Muscle-specific TGR5 overexpression improves glucose clearance in glucose-intolerant mice

Abstract

TGR5, a G protein–coupled bile acid receptor, is expressed in various tissues and regulates several physiological processes. In the skeletal muscle, TGR5 activation is known to induce muscle hypertrophy; however, the effects on glucose and lipid metabolism are not well understood, despite the fact that the skeletal muscle plays a major role in energy metabolism. Here, we demonstrate that skeletal muscle–specific TGR5 transgenic (Tg) mice exhibit increased glucose utilization, without altering the expression of major genes related to glucose and lipid metabolism. Metabolite profiling analysis by capillary electrophoresis time-of-flight mass spectrometry showed that glycolytic flux was activated in the skeletal muscle of Tg mice, leading to an increase in glucose utilization. Upon long-term, high-fat diet challenge, blood glucose clearance was improved in Tg mice without an accompanying increase in insulin sensitivity in skeletal muscle and a reduction of body weight. Moreover, Tg mice showed improved age-associated glucose intolerance. These results strongly suggest that TGR5 ameliorated glucose metabolism disorder that is caused by diet-induced obesity and aging by enhancing the glucose metabolic capacity of the skeletal muscle. Our study demonstrates that TGR5 activation in the skeletal muscle is effective in improving glucose metabolism and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia. TGR5, a G protein–coupled bile acid receptor, is expressed in various tissues and regulates several physiological processes. In the skeletal muscle, TGR5 activation is known to induce muscle hypertrophy; however, the effects on glucose and lipid metabolism are not well understood, despite the fact that the skeletal muscle plays a major role in energy metabolism. Here, we demonstrate that skeletal muscle–specific TGR5 transgenic (Tg) mice exhibit increased glucose utilization, without altering the expression of major genes related to glucose and lipid metabolism. Metabolite profiling analysis by capillary electrophoresis time-of-flight mass spectrometry showed that glycolytic flux was activated in the skeletal muscle of Tg mice, leading to an increase in glucose utilization. Upon long-term, high-fat diet challenge, blood glucose clearance was improved in Tg mice without an accompanying increase in insulin sensitivity in skeletal muscle and a reduction of body weight. Moreover, Tg mice showed improved age-associated glucose intolerance. These results strongly suggest that TGR5 ameliorated glucose metabolism disorder that is caused by diet-induced obesity and aging by enhancing the glucose metabolic capacity of the skeletal muscle. Our study demonstrates that TGR5 activation in the skeletal muscle is effective in improving glucose metabolism and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia. Bile acids, the primary component of bile, are released from the gallbladder after meals to promote the absorption of lipids and fat-soluble vitamins in the small intestine. Almost 95% of bile acids are reabsorbed in the ileum and transported back to the liver through the portal blood and recycled. Therefore, the concentration of blood bile acids temporarily reaches high levels in the postprandial state (1Schalm S.W. LaRusso N.F. Hofmann A.F. Hoffman N.E. van Berge-Henegouwen G.P. Korman M.G. Diurnal serum levels of primary conjugated bile acids. Assessment by specific radioimmunoassays for conjugates of cholic and chenodeoxycholic acid.Gut. 1978; 19: 1006-1014Crossref PubMed Scopus (63) Google Scholar). Interestingly, blood bile acids have been reported to function as metabolic regulators by activating several bile acid receptors. TGR5 (also known as G protein–coupled bile acid receptor 1) is a G protein–coupled receptor that exists in the plasma membrane and recognizes bile acid as its ligand (2Maruyama T. Miyamoto Y. Nakamura T. Tamai Y. Okada H. Sugiyama E. Itadani H. Tanaka K. Identification of membrane-type receptor for bile acids (M-BAR).Biochem. Biophys. Res. Commun. 2002; 298: 714-719Crossref PubMed Scopus (726) Google Scholar, 3Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. Hinuma S. Fujisawa Y. Fujino M. A G protein-coupled receptor responsive to bile acids.J. Biol. Chem. 2003; 278: 9435-9440Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar). Ligand-bound TGR5 interacts with Gαs subunit and then activates the cAMP signaling pathway. TGR5 is expressed in various tissues, such as brown adipose tissue (BAT), white adipose tissue (WAT), and intestinal L cells. In BAT and WAT, TGR5 promotes energy expenditure, causing the amelioration of obesity (4Watanabe M. Houten S.M. Mataki C. Christoffolete M.A. Kim B.W. Sato H. Messaddeq N. Harney J.W. Ezaki O. Kodama T. Schoonjans K. Bianco A.C. Auwerx J. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.Nature. 2006; 439: 484-489Crossref PubMed Scopus (1576) Google Scholar, 5Broeders E.P. Nascimento E.B. Havekes B. Brans B. Roumans K.H. Tailleux A. Schaart G. Kouach M. Charton J. Deprez B. Bouvy N.D. Mottaghy F. Staels B. van Marken Lichtenbelt W.D. Schrauwen P. The bile acid chenodeoxycholic acid increases human Brown adipose tissue activity.Cell Metab. 2015; 22: 418-426Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 6Velazquez-Villegas L.A. Perino A. Lemos V. Zietak M. Nomura M. Pols T.W.H. Schoonjans K. TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue.Nat. Commun. 2018; 9: 245Crossref PubMed Scopus (117) Google Scholar). In contrast, TGR5 activation in intestinal L cells enhances GLP-1 secretion and improves diabetes in mice (7Thomas C. Gioiello A. Noriega L. Strehle A. Oury J. Rizzo G. Macchiarulo A. Yamamoto H. Mataki C. Pruzanski M. Pellicciari R. Auwerx J. Schoonjans K. TGR5-mediated bile acid sensing controls glucose homeostasis.Cell Metab. 2009; 10: 167-177Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, 8Katsuma S. Hirasawa A. Tsujimoto G. Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1.Biochem. Biophys. Res. Commun. 2005; 329: 386-390Crossref PubMed Scopus (567) Google Scholar). TGR5 is also expressed in the skeletal muscle, and its expression is increased by exercise (9Sasaki T. Kuboyama A. Mita M. Murata S. Shimizu M. Inoue J. Mori K. Sato R. The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.J. Biol. Chem. 2018; 293: 10322-10332Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). We have previously demonstrated that TGR5 activation induces muscle cell differentiation in cultured muscle cells and muscle hypertrophy in mice (9Sasaki T. Kuboyama A. Mita M. Murata S. Shimizu M. Inoue J. Mori K. Sato R. The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.J. Biol. Chem. 2018; 293: 10322-10332Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Because skeletal muscle–specific TGR5 overexpression increases muscle strength, TGR5 may be a feasible target for maintaining muscle function. In fact, several compounds possessing TGR5 agonistic activity, such as citrus limonoid nomilin and obacunone, have antiobesity, antidiabetic, and muscle hypertrophy effects (10Ono E. Inoue J. Hashidume T. Shimizu M. Sato R. Anti-obesity and anti-hyperglycemic effects of the dietary citrus limonoid nomilin in mice fed a high-fat diet.Biochem. Biophys. Res. Commun. 2011; 410: 677-681Crossref PubMed Scopus (73) Google Scholar, 11Sasaki T. Mita M. Ikari N. Kuboyama A. Hashimoto S. Kaneko T. Ishiguro M. Shimizu M. Inoue J. Sato R. Identification of key amino acid residues in the hTGR5-nomilin interaction and construction of its binding model.PLoS One. 2017; 12: e0179226Crossref PubMed Scopus (13) Google Scholar, 12Horiba T. Katsukawa M. Mita M. Sato R. Dietary obacunone supplementation stimulates muscle hypertrophy, and suppresses hyperglycemia and obesity through the TGR5 and PPARgamma pathway.Biochem. Biophys. Res. Commun. 2015; 463: 846-852Crossref PubMed Scopus (22) Google Scholar). As the skeletal muscle is not only central to the locomotor system but also the largest glucose-metabolizing organ, a higher muscle mass is associated with better glycemic control (13Srikanthan P. Karlamangla A.S. Relative muscle mass is inversely associated with insulin resistance and prediabetes. Findings from the third National Health and Nutrition Examination Survey.J. Clin. Endocrinol. Metab. 2011; 96: 2898-2903Crossref PubMed Scopus (478) Google Scholar). However, a lower skeletal muscle mass is significantly associated with type 2 diabetes (14Han T.S. Al-Gindan Y.Y. Govan L. Hankey C.R. Lean M.E.J. Associations of BMI, waist circumference, body fat, and skeletal muscle with type 2 diabetes in adults.Acta Diabetol. 2019; 56: 947-954Crossref PubMed Scopus (33) Google Scholar), suggesting that increasing the skeletal muscle mass is effective in improving diabetes. Therefore, it could be expected that TGR5 activation in the skeletal muscle improves glucose metabolism by inducing muscle hypertrophy, although this has not been verified till date. In this study, we evaluated the effects of muscle TGR5 on energy metabolism using skeletal muscle–specific hTGR5 Tg mice. Interestingly, skeletal muscle–specific overexpression of TGR5 induced an increase in the respiratory exchange ratio (RER) along with the activation of glycolytic flux in the skeletal muscle. As anticipated from these results and the fact that TGR5 induces muscle hypertrophy, Tg mice exhibited better glucose clearance under long-term high-fat diet challenge, was of in muscle insulin We also that Tg mice showed in glucose intolerance. study that muscle TGR5 activation to improving glucose by increasing muscle mass and glucose and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia caused by obesity and the of muscle TGR5 on energy we muscle and the expression of several genes related to glucose and lipid metabolism and Tg mice. As we reported Tg mice exhibited significantly increased muscle however, in expression in lipid and glucose metabolism Moreover, of muscle and Tg mice We the and energy expenditure, was from and in Tg and mice fed a diet was in and energy expenditure in and the A and and significantly higher in Tg mice in the and showed an increasing in the and the of in Tg mice with mice. with these the blood glucose levels of Tg mice lower of control in the but in the and TGR5 glycolytic we capillary electrophoresis time-of-flight mass spectrometry analysis of major in the and acid in the skeletal muscle the glycolytic the levels of glucose and significantly in Tg mice. In was a for the in was and Tg mice for several such as and however, the levels of and higher in Tg mice. These results the activation of glycolytic flux in skeletal muscle. the for the of the acid was in the levels of and a increase was in acid the of TGR5 on the acid was to be muscle TGR5 lipid we the analysis using mice and the and energy expenditure from and The was that of the energy is and As in the of was in energy expenditure in and and Tg mice and However, was also in the that was significantly higher in Tg mice under These results that the increase in glucose ratio by skeletal muscle TGR5 is induced only is glucose as an energy and that muscle TGR5 has on lipid metabolism. that TGR5 lipid metabolism in TGR5 by with acid of the TGR5 and the expression of genes in lipid metabolism was TGR5 activation in increased the of that was and by in to TGR5 activation (9Sasaki T. Kuboyama A. Mita M. Murata S. Shimizu M. Inoue J. Mori K. Sato R. The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.J. Biol. Chem. 2018; 293: 10322-10332Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). exhibited a lower expression of but the to be of TGR5 activation the expression was not by with TGR5 The expression of major lipid metabolism genes by TGR5 activation or with and for to the of TGR5 on intracellular As anticipated from that muscle TGR5 activation not energy expenditure and in lipid metabolism and intracellular was and despite the overexpression of TGR5 on results that TGR5 induces muscle hypertrophy and increases glucose utilization, we evaluated the of TGR5 on glucose the glucose not a and Tg mice fed we a of using mice. of challenge, we in body and Tg and mice A and These results with the energy expenditure Tg and mice and increases in the of and and despite the of in the body of Tg mice A and In contrast, the of the muscle, and Tg and mice Interestingly, the demonstrated improved glucose clearance in Tg mice with for with mice Moreover, the insulin that Tg mice fed for improved glucose clearance with mice the levels of plasma acids and are of insulin showed Tg and mice suggest the of muscle TGR5 in of glucose metabolism. results also from transgenic was in (9Sasaki T. Kuboyama A. Mita M. Murata S. Shimizu M. Inoue J. Mori K. Sato R. The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.J. Biol. Chem. 2018; 293: 10322-10332Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). skeletal muscle TGR5 not obesity and not liver and however, it significantly increase the skeletal muscle weight. Moreover, Tg mice exhibited improved blood glucose clearance as evaluated by the and that muscle TGR5 glucose clearance in mice with we that TGR5 activation insulin TGR5 activation improves insulin resistance in muscle we evaluated of that insulin resistance by As insulin increased was by In contrast, TGR5 activation by not insulin sensitivity in and We evaluated the effects of long-term muscle TGR5 activation on insulin sensitivity using and Tg fed for In to insulin was in the skeletal muscle of and Tg mice, but to the in muscle TGR5 activation on muscle insulin sensitivity These results that TGR5 activation in the skeletal muscle has beneficial on the of insulin resistance to Because aging is to be associated with glucose S. J. C. L. in the role in insulin 2003; PubMed Scopus Google Scholar, P. of aging on glucose and glucose Clin. PubMed Scopus Google Scholar), we the of muscle TGR5 on glucose clearance in mice. mice and Tg to was in body liver and but was a increase in skeletal muscle as well as A and we the energy expenditure, and in and Tg mice and that skeletal muscle TGR5 increased the without energy expenditure or These results that TGR5 activation in skeletal muscle enhances glucose in and mice. we an glucose in and and Tg mice. with mice, mice exhibited glucose Tg mice exhibited blood glucose levels various with mice The glucose under the of Tg mice was significantly lower that of mice, and the under the was mice and Tg mice, that muscle TGR5 activation ameliorated age-associated glucose TGR5 have been till and effects by activating in and BAT and promoting GLP-1 secretion from enteroendocrine L cells (4Watanabe M. Houten S.M. Mataki C. Christoffolete M.A. Kim B.W. Sato H. Messaddeq N. Harney J.W. Ezaki O. Kodama T. Schoonjans K. Bianco A.C. Auwerx J. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.Nature. 2006; 439: 484-489Crossref PubMed Scopus (1576) Google Scholar, 5Broeders E.P. Nascimento E.B. Havekes B. Brans B. Roumans K.H. Tailleux A. Schaart G. Kouach M. Charton J. Deprez B. Bouvy N.D. Mottaghy F. Staels B. van Marken Lichtenbelt W.D. Schrauwen P. The bile acid chenodeoxycholic acid increases human Brown adipose tissue activity.Cell Metab. 2015; 22: 418-426Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 6Velazquez-Villegas L.A. Perino A. Lemos V. Zietak M. Nomura M. Pols T.W.H. Schoonjans K. TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue.Nat. Commun. 2018; 9: 245Crossref PubMed Scopus (117) Google Scholar, C. Gioiello A. Noriega L. Strehle A. Oury J. Rizzo G. Macchiarulo A. Yamamoto H. Mataki C. Pruzanski M. Pellicciari R. Auwerx J. Schoonjans K. TGR5-mediated bile acid sensing controls glucose homeostasis.Cell Metab. 2009; 10: 167-177Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar). has been reported that the of TGR5 such as and to mice body and blood glucose levels (10Ono E. Inoue J. Hashidume T. Shimizu M. Sato R. Anti-obesity and anti-hyperglycemic effects of the dietary citrus limonoid nomilin in mice fed a high-fat diet.Biochem. Biophys. Res. Commun. 2011; 410: 677-681Crossref PubMed Scopus (73) Google Scholar, 11Sasaki T. Mita M. Ikari N. Kuboyama A. Hashimoto S. Kaneko T. Ishiguro M. Shimizu M. Inoue J. Sato R. Identification of key amino acid residues in the hTGR5-nomilin interaction and construction of its binding model.PLoS One. 2017; 12: e0179226Crossref PubMed Scopus (13) Google Scholar, 12Horiba T. Katsukawa M. Mita M. Sato R. Dietary obacunone supplementation stimulates muscle hypertrophy, and suppresses hyperglycemia and obesity through the TGR5 and PPARgamma pathway.Biochem. Biophys. Res. Commun. 2015; 463: 846-852Crossref PubMed Scopus (22) Google Scholar). We have also have previously demonstrated that TGR5 activation in skeletal muscle induces muscle hypertrophy (9Sasaki T. Kuboyama A. Mita M. Murata S. Shimizu M. Inoue J. Mori K. Sato R. The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.J. Biol. Chem. 2018; 293: 10322-10332Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). muscle–specific TGR5 Tg mice exhibited increased muscle mass and muscle and In the skeletal muscle is a major that and an increase in skeletal muscle mass is expected to glucose metabolism. However, the effects of skeletal muscle TGR5 on glucose and lipid metabolism We the role of muscle TGR5 in energy metabolism using skeletal muscle–specific TGR5 Tg mice that exhibited a increase in muscle mass The analysis under the showed an increase in in Tg mice with control mice, that TGR5 activation in skeletal muscle promotes glucose and As an increase in was not challenge, it is that the of glucose was only was dietary glucose in the diet and In contrast, was in energy expenditure and Tg mice under and and and In fact, body and of Tg mice with of mice under A and These that TGR5 not promote energy expenditure and has in obesity in the skeletal muscle. However, in the human skeletal muscle, it has been reported that TGR5 activation increases the expression of genes in energy (4Watanabe M. Houten S.M. Mataki C. Christoffolete M.A. Kim B.W. Sato H. Messaddeq N. Harney J.W. Ezaki O. Kodama T. Schoonjans K. Bianco A.C. Auwerx J. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.Nature. 2006; 439: 484-489Crossref PubMed Scopus (1576) Google Scholar). Therefore, the of muscle TGR5 on energy metabolism may be mice and In is of the metabolic for energy glucose is by a is the glycolytic that the of and and is by its M. P. of muscle type and its for the control of the PubMed Scopus Google Scholar). A study demonstrated that activation of the by increases through the of its in skeletal muscle G. M. of muscle Metab. 2003; PubMed Scopus (36) Google Scholar). Moreover, it has been reported that the by T. R. P. M. the of skeletal muscle the and muscle J. PubMed Scopus Google Scholar). These that in the skeletal muscle. Because TGR5 is known to by increasing the intracellular cAMP it is that is activated in the skeletal muscle of Tg mice. this we profiling analysis by and a in the levels of and the activation of in the skeletal muscle of Tg mice In to are known in is the of glucose by to and the is the from to The in the levels of and as well as and may be to these These results from the profiling analysis the increased in Tg mice. in the skeletal muscle with glucose from the blood or intracellular The of in levels in the skeletal muscle and Tg mice strongly that the increased in Tg mice was to the increased of blood glucose Therefore, we that activation of in Tg mice is of the for improved glucose As the skeletal muscle is a of glucose muscle hypertrophy and glucose may to an increase in glucose and hyperglycemia. this and using mice by long-term We that Tg mice exhibited improved glucose clearance with control mice and In glucose in mice is caused by insulin resistance in tissues R. G. M. 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Interestingly, we in the expression of genes related to lipid metabolism the skeletal muscle of and Tg mice results also from and as TGR5 activation on under treatment A and TGR5 activation not insulin resistance in as well as insulin resistance in mice A and the levels of plasma and are of insulin not Tg and mice after These results are with that increased glucose in Tg mice glucose is in the of Our suggest that the of glucose clearance by muscle TGR5 is of insulin sensitivity but caused by skeletal muscle hypertrophy and increased glucose utilization. study a study reported that TGR5 activation in the skeletal muscle improves insulin sensitivity and glucose in a mice S. S. M. Y. L. J. Y. TGR5 insulin resistance in the skeletal and improves glucose in 2019; Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In the study, insulin resistance was by TGR5 ligand and it was by TGR5 the for this it be to the type of the as various to a in signaling of G protein–coupled ligand of for Chem. PubMed Scopus Google Scholar). In to aging is an for glucose and insulin resistance S. J. C. L. in the role in insulin 2003; PubMed Scopus Google Scholar, P. of aging on glucose and glucose Clin. PubMed Scopus Google Scholar). Therefore, we the effects of skeletal muscle TGR5 on glucose metabolism in mice and better glucose in Tg mice In glucose is caused by body F. M. and obesity the insulin resistance of 2009; PubMed Scopus Google Scholar, M.A. resistance in aging is related to PubMed Google Scholar), F. M. and obesity the insulin resistance of 2009; PubMed Scopus Google Scholar), and skeletal muscle L.A. The of signaling in role in skeletal muscle insulin signaling and for 2018; Google Scholar). We body and in and Tg mice and that these and In contrast, increases in skeletal muscle and in Tg mice These results suggest the of muscle hypertrophy and increased glucose in the of glucose clearance in mice and mice. In study that muscle TGR5 improves glucose induced by or aging without muscle insulin The results of a of analysis and suggest that skeletal muscle hypertrophy and increased glucose through the activation of glycolytic flux to improved glucose clearance in the in that we and have various TGR5 from till T. Katsukawa M. Mita M. Sato R. Dietary obacunone supplementation stimulates muscle hypertrophy, and suppresses hyperglycemia and obesity through the TGR5 and PPARgamma pathway.Biochem. Biophys. Res. Commun. 2015; 463: 846-852Crossref PubMed Scopus (22) Google Scholar, R. as an and anti-hyperglycemic PubMed Scopus Google Scholar, C. Strehle A. C. G. A. Schoonjans K. M. Auwerx J. R. A. study of a novel and TGR5 and its in Chem. PubMed Scopus Google Scholar, H. C. Strehle A. C. A. A. C. Auwerx J. R. of a TGR5 from Biophys. Res. Commun. PubMed Scopus Google Scholar), it be of to dietary by these could glucose through muscle TGR5 activation in mice and The in this and and and and TGR5 transgenic mice as previously (9Sasaki T. Kuboyama A. Mita M. Murata S. Shimizu M. Inoue J. Mori K. Sato R. The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.J. Biol. Chem. 2018; 293: 10322-10332Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In hTGR5 was and the was with a and to and with energy by from The of mice in is in the to the of the of the of and in Tg and mice using an with as previously J. Y. K. Hashidume T. K. S. Shimizu M. R. Sato R. by increased energy but not in 2017; PubMed Scopus Google Scholar). The with The was using an the of the mice for and glucose was body mice for and glucose was body mice for and then with insulin body glucose was using a of serum and using from insulin using the from from and Tg mice and for the analysis of and compounds by the of using and and using a A. T. Y. S. F. R. T. K. K. K. of of on levels in 2011; PubMed Scopus Google Scholar). A analysis of by was as previously A. N. R. K. K. for analysis of by capillary 2011; PubMed Scopus Google Scholar). from cultured in with and induce cultured in with and for to in 95% with TGR5 overexpression with the cells with and with was from or the skeletal muscle of mice using to the The was to and from using an was to an The for the analysis are in and skeletal muscle in and with a and a The to to a membrane and then with the in the cell of the was as an and for skeletal muscle was as a control to the expression of in with and lipids by in The levels of intracellular using the and to the levels of using a to are as or to was as the are in the The that have of with the of this The to for the T.S. and the and and Y.Y. the T.S. by and the and T.S. the and the the of the was by from the T. for the of R. and T. R. and the for and R.

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