Genetic disruption of myostatin reduces the development of proatherogenic dyslipidemia and atherogenic lesions in Ldlr null mice

Powen Tu, Shalender Bhasin, Paul W Hruz, Karen L Herbst, Lawrence W Castellani, Ning Hua, James A Hamilton, Wen Guo, Powen Tu, Shalender Bhasin, Paul W Hruz, Karen L Herbst, Lawrence W Castellani, Ning Hua, James A Hamilton, Wen Guo

Abstract

Objective: Insulin-resistant states, such as obesity and type 2 diabetes, contribute substantially to accelerated atherogenesis. Null mutations of myostatin (Mstn) are associated with increased muscle mass and decreased fat mass. In this study, we determined whether Mstn disruption could prevent the development of insulin resistance, proatherogenic dyslipidemia, and atherogenesis.

Research design and methods: C57BL/6 Ldlr(-/-) mice were cross-bred with C57BL/6 Mstn(-/-) mice for >10 generations to generate Mstn(-/-)/Ldlr(-/-) double-knockout mice. The effects of high-fat/high-cholesterol diet on body composition, plasma lipids, systemic and tissue-specific insulin sensitivity, hepatic steatosis, as well as aortic atheromatous lesion were characterized in Mstn(-/-)/Ldlr(-/-) mice in comparison with control Mstn(+/+)/Ldlr(-/-) mice.

Results: Compared with Mstn(+/+)/Ldlr(-/-) controls, Mstn(-/-)/ Ldlr(-/-) mice were resistant to diet-induced obesity, and had greatly improved insulin sensitivity, as indicated by 42% higher glucose infusion rate and 90% greater muscle [(3)H]-2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp. Mstn(-/-)/Ldlr(-/-) mice were protected against diet-induced hepatic steatosis and had 56% higher rate of hepatic fatty acid beta-oxidation than controls. Mstn(-/-)/Ldlr(-/-) mice also had 36% lower VLDL secretion rate and were protected against diet-induced dyslipidemia, as indicated by 30-60% lower VLDL and LDL cholesterol, free fatty acids, and triglycerides. Magnetic resonance angiography and en face analyses demonstrated 41% reduction in aortic atheromatous lesions in Ldlr(-/-) mice with Mstn deletion.

Conclusions: Inactivation of Mstn protects against the development of insulin resistance, proatherogenic dyslipidemia, and aortic atherogenesis in Ldlr(-/-) mice. Myostatin may be a useful target for drug development for prevention and treatment of obesity and its associated type 2 diabetes and atherosclerosis.

Figures

FIG. 1.
FIG. 1.
Effects of Mstn disruption on body fat accumulation in Ldlr−/− mice. A: Representative gross appearance (top panel) and micro-CT image of visceral and subcutaneous fat (bottom panel) of mice after 12 weeks of HFD (HF-diet). c, cecum; vf, visceral fat, sf, subcutaneous fat. B: NMR analysis of total fat at baseline (time 0) and after 5 and 10 weeks of HFD. Mstn+/+/Ldlr+/+, ♦; Mstn+/+/Ldlr−/−, ○; Mstn+−/Ldlr−/−, ▲; Mstn+−/Ldlr−/−, ●. C: Inguinal, epididymal, perirenal, and intrascapular brown fat weights of mice after 12 weeks of HFD. D: Representative gross appearance of hind-limb muscles (left panel) and quadriceps muscle weights (right panel) of mice after 12 weeks of HFD. ++/++, Mstn+/+/Ldlr+/+. ++/−−, Mstn+/+/Ldlr−/−. +−/−−, Mstn+−/Ldlr−/−. −−/−−, Mstn−/−/Ldlr−/−. Data are expressed as means ± SE (n = 11–21). **P < 0.01 compared with all other genotypes. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Effects of Mstn disruption on atherogenesis progression in Ldlr−/− mice. A: Representative MRA of aortic arch and its major branches. BCA, brachiocephalic artery. B: Cross-sectional image of BCA. C and D: Symmetry coefficient (ratio of the largest to the smallest diameter) (C), and cross-sectional area (D) of BCA lumen (n = 4). E: Oil Red O staining of atherosclerotic lesions in aortic root at the level of the aortic valves (top panel). Magnification 40×. Gross aortic arch and branches (bottom panel) (n = 9–20). F and G: Sudan IV staining of en face aortas (F) and quantitative analyses of atherosclerotic lesion areas (percent of total aortic surface area) (G) (n = 9–20). ++/++, Mstn+/+/Ldlr+/+. ++/−−, Mstn+/+/Ldlr−/−. +−/−−, Mstn+−/Ldlr−/−. −−/−−, Mstn−/−/Ldlr−/−. Data are expressed as means ± SE (n = 11–21). **P < 0.05. **P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 3.
FIG. 3.
Atherogenic lipid profile in Ldlr−/− mice with Mstn deletion. A and B: Fasting plasma FFA, triglycerides, and cholesterol levels of mice at baseline (A) and after 10 weeks of HFD (HF-diet) (B). C and D: Lipoprotein profile in Mstn+/+/Ldlr−/− (○) and Mstn−/−/Ldlr−/− (●) mice after 11 weeks of HFD. Data are presented as average cholesterol (C) and triglycerides (D) distribution for each group. E: Plasma apoA1- and apoB100-containing lipoprotein particles before and after induction of HFD. The graphs demonstrate the quantification of each molecule, displayed as apoB100/apoA1 ratio. Averages were taken from four different gels. Blood was drawn from mice after 10 weeks of HFD. ++/−−, Mstn+/+/Ldlr−/−. −−/−−, Mstn−/−/Ldlr−/−. Data are shown as the means ± SE (n = 7–10). *P < 0.05, **P < 0.01.
FIG. 4.
FIG. 4.
Metabolic studies in Ldlr−/− mice with Mstn deletion. A–C: Fasting blood glucose (A), plasma insulin (B), and glucose (mg/dl) × insulin product (μIU/ml) ratio (C) of Mstn+/+/Ldlr−/− and Mstn−/−/Ldlr−/− mice after 8 weeks of HFD. D–G: Hyperinsulinemic-euglycemic clamp studies in mice after 12 weeks of HFD. Trace of blood glucose and GIR during the 2-h clamp (D) (○ and ●, blood glucose; □ and ■, GIR), average GIR (E), glucose uptake in quadriceps muscle (F), and average plasma insulin during clamp period (G). H: Akt serine-473 and GSKα/β serine-21/9 phosphorylation in the quadriceps muscle of mice. The graphs demonstrate the quantification of phosphorylated form of each molecule. Averages were taken from three different experiments. ++/−−, Mstn+/+/Ldlr−/−. −−/−−, Mstn−/−/Ldlr−/−. AU, arbitrary units. Data are shown as the means ± SE (n = 7–10). *P < 0.05, **P < 0.01.
FIG. 5.
FIG. 5.
Effects of Mstn deletion on liver of Ldlr−/− mice. A: Hematoxylin and eosin (H&E) and Oil Red O staining of the liver of Mstn+/+/Ldlr−/− and Mstn−/−/Ldlr−/− mice after 12 weeks of HFD (HF-diet). B and C: mRNA expression of Srebf1 (B) and fatty acid synthase (Fasn) (C) in the liver of mice before and after 12 weeks of HFD. Values are expressed with respect to Mstn+/+/Ldlr−/− controls. D: Protein expression of fatty acid synthase. Averages were taken from three different gels. E: Plasma apoB100 at 0 and 180 min after injection of Triton WR1339, a lipoprotein lipase inhibitor. VLDL secretion is determined as percent increase of apoB100 from baseline. Averages were taken from three different gels. F: Akt serine-473 and GSKα/β serine-21/9 phosphorylation in the liver of mice after 12 weeks of HFD. The graphs demonstrate the quantification of phosphorylation of each molecule. ++/−−, Mstn+/+/Ldlr−/−. −−/−−, Mstn−/−/Ldlr−/−. AU, arbitrary units. Data are shown as the means ± SE (n = 7–10). *P < 0.05, **P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)

References

    1. Sloan FA, Bethel MA, Ruiz DJ, Shea AM., MN F: The growing burden of diabetes mellitus in the US elderly population. Arch Intern Med 2008; 168: 192– 199
    1. Ding J, Kritchevsky SB, Newman AB, Taaffe DR, Nicklas BJ, Visser M, Lee JS, Nevitt M, Tylavsky FA, Rubin SM, Pahor M, Harris TB: Effects of birth cohort and age on body composition in a sample of community-based elderly. Am J Clin Nutr 2007; 85: 405– 410
    1. McPherron AC, Lawler AM, Lee SJ: Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 1997; 387: 83– 90
    1. Amthor H, Huang R, McKinnell I, Christ B, Kambadur R, Sharma M, Patel K: The regulation and action of myostatin as a negative regulator of muscle development during avian embryogenesis. Dev Biol 2002; 251: 241– 257
    1. Artaza JN, Bhasin S, Magee TR, Reisz-Porszasz S, Shen R, Groome NP, Meerasahib MF, Gonzalez-Cadavid NF: Myostatin inhibits myogenesis and promotes adipogenesis in C3H 10T(1/2) mesenchymal multipotent cells. Endocrinology 2005; 146: 3547– 3557
    1. McPherron AC, Lee SJ: Suppression of body fat accumulation in myostatin-deficient mice. J Clin Invest 2002; 109: 595– 601
    1. Lee SJ: Regulation of muscle mass by myostatin. J Clin Invest 2004; 20: 61– 86
    1. Breslow JL: Mouse models of atherosclerosis. Science 1996; 272: 685– 688
    1. Wilkes JJ, Lloyd DJ, Gekakis N: A loss-of-function mutation in myostatin reduces TNF-α production and protects liver against obesity-induced insulin resistance. Diabetes 2009; 58: 1133– 1143
    1. Guo T, Jou W, Chanturiya T, Portas J, Gavrilova O, McPherron AC: Myostatin inhibition in muscle, but not adipose tissue, decreases fat mass and improves insulin sensitivity. PLoS ONE 2009; 4: e4937.
    1. Izumiya Y, Hopkins T, Morris C, Sato K, Zeng L, Viereck J, Hamilton JA, Ouchi N, LeBrasseur NK, Walsh K: Fast/Glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese mice. Cell Metab 2008; 7: 159– 172
    1. Furuhashi M, Tuncman G, Gorgun CZ, Makowski L, Atsumi G, Vaillancourt E, Kono K, Babaev VR, Fazio S, Linton MF, Sulsky R, Robl JA, Parker RA, Hotamisligil GS: Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2. Nature 2007; 447: 959– 965
    1. Hedrick CC, Castellani LW, Warden CH, Puppione DL, Lusis AJ: Influence of mouse apolipoprotein A-II on plasma lipoproteins in transgenic mice. J Biol Chem 1993; 268: 20676– 20682
    1. Innis-Whitehouse W, Li X, Brown WV, Le NA: An efficient chromatographic system for lipoprotein fractionation using whole plasma. J Lipid Res 1998; 39: 679– 690
    1. Hruz PW, Murata H, Qiu H, Mueckler M: Indinavir induces acute and reversible peripheral insulin resistance in rats. Diabetes 2002; 51: 937– 942
    1. Folch J, Ascoli I, Lees M, Meath JA, Le BN: Preparation of lipide extracts from brain tissue. J Biol Chem 1951; 191: 833– 841
    1. Kuipers F, Jong MC, Lin Y, Eck M, Havinga R, Bloks V, Verkade HJ, Hofker MH, Moshage H, Berkel TJ, Vonk RJ, Havekes LM: Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E-deficient mouse hepatocytes. J Clin Invest 1997; 100: 2915– 2922
    1. Lamarche B, Moorjani S, Lupien PJ, Cantin B, Bernard PM, Dagenais GR, Despres JP: Apolipoprotein A-I and B levels and the risk of ischemic heart disease during a five-year follow-up of men in the Quebec cardiovascular study. Circulation 1996; 94: 273– 278
    1. Walldius G, Jungner I, Holme I, Aastveit AH, Kolar W, Steiner E: High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet 2001; 358: 2026– 2033
    1. Wellen KE, Hotamisligil GS: Inflammation, stress, and diabetes. J Clin Invest 2005; 115: 1111– 1119
    1. Ginsberg HN: Very low density lipoprotein metabolism in diabetes mellitus. Diabetes Metab Rev 1987; 3: 571– 589
    1. Malmstrom R, Packard CJ, Caslake M, Bedford D, Stewart P, Yki-Jarvinen H, Shepherd J, Taskinen MR: Defective regulation of triglyceride metabolism by insulin in the liver in NIDDM. Diabetologia 1997; 40: 454– 462
    1. Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL: Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell 2000; 6: 77– 86
    1. Leow MK, Addy CL, Mantzoros CS: Clinical review 159: Human immunodeficiency virus/highly active antiretroviral therapy-associated metabolic syndrome: clinical presentation, pathophysiology, and therapeutic strategies. J Clin Endocrinol Metab 2003; 88: 1961– 1976
    1. Lin J, Yang R, Tarr PT, Wu PH, Handschin C, Li S, Yang W, Pei L, Uldry M, Tontonoz P, Newgard CB, Spiegelman BM: Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP. Cell 2005; 120: 261– 273
    1. Stefanovic-Racic M, Perdomo G, Mantell BS, Sipula IJ, Brown NF, RM OD: A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels. Am J Physiol Endocrinol Metab 2008; 294: E969– E977
    1. Fritsche L, Weigert C, Haring HU, Lehmann R: How insulin receptor substrate proteins regulate the metabolic capacity of the liver–implications for health and disease. Curr Med Chem 2008; 15: 1316– 1329
    1. Kabir M, Catalano KJ, Ananthnarayan S, Kim SP, Van Citters GW, Dea MK, Bergman RN: Molecular evidence supporting the portal theory: a causative link between visceral adiposity and hepatic insulin resistance. Am J Physiol Endocrinol Metab 2005; 288: E454– E461
    1. Samuel VT, Liu ZX, Qu X, Elder BD, Bilz S, Befroy D, Romanelli AJ, Shulman GI: Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease. J Biol Chem 2004; 279: 32345– 32353
    1. den Boer M, Voshol PJ, Kuipers F, Havekes LM, Romijn JA: Hepatic steatosis: a mediator of the metabolic syndrome. Lessons from animal models. Arterioscler Thromb Vasc Biol 2004; 24: 644– 649
    1. Guo W, Flanagan J, Jasuja R, Kirkland J, Jiang L, Bhasin S: The effects of myostatin on adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are mediated through cross-communication between Smad3 and Wnt/beta-catenin signaling pathways. J Biol Chem 2008; 283: 9136– 9145
    1. Feldman BJ, Streeper RS, Farese RV, Jr, Yamamoto KR: Myostatin modulates adipogenesis to generate adipocytes with favorable metabolic effects. Proc Natl Acad Sci U S A 2006; 103: 15675– 15680
    1. Musaro A, McCullagh K, Paul A, Houghton L, Dobrowolny G, Molinaro M, Barton ER, Sweeney HL, Rosenthal N: Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet 2001; 27: 195– 200
    1. Thorens B, Charron MJ, HF L: Molecular physiology of glucose transporters Diabetes Care 1990; 3: 209– 218
    1. Abu-Elheiga L, Oh W, Kordari P, Wakil SJ: Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets. Proc Natl Acad Sci U S A 2003; 100: 10207– 10212
    1. Pedersen BK, Akerstrom TC, Nielsen AR, Fischer CP: Role of myokines in exercise and metabolism. J Appl Physiol 2007; 103: 1093– 1098
    1. Izumiya Y, Bina HA, Ouchi N, Akasaki Y, Kharitonenkov A, Walsh K: FGF21 is an Akt-regulated myokine. FEBS Lett 2008; 582: 3805– 3810
    1. Ouchi N, Oshima Y, Ohashi K, Higuchi A, Ikegami C, Izumiya Y, Walsh K: Follistatin-like 1, a secreted muscle protein, promotes endothelial cell function and revascularization in ischemic tissue through a nitric-oxide synthase-dependent mechanism. J Biol Chem 2008; 283: 32802– 32811
    1. Wagner KR, Fleckenstein JL, Amato AA, Barohn RJ, Bushby K, Escolar DM, Flanigan KM, Pestronk A, Tawil R, Wolfe GI, Eagle M, Florence JM, King WM, Pandya S, Straub V, Juneau P, Meyers K, Csimma C, Araujo T, Allen R, Parsons SA, Wozney JM, Lavallie ER, JR: M. Phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy. Ann Neurol 2008; 65: 543– 545

Source: PubMed

Sponsorzy i współpracownicy

Warunki medyczne

Interwencje lekowe

3
Subskrybuj