipoprotein (LDL) receptor deficiency. Methods: LDL receptor deficient mice and C57BL/6 controls were fed a standard diet or a high fat (HF) diet. Plasma total cholesterol (pTC) was determined. Eyes were examined by transmission electron microscopy. Immunohistochemical staining for VEGF was performed. Results: pTC were highest in LDL receptor deficient mice after HF diet and elevated after standard diet compared to controls with and without HF diet. While BrM of controls did not exhibit any visible changes, membrane bound translucent particles were seen in all BrM of knockout mice. The amount of these particles was substantially increased and membranes were thickened after HF diet. VEGF staining was positive in knockout mice only and was located in retinal pigment epithelial cells, the outer plexiform layer, and photoreceptor inner segments. Most intensive VEGF expression was documented after HF diet. Conclusion: LDL receptor deficient mice exhibit an accumulation of lipid particles in BrM which is further increased after fat intake. VEGF expression is found in the outer retinal layers of LDL receptor deficient mice and appears to correlate with the amount of lipid particles present in BrM.

Orexin-A (OXA) regulates food intake and energy homeostasis. It increases insulin secretion in vivo and in vitro, although controversial effects of OXA on plasma glucagon are reported. We characterized the effects of OXA on glucagon secretion and identify intracellular target molecules in glucagon-producing cells. Glucagon secretion from in situ perfused rat pancreas, isolated rat pancreatic islets, and clonal pancreatic A-cells (InR1-G9) were measured by RIA. The expression of orexin receptor 1 (OXR1) was detected by Western blot and immunofluorescence. The effects of OXA on cAMP, adenylate-cyclase-kinase (AKT), phosphoinositide-dependent kinase (PDK)-1, forkhead box O-1 (Foxo1), and cAMP response element-binding protein were measured by ELISA and Western blot. Intracellular calcium (Ca(2+)(i)) concentration was detected by fura-2and glucagon expression by real-time PCR. Foxo1 was silenced in InR1-G9 cells by transfecting cells with short interfering RNA. OXR1 was expressed on pancreatic A and InR1-G9 cells. OXA reduced glucagon secretion from perfused rat pancreas, isolated rat pancreatic islets, and InR1-G9 cells. OXA inhibited proglucagon gene expression via the phosphatidylinositol 3-kinase-dependent pathway. OXA decreased cAMP and Ca(2+)(i) concentration and increased AKT, PDK-1, and Foxo1 phosphorylation. Silencing of Foxo1 caused a reversal of the inhibitory effect of OXA on proglucagon gene expression. Our study provides the first in vitro evidence for the interaction of OXA with pancreatic A cells. OXA inhibits glucagon secretion and reduces intracellular cAMP and Ca(2+)(i) concentration. OXA increases AKT/PDK-1 phosphorylation and inhibits proglucagon expression via phosphatidylinositol 3-kinase- and Foxo-1-dependent pathways. As a physiological inhibitor of glucagon secretion, OXA may have a therapeutic potential to reduce hyperglucagonemia in type 2 diabetes.

a b s t r a c tMetabolic activities of orexin A (OXA) in mature adipocytes are mediated via PI3K/PKB and PPARc. However, the effects of OXA on preadipocytes are largely unknown. We report here that OXA stimulates the proliferation and viability of 3T3-L1 preadipocytes and protects them from apoptosis via ERK1/2, but not through PKB. OXA reduces proapoptotic activity of caspase-3 via ERK1/2. Inhibition of ERK1/2 prevents the differentiation of preadipocytes into adipocytes. Unlike insulin, neither short-term nor prolonged exposure of 3T3-L1 preadipocytes to OXA induces preadipocyte differentiation to adipocytes, despite increased ERK1/2 phosphorylation. Unlike insulin, OXA fails to activate PKB, which explains its inability to induce the differentiation of preadipocytes.

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