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Overexpression of Sphingosine Kinase 1 Prevents Ceramide Accumulation and Ameliorates Muscle Insulin Resistance in High Fat–Fed Mice Clinton R

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Overexpression of Sphingosine Kinase 1 Prevents Ceramide Accumulation and Ameliorates Muscle Insulin Resistance in High Fat–Fed Mice Clinton R ORIGINAL ARTICLE Overexpression of Sphingosine Kinase 1 Prevents Ceramide Accumulation and Ameliorates Muscle Insulin Resistance in High Fat–Fed Mice Clinton R. Bruce,1,2 Steve Risis,1 Joanne R. Babb,1 Christine Yang,1 Greg M. Kowalski,2 Ahrathy Selathurai,2 Robert S. Lee-Young,1 Jacquelyn M. Weir,3 Kazuaki Yoshioka,4 Yoh Takuwa,4 Peter J. Meikle,3 Stuart M. Pitson,5 and Mark A. Febbraio1 fi The sphingolipids sphingosine-1-phosphate (S1P) and ceramide delta haploinsuf ciency display increased DAG content are important bioactive lipids with many cellular effects. In- and reduced peripheral insulin sensitivity (5). tracellular ceramide accumulation causes insulin resistance, but Ceramide is a potent lipid-signaling molecule that can sphingosine kinase 1 (SphK1) prevents ceramide accumulation, cause insulin resistance by inhibiting the ability of insulin in part, by promoting its metabolism into S1P. Despite this, the to activate Akt (6) and/or via the activation of c-jun amino role of SphK1 in regulating insulin action has been largely terminal kinase (JNK) (7,8). Importantly, preventing overlooked. Transgenic (Tg) mice that overexpress SphK1were ceramide accumulation by inhibiting de novo ceramide fed a standard chow or high-fat diet (HFD) for 6 weeks before undergoing several metabolic analyses. SphK1 Tg mice fed an synthesis protects against the development of insulin re- HFD displayed increased SphK activity in skeletal muscle, which sistance (9,10). These observations support the hypothesis was associated with an attenuated intramuscular ceramide that increases in ceramide are an important mechanism accumulation compared with wild-type (WT) littermates. This underlying the development of muscle insulin resistance, was associated with a concomitant reduction in the phosphory- and therefore, targeting pathways to prevent ceramide lation of c-jun amino-terminal kinase, a serine threonine kinase accumulation may be a viable therapeutic approach. associated with insulin resistance. Accordingly, skeletal muscle One such approach is to increase ceramide degradation and whole body insulin sensitivity were improved in SphK1 Tg, and clearance. Two important enzymes in this pathway are compared with WT mice, when fed an HFD. We have identified that the enzyme SphK1 is an important regulator of lipid ceramidase and sphingosine kinase (SphK). Ceramidase is partitioning and insulin action in skeletal muscle under condi- responsible for converting ceramide to sphingosine, and tions of increased lipid supply. SphK phosphorylates sphingosine to sphingosine 1 phos- phate (S1P). Because breakdown of S1P is the only way for cellular lipids to exit the sphingolipid pathway, SphK is important in regulating sphingolipid metabolism (11). besity is associated with the development of SphK exists in two isoforms, SphK1 and SphK2. Despite insulin resistance and type 2 diabetes. The clear evidence that SphK1 activation reduces ceramide pathogenesis of insulin resistance is a well- (12,13), the role of SphK1 in regulating insulin action has Oinvestigated area, yet the precise interplay be- been largely overlooked. Activation of SphK1 prevents tween the molecular pathways that leads to this disorder is ceramide accumulation by promoting its metabolism into not fully understood (1). Extensive evidence, however, S1P. SIP is a molecule with many complex functions: it not suggests that defects in fatty acid (FA) metabolism and only activates five specific G-coupled protein receptors subsequent lipid accumulation in liver and skeletal muscle that subsequently activate many downstream signaling play a major role (2,3). Although the increase in lipid pathways but also has important second messenger manifests as an increase in triacylglycerol (TAG), it is actions (14). SIP is generally thought to promote activation likely that this is a marker of dysfunctional FA metabolism of inhibitor of k kinase-b (IKK-b) and JNK via upstream and that accumulation of bioactive lipids, such as ceram- activation of transforming growth factor-b–activated ki- ide and diacylglycerol (DAG), impair insulin action. DAG nase 1 (14), however, SphK1 can block JNK activation accumulation in muscle is associated with insulin re- (13,15) and prevent tissue inflammation (15), which is sistance in humans (4), whereas mice with DAG kinase linked to insulin resistance (16). In contrast, inhibiting SphK1 leads to JNK activation (17). Interestingly, S1P itself From the 1Cellular and Molecular Metabolism Laboratory, Baker IDI Heart and opposes the effects of ceramide. For example, S1P has Diabetes Institute, Melbourne, Victoria, Australia; the 2Department of Phys- been shown to counteract ceramide-induced activation of iology, Monash University, Clayton, Victoria, Australia; the 3Lipidomics Lab- oratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, JNK (18). Thus, it has been proposed that the ceramide-to- Australia; the 4Department of Physiology, Kanazawa University, Kanazawa, S1P ratio may function as an intracellular rheostat (19,20). Japan; and the 5Molecular Signalling Laboratory, Centre for Cancer Biology, Although not well studied in the context of metabolic Adelaide, South Australia, Australia. Corresponding author: Mark A. Febbraio, [email protected]; or disease, evidence is emerging to suggest that the sphin- Clinton R. Bruce, [email protected]. golipid rheostat is important in regulating insulin action. Received 9 January 2012 and accepted 21 June 2012. Adiponectin has been thought to exert insulin-sensitizing DOI: 10.2337/db12-0029 This article contains Supplementary Data online at http://diabetes effects via activation of AMP-activated protein kinase .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0029/-/DC1. (AMPK) (21). However, Scherer and colleagues (22) re- Ó 2012 by the American Diabetes Association. Readers may use this article as cently provided compelling evidence that adiponectin, by long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by stimulating ceramidase activity, decreases intracellular -nc-nd/3.0/ for details. ceramide and concomitantly increases S1P. Hence, the diabetes.diabetesjournals.org DIABETES 1 Diabetes Publish Ahead of Print, published online September 6, 2012 SPHK1 PREVENTS INSULIN RESISTANCE dynamic balance between the levels of ceramide and S1P catheter. Blood (10 mL) was taken at 2, 15, 25, and 35 min for the de- 14 may have implications for the development of obesity- termination of [ C]2DG. Mice were then anesthetized and tissues collected. induced insulin resistance. The aim of the current study Plasma insulin and FFA levels were determined from samples obtained from cardiac puncture on anesthetized mice at the end of the clamp procedure. was, therefore, to examine the role of SphK in regulating Determination of plasma and tissue radioactivity. Blood samples (10 mL) skeletal muscle ceramide content and insulin sensitivity were deproteinized with barium hydroxide (0.3 N) and zinc sulfate (0.3 N), and in conditions of lipid oversupply. We hypothesized that [3-3H]glucose and 2[14C]DG radioactivity was determined by liquid scintillation SphK1 overexpression would promote flux through the counting. The rate of glucose appearance (endogenous Ra) and disappearance sphingolipid degradative pathway, which would prevent (Rd) were determined using steady-state equations. Endogenous glucose high-fat diet (HFD)–induced ceramide accumulation and, production during the clamp was calculated by subtracting the glucose in- fusion rate (GIR) from R . The insulin-stimulated component of the total R therefore, enhance muscle insulin action. d d (IS-Rd) is equal to clamp Rd minus the basal glucose turnover rate. Accumu- lation of [14C]2DG was determined in an aqueous extract of tissue after ho- mogenization. Free and phosphorylated [14C]2DG were separated by ion RESEARCH DESIGN AND METHODS exchange chromatography on Dowex l-X8 columns (acetate form). The area Animals. All experimental procedures were approved by the Alfred Medical under the tracer disappearance curve of [14C]2DG, together with the radio- Research and Education Precinct Animal Ethics Committee and were in ac- activity for the phosphorylated [14C]2DG from individual tissues, were used to cordance with the National Health and Medical Research Council (NHMRC) of calculate the glucose metabolic index (Rg’) (28). Australia Guidelines on Animal Experimentation. Generation of the moderate- Western blotting. Tissues were lysed, and protein concentration was de- level or low-level SphK1a transgenic (SphK1 Tg) mice has been described termined. Lysates were solubilized, 40 mg protein was loaded and resolved by elsewhere (23,24). Mice were maintained on a C57Bl/6 background (back- SDS-PAGE on polyacrylamide gels, transferred to membranes, and blocked crossed $9 times). All experiments commenced when mice were 8 weeks old. with 5% milk. Immunoblotting was performed using the following primary Male SphK1 Tg mice and their wild-type (WT) littermates were kept in antibodies: phosphorylated (p)Akt serine 473 and total (t)Akt (Cell Signaling a temperature controlled room (22 6 1°C) on a 12-h light/dark cycle with free Technology, Danvers, MA), pJNK (Thr183/Tyr185) and tJNK (Cell Signaling access to food and water. Mice were fed a standard chow diet (14.3 MJ/kg, 76% Technology), as well as pIKK-b (Ser181), tIKK-b (Cell Signaling Technology). of kJ from carbohydrate, 5% fat, 19% protein, Specialty
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