Autotaxin-LPA signaling contributes to obesity-induced insulin resistance in muscle and impairs mitochondrial metabolism
2018; Elsevier BV; Volume: 59; Issue: 10 Linguagem: Inglês
10.1194/jlr.m082008
ISSN1539-7262
AutoresKenneth D’Souza, Carine Nzirorera, Andrew Cowie, Geena V. Paramel, Purvi Trivedi, Thomas O. Eichmann, Dipsikha Biswas, Mohamed Touaibia, Andrew J. Morris, Vassilis Aidinis, Daniel A. Kane, Thomas Pulinilkunnil, Petra C. Kienesberger,
Tópico(s)Paraoxonase enzyme and polymorphisms
ResumoAutotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid (LPA). ATX-LPA signaling has been implicated in diet-induced obesity and systemic insulin resistance. However, it remains unclear whether the ATX-LPA pathway influences insulin function and energy metabolism in target tissues, particularly skeletal muscle, the major site of insulin-stimulated glucose disposal. The objective of this study was to test whether the ATX-LPA pathway impacts tissue insulin signaling and mitochondrial metabolism in skeletal muscle during obesity. Male mice with heterozygous ATX deficiency (ATX+/−) were protected from obesity, systemic insulin resistance, and cardiomyocyte dysfunction following high-fat high-sucrose (HFHS) feeding. HFHS-fed ATX+/− mice also had improved insulin-stimulated AKT phosphorylation in white adipose tissue, liver, heart, and skeletal muscle. Preserved insulin-stimulated glucose transport in muscle from HFHS-fed ATX+/− mice was associated with improved mitochondrial pyruvate oxidation in the absence of changes in fat oxidation and ectopic lipid accumulation. Similarly, incubation with LPA decreased insulin-stimulated AKT phosphorylation and mitochondrial energy metabolism in C2C12 myotubes at baseline and following palmitate-induced insulin resistance. Taken together, our results suggest that the ATX-LPA pathway contributes to obesity-induced insulin resistance in metabolically relevant tissues. Our data also suggest that LPA directly impairs skeletal muscle insulin signaling and mitochondrial function. Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid (LPA). ATX-LPA signaling has been implicated in diet-induced obesity and systemic insulin resistance. However, it remains unclear whether the ATX-LPA pathway influences insulin function and energy metabolism in target tissues, particularly skeletal muscle, the major site of insulin-stimulated glucose disposal. The objective of this study was to test whether the ATX-LPA pathway impacts tissue insulin signaling and mitochondrial metabolism in skeletal muscle during obesity. Male mice with heterozygous ATX deficiency (ATX+/−) were protected from obesity, systemic insulin resistance, and cardiomyocyte dysfunction following high-fat high-sucrose (HFHS) feeding. HFHS-fed ATX+/− mice also had improved insulin-stimulated AKT phosphorylation in white adipose tissue, liver, heart, and skeletal muscle. Preserved insulin-stimulated glucose transport in muscle from HFHS-fed ATX+/− mice was associated with improved mitochondrial pyruvate oxidation in the absence of changes in fat oxidation and ectopic lipid accumulation. Similarly, incubation with LPA decreased insulin-stimulated AKT phosphorylation and mitochondrial energy metabolism in C2C12 myotubes at baseline and following palmitate-induced insulin resistance. Taken together, our results suggest that the ATX-LPA pathway contributes to obesity-induced insulin resistance in metabolically relevant tissues. Our data also suggest that LPA directly impairs skeletal muscle insulin signaling and mitochondrial function. Autotaxin (ATX) is a lysophospholipase D that is also known as ecto-nucleotide pyrophosphatase/phosphodiesterase family member 2 (ENPP2) (1.Benesch M.G. Ko Y.M. McMullen T.P. Brindley D.N. Autotaxin in the crosshairs: taking aim at cancer and other inflammatory conditions.FEBS Lett. 2014; 588: 2712-2727Crossref PubMed Scopus (87) Google Scholar), and generates the majority of extracellular lysophosphatidic acid (LPA) by hydrolyzing the lysophosphatidylcholine (LPC) contained in lipoproteins and activated platelets (1.Benesch M.G. Ko Y.M. McMullen T.P. Brindley D.N. Autotaxin in the crosshairs: taking aim at cancer and other inflammatory conditions.FEBS Lett. 2014; 588: 2712-2727Crossref PubMed Scopus (87) Google Scholar–3.Aoki J. Taira A. Takanezawa Y. Kishi Y. Hama K. Kishimoto T. Mizuno K. Saku K. Taguchi R. Arai H. Serum lysophosphatidic acid is produced through diverse phospholipase pathways.J. Biol. Chem. 2002; 277: 48737-48744Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Despite its ubiquitous expression, a substantial proportion of circulating ATX emanates from the adipose tissue. Indeed, adipose-specific ATX knockout mice exhibit ∼50% reduced plasma LPA (4.Dusaulcy R. Rancoule C. Gres S. Wanecq E. Colom A. Guigne C. van Meeteren L.A. Moolenaar W.H. Valet P. Saulnier-Blache J.S. Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.J. Lipid Res. 2011; 52: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar). ATX-LPA signaling through G protein-coupled receptors (LPA1–6) influences many biological processes, including brain development, embryo implantation, vasculogenesis, and hair follicle formation (6.Yung Y.C. Stoddard N.C. Chun J. LPA receptor signaling: pharmacology, physiology, and pathophysiology.J. Lipid Res. 2014; 55: 1192-1214Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Signaling effectors downstream of LPA receptors include PI3Kinase-AKT-Ras-related C3 botulinum toxin substrate 1 activation, modulation of adenylate cyclase, stimulation of the mitogen-activated protein kinase pathway, Rho and Rho kinase activation, and stimulation of the phospholipase C-protein kinase C pathway (7.Lin M.E. Herr D.R. Chun J. Lysophosphatidic acid (LPA) receptors: signaling properties and disease relevance.Prostaglandins Other Lipid Mediat. 2010; 91: 130-138Crossref PubMed Scopus (294) Google Scholar). The ATX-LPA signaling axis plays an important role in many disease states, such as cancer, cardiovascular disease, neuropathic pain, pulmonary fibrosis, and arthritis (2.Okudaira S. Yukiura H. Aoki J. Biological roles of lysophosphatidic acid signaling through its production by autotaxin.Biochimie. 2010; 92: 698-706Crossref PubMed Scopus (132) Google Scholar, 8.Abdel-Latif A. Heron P.M. Morris A.J. Smyth S.S. 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Karouzakis E. Sevastou I. Nikolaidou-Katsaridou N. Zhao Z. Mersinias V. Armaka M. Xu Y. Masu M. et al.Autotaxin expression from synovial fibroblasts is essential for the pathogenesis of modeled arthritis.J. Exp. Med. 2012; 209: 925-933Crossref PubMed Scopus (124) Google Scholar). Notably, the ATX-LPA axis has been explored as a therapeutic target in diseases associated with chronic inflammation (13.Stoddard N.C. Chun J. Promising pharmacological directions in the world of lysophosphatidic Acid signaling.Biomol. Ther. (Seoul). 2015; 23: 1-11Crossref PubMed Scopus (91) Google Scholar, 14.Barbayianni E. Kaffe E. Aidinis V. Kokotos G. Autotaxin, a secreted lysophospholipase D, as a promising therapeutic target in chronic inflammation and cancer.Prog. Lipid Res. 2015; 58: 76-96Crossref PubMed Scopus (88) Google Scholar). At least three compounds targeting the ATX-LPA pathway have passed phase I and II clinical trials for the treatment of idiopathic pulmonary fibrosis and systemic sclerosis (13.Stoddard N.C. Chun J. Promising pharmacological directions in the world of lysophosphatidic Acid signaling.Biomol. Ther. (Seoul). 2015; 23: 1-11Crossref PubMed Scopus (91) Google Scholar, 15.Kihara Y. Mizuno H. Chun J. Lysophospholipid receptors in drug discovery.Exp. Cell Res. 2015; 333: 171-177Crossref PubMed Scopus (142) Google Scholar), demonstrating that modulators of ATX-LPA receptor signaling have promising therapeutic potential. A recent study showed that genetic deletion or long-term pharmacologic inhibition of ATX in adult mice is well-tolerated, alleviating concerns that targeting the ATX-LPA pathway could elicit toxicity (16.Katsifa A. Kaffe E. Nikolaidou-Katsaridou N. Economides A.N. Newbigging S. McKerlie C. Aidinis V. The bulk of autotaxin activity is dispensable for adult mouse life.PLoS One. 2015; 10: e0143083Crossref PubMed Scopus (50) Google Scholar). The ATX-LPA pathway has also been implicated in obesity and metabolic disease, specifically insulin resistance and impaired glucose homeostasis (4.Dusaulcy R. Rancoule C. Gres S. Wanecq E. Colom A. Guigne C. van Meeteren L.A. Moolenaar W.H. Valet P. Saulnier-Blache J.S. Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.J. Lipid Res. 2011; 52: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar, 17.Ferry G. Tellier E. Try A. Gres S. Naime I. Simon M.F. Rodriguez M. Boucher J. Tack I. Gesta S. et al.Autotaxin is released from adipocytes, catalyzes lysophosphatidic acid synthesis, and activates preadipocyte proliferation. Up-regulated expression with adipocyte differentiation and obesity.J. Biol. Chem. 2003; 278: 18162-18169Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 18.Boucher J. Quilliot D. Praderes J.P. Simon M.F. Gres S. Guigne C. Prevot D. Ferry G. Boutin J.A. Carpene C. et al.Potential involvement of adipocyte insulin resistance in obesity-associated up-regulation of adipocyte lysophospholipase D/autotaxin expression.Diabetologia. 2005; 48: 569-577Crossref PubMed Scopus (91) Google Scholar, 19.Rachakonda V.P. Reeves V.L. Aljammal J. Wills R.C. Trybula J.S. DeLany J.P. Kienesberger P.C. Kershaw E.E. Serum autotaxin is independently associated with hepatic steatosis in women with severe obesity.Obesity (Silver Spring). 2015; 23: 965-972Crossref PubMed Scopus (32) Google Scholar, 20.Reeves V.L. Trybula J.S. Wills R.C. Goodpaster B.H. Dube J.J. Kienesberger P.C. Kershaw E.E. Serum autotaxin/ENPP2 correlates with insulin resistance in older humans with obesity.Obesity (Silver Spring). 2015; 23: 2371-2376Crossref PubMed Scopus (43) Google Scholar, 21.Sun S. Wang R. Song J. Guan M. Li N. Zhang X. Zhao Z. Zhang J. Blocking gp130 signaling suppresses autotaxin expression in adipocytes and improves insulin sensitivity in diet-induced obesity.J. Lipid Res. 2017; 58: 2102-2113Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 22.D'Souza K. Kane D.A. Touaibia M. Kershaw E.E. Pulinilkunnil T. Kienesberger P.C. Autotaxin is regulated by glucose and insulin in adipocytes.Endocrinology. 2017; 158: 791-803Crossref PubMed Scopus (26) Google Scholar). In humans, serum ATX correlates with measures of obesity, impaired glucose homeostasis, and insulin resistance (19.Rachakonda V.P. Reeves V.L. Aljammal J. Wills R.C. Trybula J.S. DeLany J.P. Kienesberger P.C. Kershaw E.E. Serum autotaxin is independently associated with hepatic steatosis in women with severe obesity.Obesity (Silver Spring). 2015; 23: 965-972Crossref PubMed Scopus (32) Google Scholar, 20.Reeves V.L. Trybula J.S. Wills R.C. Goodpaster B.H. Dube J.J. Kienesberger P.C. Kershaw E.E. Serum autotaxin/ENPP2 correlates with insulin resistance in older humans with obesity.Obesity (Silver Spring). 2015; 23: 2371-2376Crossref PubMed Scopus (43) Google Scholar). In addition, serum ATX is independently associated with hepatic steatosis, a metabolic complication of obesity and diabetes, in severely obese women (19.Rachakonda V.P. Reeves V.L. Aljammal J. Wills R.C. Trybula J.S. DeLany J.P. Kienesberger P.C. Kershaw E.E. Serum autotaxin is independently associated with hepatic steatosis in women with severe obesity.Obesity (Silver Spring). 2015; 23: 965-972Crossref PubMed Scopus (32) Google Scholar). Studies in mice showed that ATX-LPA signaling influences adiposity, although it remains incompletely understood whether the ATX-LPA pathway promotes or protects from diet-induced obesity and adipocyte hypertrophy (4.Dusaulcy R. Rancoule C. Gres S. Wanecq E. Colom A. Guigne C. van Meeteren L.A. Moolenaar W.H. Valet P. Saulnier-Blache J.S. Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.J. Lipid Res. 2011; 52: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar, 23.Federico L. Ren H. Mueller P.A. Wu T. Liu S. Popovic J. Blalock E.M. Sunkara M. Ovaa H. Albers H.M. et al.Autotaxin and its product lysophosphatidic acid suppress brown adipose differentiation and promote diet-induced obesity in mice.Mol. Endocrinol. 2012; 26: 786-797Crossref PubMed Scopus (54) Google Scholar). Dusaulcy et al. (4.Dusaulcy R. Rancoule C. Gres S. Wanecq E. Colom A. Guigne C. van Meeteren L.A. Moolenaar W.H. Valet P. Saulnier-Blache J.S. Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.J. Lipid Res. 2011; 52: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) showed that high-fat-fed adipose-specific ATX knockout mice have increased fat mass and adipocyte size. On the other hand, Nishimura et al. (5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar) demonstrated that adipose-specific and global heterozygous ATX knockout (ATX+/−) mice are protected from high-fat diet-induced obesity (5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar). Brown adipose tissue (BAT) was functionally more active, resulting in increased energy expenditure in ATX-deficient mice (5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar). Moreover, ATX overexpression in mice led to augmented obesity following high-fat feeding (5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar, 23.Federico L. Ren H. Mueller P.A. Wu T. Liu S. Popovic J. Blalock E.M. Sunkara M. Ovaa H. Albers H.M. et al.Autotaxin and its product lysophosphatidic acid suppress brown adipose differentiation and promote diet-induced obesity in mice.Mol. Endocrinol. 2012; 26: 786-797Crossref PubMed Scopus (54) Google Scholar). Regardless of the effect of ATX-LPA on adiposity, studies using global heterozygous or fat-specific ATX knockout mice suggest that the ATX-LPA pathway contributes to high-fat diet-induced systemic insulin resistance and impaired glucose homeostasis (4.Dusaulcy R. Rancoule C. Gres S. Wanecq E. Colom A. Guigne C. van Meeteren L.A. Moolenaar W.H. Valet P. Saulnier-Blache J.S. Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.J. Lipid Res. 2011; 52: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 5.Nishimura S. Nagasaki M. Okudaira S. Aoki J. Ohmori T. Ohkawa R. Nakamura K. Igarashi K. Yamashita H. Eto K. et al.ENPP2 contributes to adipose tissue expansion in diet-induced obesity.Diabetes. 2014; 63: 4154-4164Crossref PubMed Scopus (65) Google Scholar). Consistent with these studies, acute LPA injection resulted in impaired glucose tolerance in chow- and high-fat diet-fed mice through inhibition of glucose-induced insulin secretion, an effect that was blunted by Ki16425, a preferential LPA1/3 antagonist (24.Rancoule C. Attane C. Gres S. Fournel A. Dusaulcy R. Bertrand C. Vinel C. Treguer K. Prentki M. Valet P. et al.Lysophosphatidic acid impairs glucose homeostasis and inhibits insulin secretion in high-fat diet obese mice.Diabetologia. 2013; 56: 1394-1402Crossref PubMed Scopus (58) Google Scholar). Prolonged administration of Ki16425 for 3 weeks enhanced glucose tolerance and insulin sensitivity in high-fat diet-fed mice in the absence of changes in adiposity, which was mainly ascribed to increased islet cell number, glycogen storage in the liver, and muscle glucose oxidation (24.Rancoule C. Attane C. Gres S. Fournel A. Dusaulcy R. Bertrand C. Vinel C. Treguer K. Prentki M. Valet P. et al.Lysophosphatidic acid impairs glucose homeostasis and inhibits insulin secretion in high-fat diet obese mice.Diabetologia. 2013; 56: 1394-1402Crossref PubMed Scopus (58) Google Scholar). Despite growing evidence suggesting that the ATX-LPA pathway impacts glucose homeostasis and global insulin sensitivity in mice and humans, it remains unclear whether ATX-LPA signaling influences insulin sensitivity through direct or indirect effects of LPA on insulin target tissues. In particular, it is incompletely understood whether the ATX-LPA pathway modulates insulin function and energy metabolism in skeletal muscle, which accounts for the majority of insulin-mediated glucose disposal (25.Abdul-Ghani M.A. DeFronzo R.A. Pathogenesis of insulin resistance in skeletal muscle.J. Biomed. Biotechnol. 2010; 2010: 476279Crossref PubMed Scopus (399) Google Scholar). Therefore, we examined tissue insulin signaling in mice with global heterozygous ATX deficiency subjected to diet-induced obesity and tested to determine whether the ATX-LPA pathway influences glucose and lipid metabolism in skeletal muscle. Our study shows that: 1) partial ATX deficiency protects from impaired insulin signaling in white adipose tissue, liver, heart, and skeletal muscle following high-fat high-sucrose (HFHS) feeding, as is reflected in reduced weight gain, improved glucose and insulin tolerance, and blunted cardiomyocyte dysfunction in male HFHS-fed ATX+/− mice; 2) HFHS-fed ATX+/− mice are resistant to impaired insulin-stimulated glucose transport in skeletal muscle and have improved mitochondrial pyruvate oxidation; and 3) in C2C12 myotubes, incubation with LPA reduces insulin-stimulated AKT phosphorylation at baseline and exacerbates palmitate-induced insulin resistance, coincident with impaired mitochondrial respiration. Taken together, these data suggest that the ATX-LPA pathway negatively regulates muscle insulin signaling and mitochondrial function. Unless otherwise stated, chemicals and reagents were obtained from Sigma. The generation of ATX+/− mice (C57Bl/6-Enpp2 /FLMG) and genotyping instructions have been previously reported (26.Fotopoulou S. Oikonomou N. Grigorieva E. Nikitopoulou I. Paparountas T. Thanassopoulou A. Zhao Z. Xu Y. Kontoyiannis D.L. Remboutsika E. et al.ATX expression and LPA signalling are vital for the development of the nervous system.Dev. Biol. 2010; 339: 451-464Crossref PubMed Scopus (118) Google Scholar). Briefly, loxP-flanked neomycin selection cassettes were inserted upstream of exon 1 and downstream of exon 2 (26.Fotopoulou S. Oikonomou N. Grigorieva E. Nikitopoulou I. Paparountas T. Thanassopoulou A. Zhao Z. Xu Y. Kontoyiannis D.L. Remboutsika E. et al.ATX expression and LPA signalling are vital for the development of the nervous system.Dev. Biol. 2010; 339: 451-464Crossref PubMed Scopus (118) Google Scholar). Transgenic expression of Cre recombinase in mice bearing this allele resulted in excision of both exons, thus abolishing protein expression. Mice were housed on a 12 h light:12 h dark cycle with ad libitum access to chow diet (LD5001 from LabDiet with 13.5 kcal% from fat) or HFHS diet (12451 from Research Diets with 45 kcal% from fat and 17 kcal% from sucrose) and water. Male and female mice (7–9 weeks old) were randomly assigned to chow or HFHS cohorts and fed for 20 weeks. For food intake studies, mice were individually housed and food consumption was monitored daily over a 5 day period 2 weeks post diet start. Mice were subjected to an insulin tolerance test (ITT) or glucose tolerance test (GTT) at 15 and 17 weeks post diet start, respectively. Peripheral fat accumulation in isofluorane-anesthetized mice was determined by X-ray imaging using a Bruker In-Vivo Xtreme imager 18 weeks post diet start. Planar X-ray images were analyzed using ImageJ software (National Institutes of Health) and area of peripheral fat was expressed as percent of total body area. Mice were euthanized by decapitation following a 3 h food withdrawal and tissues were collected. Perigonadal adipose tissue (PGAT) and BAT were weighed prior to being flash-frozen. EDTA-plasma was collected and spun at 15,600 g for 10 min at 4°C. For serum collection, blood was spun at 2,000 g for 15 min at 4°C. Plasma and serum were frozen and stored at −80°C until further use. All protocols involving mice were approved by the Dalhousie University Institutional Animal Care and Use Committee. ITTs and GTTs were performed as previously described (27.Kienesberger P.C. Lee D. Pulinilkunnil T. Brenner D.S. Cai L. Magnes C. Koefeler H.C. Streith I.E. Rechberger G.N. Haemmerle G. et al.Adipose triglyceride lipase deficiency causes tissue-specific changes in insulin signaling.J. Biol. Chem. 2009; 284: 30218-30229Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). For ITTs, awake mice were injected intraperitoneally with 1 U human insulin (HumulinR; Eli Lilly) per kilogram body weight following a 3 h food withdrawal. For GTTs, awake 16 h-fasted mice were injected intraperitoneally with 20% (w/v) D-glucose at 2 g/kg body weight. Blood glucose was measured using an Aviva Nano glucometer (Accu-Chek). For insulin signaling studies performed in vivo, mice were injected intraperitoneally with 10 U of insulin per kilogram body weight or an equal volume of saline. After 10 min, mice were euthanized and tissues were collected. For ex vivo insulin signaling studies in muscle, isolated soleus muscles were preincubated for 30 min in pregassed (95% O2, 5% CO2) Krebs-Henseleit bicarbonate buffer (KHB) (pH 7.4) (118.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 25 mM NaCHO3, 2.5 mM CaCl2, 1.2 mM MgSO4, and 5 mM HEPES) at 37°C. Following subsequent incubation in buffer with or without 33 nM insulin for 10 min, muscles were removed; connective tissue, fat, and tendons were excised; and tissues were blotted dry and snap-frozen in liquid nitrogen. Glucose transport in soleus muscle was determined ex vivo as previously described (27.Kienesberger P.C. Lee D. Pulinilkunnil T. Brenner D.S. Cai L. Magnes C. Koefeler H.C. Streith I.E. Rechberger G.N. Haemmerle G. et al.Adipose triglyceride lipase deficiency causes tissue-specific changes in insulin signaling.J. Biol. Chem. 2009; 284: 30218-30229Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), with slight modifications. Soleus muscles from mice were rapidly dissected, preincubated for 1 h in KHB containing 10 mM D-glucose at 37°C, and rinsed by incubation in KHB supplemented with 10 mM D-mannitol for 10 min. Glucose transport was assessed by incubation in KHB with 1 mM 2-deoxyglucose, 9 mM mannitol, 1.5 μCi/ml 2-deoxy-D-[1,2-3H]glucose (Perkin Elmer), and 0.3 μCi/ml D-[1-14C]mannitol (Perkin Elmer) for 20 min at 37°C. All buffers were pregassed with 95% O2, 5% CO2 and were supplemented with saline or 33 nM insulin. Basal glucose transport and insulin-stimulated glucose transport were determined in contralateral muscles. Following the final incubation, muscles were cleaned by excising connective tissue, fat, and tendons, and tissues were blotted dry and snap-frozen in liquid nitrogen. Muscles were weighed and digested for 30 min in 300 μl of 1 N NaOH at 65°C and centrifuged at 13,000 g for 10 min. Radioactivity in the supernatant was determined by liquid scintillation counting and glucose transport into tissues was calculated. RNA isolation, reverse transcription, and real-time quantitative PCR were performed as previously described (22.D'Souza K. Kane D.A. Touaibia M. Kershaw E.E. Pulinilkunnil T. Kienesberger P.C. Autotaxin is regulated by glucose and insulin in adipocytes.Endocrinology. 2017; 158: 791-803Crossref PubMed Scopus (26) Google Scholar, 28.Perez L.J. Rios L. Trivedi P. D'Souza K. Cowie A. Nzirorera C. Webster D. Brunt K. Legare J.F. Hassan A. et al.Validation of optimal reference genes for quantitative real time PCR in muscle and adipose tissue for obesity and diabetes research.Sci. Rep. 2017; 7: 3612Crossref PubMed Scopus (31) Google Scholar) using the following primer sequences: Glut1-forward (fwd) 5′-GGTGTGCAGCAGCCTGTGTACG-3′, Glut1-reverse (rev) 5′-TAGGACATCCAAGGCAGCCGTTC-3′, Glut4-fwd 5′-ACCGGCAGCCTCTGATCATCG-3′, Glut4-rev 5′-GAGTGTCCGTCGTCCAGCTCGTT-3′, Rpl27-fwd 5′-ACGGTGGAGCCTTATGTGAC-3′, Rpl27-rev 5′-TCCGTCAGAGGGACTGTCTT-3′, Rpl41-fwd 5′-GCCATGAGAGCGAAGTGG-3′, and Rpl41-rev 5′-CTCCTGCAGGCGTCGTAG-3′. Serum insulin was determined using an ELISA kit assay (Crystal Chem). NEFA (WAKO Chemicals) and triacylglycerol (TG) (Thermo Fisher Scientific) analyses were performed using colorimetric kit assays, as per the manufacturer's instructions. Plasma LPA levels were determined by HPLC/ESI/MS/MS analysis as previously described (29.Kraemer M.P. Halder S. Smyth S.S. Morris A.J. Measurement of lysophosphatidic acid and sphingosine-1-phosphate by liquid chromatography-coupled electrospray ionization tandem mass spectrometry.Methods Mol. Biol. 2018; 1697: 31-42Crossref PubMed Scopus (5) Google Scholar). Insulin resistance was induced in C2C12 cells as previously described (28.Perez L.J. Rios L. Trivedi P. D'Souza K. Cowie A. Nzirorera C. Webster D. Brunt K. Legare J.F. Hassan A. et al.Validation of optimal reference genes for quantitative real time PCR in muscle and adipose tissue for obesity and diabetes research.Sci. Rep. 2017; 7: 3612Crossref PubMed Scopus (31) Google Scholar). Briefly, cells were incubated in medium containing 2% (w/v) fatty acid-free (FAF)-BSA and 0.8 mM sodium palmitate for 16–18 h. Myotubes were cultured with 2% FAF-BSA in the absence of palmitate to mimic an insulin-sensitive state. To examine insulin signaling, cells were incubated with 100 nM insulin or PBS for 15 min. Cells were washed once and harvested in ice-cold PBS, followed by centrifugation at 20,000 g for 10 min at 4°C. Cell pellets were flash-frozen in liquid nitrogen and stored at −80°C until further use. For experiments involving LPA, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (18:1 LPA; Avanti) was dissolved in PBS with 0.1% FAF-BSA, gently shaken, and mixed with DMEM-1X supplemented with 5 mM glucose prior to its addition to C2C12 myotubes. Cells were cultured in the absence of serum during LPA treatment to avoid potential additional sources of LPA, LPC, and ATX. ATX activity in plasma and serum was quantified as previously described (22.D'Souza K. Kane D.A. Touaibia M. Kershaw E.E. Pulinilkunnil T. Kienesberger P.C. Autotaxin is regulated by glucose and insulin in adipocytes.Endocrinology. 2017; 158: 791-803Crossref PubMed Scopus (26) Google Scholar, 30.Benesch M.G. Zhao Y.Y. Curtis J.M. McMullen T.P. Brindley D.N. Regulation of autotaxin expression and secretion by lysophosphatidate and sphingosine 1-phosphate.J. Lipid Res. 2015; 56: 1134-1144Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, 2 μl of plasma or serum were added to 18 μl of buffer A containing 100 mM Tris-HCl (pH 9.0), 500 mM NaCl, 5 mM MgCl2, and 0.05% v/v Triton X-100. For samples examined in the presence of the ATX inhibitor, PF-8380 (31.St-Cœur P.D. Ferguson D. Morin P. Touaibia M. PF-8380 and closely related analogs: synthesis and structure-activity relationship towards autotaxin inhibition and glioma cell viability.Arch. Pharm. (Weinheim). 2013; 346: 91-97Crossref PubMed Scopus (26) Google Scholar), 5 μl of buffer A containing 10% DMSO or 5 mM of PF-8380 were added. Samples were preincubated at 37°C for 30 min and 25 μl of 6 mM 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 LPC; Avanti) were added. The reaction mixture was incubated at 37°C for 6 h to allow for ATX-mediated choline release. Subsequently, 20 μl of sample were incubated with 90 μl of buffer C {9.65 ml of buffer B [100 mM Tris-HCl (pH 8.5) and 5 mM CaCl2], 110 μl of 30 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS; Cedarlane), 110 μl of 50 mM 4-aminotipyrine, 6.6 μl of 1,000 U/ml horseradish peroxidase, and 110 μl of 300 U/ml choline oxidase} at 37°C for 20 min and choline oxidation was reco
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