Central Leptin Regulates Total Ceramide Content and Sterol Regulatory Element Binding Protein-1C Proteolytic Maturation in Rat White Adipose Tissue

ENERGY BALANCE-OBESITY Central Leptin Regulates Total Ceramide Content and Sterol Regulatory Element Binding Protein-1C Proteolytic Maturation in Rat White Adipose Tissue Elena Bonzón-Kulichenko, Dominik Schwudke, Nilda Gallardo, Eduardo Moltó, Teresa Fernández-Agulló, Andrej Shevchenko, and Antonio Andrés Biochemistry Section (E.B.-K., N.G., E.M., A.A.), Faculty of Chemistry, and Regional Centre for Biomedical Research, University of Castilla-La Mancha, 13071 Ciudad Real, Spain; Max Plank Institute of Molecular Cell Biology and Genetics (D.S., A.S.), 01307 Dresden, Germany; and Health Sciences Faculty (T.F.-A.), University Rey Juan Carlos, Alcorcón, 28922 Madrid, Spain Obesity and type 2 diabetes are associated with insulin and leptin resistance, and increased ceramide contents in target tissues. Because the adipose tissue has become a central focus in these diseases, and leptin-induced increases in insulin sensitivity may be related to effects of leptin on lipid metabolism, we investigated herein whether central leptin was able to regulate total ceramide levels and the expression of enzymes involved in ceramide metabolism in rat white adipose tissue (WAT). After 7 d central leptin treatment, the total content of ceramides was analyzed by quantitative shotgun lipidomics mass spectrometry. The effects of leptin on the expression of several enzymes of the sphingolipid metabolism, sterol regulatory element binding protein (SREBP)-1c, and insulin-induced gene 1 (INSIG-1) in this tissue were studied. Total ceramide levels were also determined after surgical WAT denervation. Central leptin infusion significantly decreased both total ceramide content and the long-chain fatty acid ceramide species in WAT. Concomitant with these results, leptin decreased the mRNA levels of enzymes involved in de novo ceramide synthesis (SPT-1, LASS2, LASS4) and ceramide production from sphingomyelin (SMPD-1/ 2). The mRNA levels of enzymes of ceramide degradation (Asah1/2) and utilization (sphingomyelin synthase, ceramide kinase, glycosyl-ceramide synthase, GM3 synthase) were also down-regulated. Ceramide-lowering effects of central leptin were prevented by local autonomic nervous system denervation of WAT. Finally, central leptin treatment markedly increased INSIG-1 mRNA expression and impaired SREBP-1c activation in epididymal WAT. These observations indicate that in vivo central leptin, acting through the autonomic nervous system, regulates total ceramide levels and SREBP-1c proteolytic maturation in WAT, probably contributing to improve the overall insulin sensitivity. (Endocrinology 150: 169 –178, 2009) eptin, an adipocyte-derived hormone, is actively involved in the control of body weight and food intake (1). Dysregulations of leptin action result in obesity, insulin resistance, and type 2 diabetes (2– 4). Over-accumulation of triacylglycerides (TAGs) in lean tissues precedes the development of insulin resistance and type 2 diabetes (3). However, increased content of TAG in lean L tissues may only be a marker of dysfunctional fatty acid (FA) metabolism, meanwhile more biologically active lipids, such as ceramides, which could be produced from unoxidized FAs, could be responsible for the impaired insulin signaling (4). Thus, ceramide accumulates in insulin-resistant nonadipose tissues from rodent and humans (5, 6), impairs the insulin-stimulated glucose ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-0505 Received April 9, 2008. Accepted September 10, 2008. First Published Online September 18, 2008 Abbreviations: AUC, Area under the curve; Chol, cholesterol; ER, endoplasmic reticulum; eWAT, epididymal white adipose tissue; FA, fatty acid; INSIG-1, insulin-induced gene 1; MS/MS, Tandem mass spectrometry; PC, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3phosphocholine; PM, plasma membrane; pSTAT3, phosphorylated signal transducer and activator of transcription-3; rWAT, retroperitoneal white adipose tissue; SCAP, sterol regulatory element binding protein cleavage-activating protein; SM, sphingomyelin; SMS, sphingomyelin synthase; SPT, serine palmitoyl transferase; SREBP, sterol regulatory element binding protein; STAT3, signal transducer and activator of transcription-3; TAG, triacylglyceride; TLC, thin-layer chromatography; TOF MS, time-of-flight mass spectrometry; WAT, white adipose tissue. Endocrinology, January 2009, 150(1):169 –178 endo.endojournals.org 169 170 Bonzón-Kulichenko et al. Central Leptin and WAT Sphingolipid Metabolism uptake through the inhibition of the enzyme Akt (7, 8), and is involved in the lipotoxicity of the heart (9) and pancreatic ␤-cells (10). In addition, ceramide is a pivotal substrate for the synthesis of sphingomyelin (SM) and cell surface glycosphingolipids (11). Recent studies have shown that a low-grade inflammation in adipose tissue contributes to development of obesity related insulin resistance (12). Moreover, increased ceramide content in adipose tissue and inflammation characterize humans with high liver fat content independently of obesity, suggesting that ceramides could contribute to induce both insulin resistance and inflammation (13). In this sense it has been recently reported that ceramide content is increased in adipocytes from insulin-resistant old mice, and suggested that adipocytes are the major contributor to the age-related increase in adipose tissue inflammatory cytokines (14). A treatment with leptin increases overall insulin sensitivity (15, 16) and down-regulates de novo ceramide synthesis pathway in pancreatic ␤-cells, by decreasing the expression of serine palmitoyl transferase (SPT) (10). In addition, adenovirus-induced hyperleptinemia prevents ceramide-mediated lipotoxicity in cardiomyocytes from acyl coenzyme A synthase transgenic mice (17). These studies indicate that sphingolipid metabolism could be regulated by central and/or peripheral leptin acting through leptin receptors. Nevertheless, several experimental results suggest that leptin exerts its effects on lipid metabolism predominantly through the hypothalamus, where leptin receptor expression is higher than in other brain regions and tissues (18). In addition, leptin-mediated effects on sphingolipid metabolism in white adipose tissue (WAT) have not been investigated. Here, we investigated the effects of centrally administered leptin on ceramide contents and on the expression of several enzymes of the sphingolipid metabolism in rat WAT. The role of the autonomic nervous system, as mediator of central leptin effects on WAT, was analyzed. In addition, because changes in the sphingolipid metabolism regulate the activation of the lipogenic transcription factor, sterol regulatory element binding protein (SREBP)-1c (19, 20), we further examined if central leptin regulated SREBP-1c activation, and whether a ceramide-dependent pathway could be involved in this process. Endocrinology, (...truncated)