rspНаучно-практическая ревматологияRheumatology Science and Practice1995-44841995-4492IMA-PRESS, LLC10.14412/1995-4484-2012-1290rsp-1178Research ArticleСтатьиArticlesНАРУШЕНИЕ РЕГУЛЯТОРНЫХ МЕХАНИЗМОВ СИГНАЛЬНОГО ПУТИ mTOR ПРИ ОСТЕОАРТРОЗЕIMPAIRED REGULATORY MECHANISMS OF THE mTOR SIGNALING PATHWAY IN OSTEOARTHROSISChetinaElena VasilyevnaChetinaElena Vasilyevna

Laboratory of Genetics

Laboratory of Genetics

etchetina@mail.ruBratyginaE ABratyginaE A

Laboratory of Genetics

Laboratory of Genetics

-ЗайцеваЕ. М.ZaitsevaE M

Laboratory of Genetics

Laboratory of Genetics

-ШараповаЕ. П.SharapovaE P

Laboratory of Genetics

Laboratory of Genetics

-AlekseyevaA LAlekseyevaA L

Laboratory of Genetics

Laboratory of Genetics

-DeminN VDeminN V

Laboratory of Genetics

Laboratory of Genetics

-МакаровС. А.MakarovS A

Laboratory of Genetics

Laboratory of Genetics

-201215122012506№6 (2012)3337Copyright © Chetina E.V., Bratygina E.A., Зайцева Е.М., Шарапова Е.П., Alekseyeva A.L., Demin N.V., Макаров С.А., 20122012Chetina E.V., Bratygina E.A., Зайцева Е.М., Шарапова Е.П., Alekseyeva A.L., Demin N.V., Макаров С.А.Chetina E.V., Bratygina E.A., Zaitseva E.M., Sharapova E.P., Alekseyeva A.L., Demin N.V., Makarov S.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://rsp.mediar-press.net/rsp/article/view/1178

Цель — изучить характер нарушения регуляторных механизмов сигнального пути mTOR (mammalian target of rapamycin) посредством мониторинга экспрессии генов в крови больных остеоартрозом (ОА) на разных стадиях заболевания. Материал и методы. В исследование включено 33 амбулаторных больных ОА, 14 больных ОА перед эндопротезированием коленного сустава и 27 здоровых людей — контроль (средний возраст 58,0+7,4; 56,5+8,9 и 55,0+8,3 года соответственно). Общую РНК выделяли из крови и использовали для определения уровня экспрессии генов в полимеразной цепной реакции в режиме реального времени для АМФ-активируемой протеинкиназы (АМПК); фактора, индуцируемого гипоксией 1а (ФИГ1а); белков, лимитирующих скорость гексозаминового сигнального пути, — глутамин-фруктозо-6-фосфатамидотрансферазы и ацетил-глюкозаминилтрансферазы, а также транспортера глюкозы GLUT1 и компонентов 6-й и 7-й ступеней гликолитического пути — глюкозо-6-фосфатдегидрогеназы и фосфоглицерат киназы 1 соответственно; генов, связанных с липогенезом, — синтазы жирных кислот (СЖК) и активностью пентозофосфатного пути — глюкозо-6-фосфат дегидрогеназы в крови больных ОА на разных стадиях заболевания. Результаты. Анализ экспрессии генов показал, что у больных ОА с низкой экспрессией гена mTOR (подгруппа LOW) экспрессия генов AIT и GLUT1 оказалась существенно ниже, а гена АМПК — выше, чем у здоровых лиц. У больных ОА с высокой экспрессией гена mTOR (подгруппа HIGH) экспрессия всех исследуемых генов значительно повышена, за исключением гена СЖК, причем наибольшее превышение экспрессии по сравнению с контрольными лицами наблюдалось в случае генов АМПК и ФИГ1α. У больных с поздней стадией заболевания (подгруппа ES) экспрессия всех исследованных генов, включая ген СЖК, оказалась повышенной по сравнению со здоровыми лицами. Заключение. Развитие ОА сопровождается значительным снижением эффективности энергетического метаболизма. При этом у больных с низкой экспрессией гена mTOR недостаток энергии может быть связан с ослаблением транспорта метаболитов в клетки. У больных с высокой экспрессией mTOR он может быть обусловлен дефицитом конечного акцептора электронов — кислорода, а у больных на поздней стадии заболевания не исключено патологическое перераспределение энергетических субстратов в пользу липогенеза.

Objective: to study the pattern of impaired regulatory mechanisms of the mammalian target of rapamycin (TOR) signaling pathway, by monitoring gene expression in the blood of patients with osteoarthrosis (OA) at different stages of the disease. Subjects and methods. The study covered 33 outpatients with OA, 14 patients with this condition prior to knee joint endoprosthesis, and 27 healthy individuals (controls) (mean age 58.0+7.4, 56.5+8.9, and 55.0+8.3 years, respectively). Total RNA was isolated from their blood and used to determine the level of gene expression by a real-time polymerase chain reaction for AMP-activated protein kinase (AMPK), hypoxia-inducible factor-1α (HIF1α), the rate-limiting proteins of the hexosamine signaling pathway — glutamine-fructose-6-phosphate amidotransferase and acetylglucosaminyltransferase, as well as the glucose transporter GLUT1 and steps 6 and 7 glycolytic pathway components — glucose-6-phosphate dehydrogenase and phosphoglycerate kinase-1, respectively; the lipogenesis-related genes — fatty acid synthase (FAS) and the activity of the pentose phosphate pathway — glucose-6-phosphate dehydrogenase in the blood of patients with OA at different stages of the disease. Results. Analysis of gene expressions showed that in the OA patients with a low expression of the mTOR gene (a LOW subgroup), the expression of AGT and GLUT1 genes proved to be significantly lower and that of the AMPK gene was higher than in the healthy individuals. In the OA patients with a high expression of the mTOR gene (a HIGH subgroup), the expression of all the genes under study was much higher, except for the FAS gene; moreover, the greatest expression excess as compared to the controls was observed for the AMPK and HIFlα genes. In the patients with endstage disease (an ES subgroup), the expression of all the study genes, including the FAS gene, turned out to be higher than in the healthy individuals. Conclusion. The development of OA is accompanied by a considerable decrease in the efficiency of energy metabolism. At the same time, in the patients with a low mTOR gene expression, energy deficiency may be due to decreased cellular metabolite transport. It may be caused by the deficiency of the end electron acceptor oxygen in the patients with a high mTOR gene expression and the pathological redistribution of energy substrate in favor of lipogenesis cannot be ruled out in those with end-stage disease.

остеоартрозэкспрессия геновэнергетический метаболизмкровьmTORosteoarthrosisgene expressionmTORenergy metabolismbloodReferences<div><p>Henrotin Y., Lambert C., Couchourel D. et al. Nutraceuticals: do they represent a new era in the management of osteoarthritis? — А narrative review from the lessons taken with five products. Osteoarthr Cartilage 2011; 19: 1—21.</p><p>Marshall K.W., Zhang H., Nossova N. Chondrocyte genomics: implications for disease modification in osteoarthritis. Drug Discov Today 2006; 11: 825—32.</p><p>Четина Е.В. Сигнальные пути нутриентов и ревматические заболевания. Науч.-практич. ревматол. (в печати).</p><p>Raught B., Gingras A.C., Sonenberg N. The target of rapamycin (TOR) proteins. Proc Natl Аcad Sci USA 2005; 98: 7037—44.</p><p>Nicklin P., Bergman P., Zhang B. et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 2009; 136: 521—34.</p><p>Sofer A., Lei K., Johannessen C.M. et al. Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol Cell Biol 2005; 25: 5834—45.</p><p>Hara K., Yonezawa K., Weng Q.P. et al. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 1998; 273: 14484—94.</p><p>Мецлер Д. Биохимия. Химические реакции в живой клетке. М.: Мир, 1980.</p><p>Love D.C., Hanover J.A. The hexosamine signaling pathway: Deciphering the O-GlcNAc code. Sci. STKE 2005; 2005: re13.</p><p>Smith S., Witkowski A., Joshi A.K. Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res 2003; 42: 289—317.</p><p>Kahn B.B., Alquier T., Carling D. et al. AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 2005; 1: 15—25.</p><p>Carling D. The AMP-activated protein kinase cascade, а unifying system for energy control. Trends Biochem Sci 2004; 29: 18—24.</p><p>Четина Е.В., Братыгина Е.А., Зайцева Е.М. и др. Прогнозирование течения остеоартроза по экспрессии гена mTOR (mammalian target of rapamycin). Науч.-практич. ревматол. 2012; 1: 27—32.</p><p>Четина Е.В. Ингибирование активности расщепления коллагена в хряще больных остеоартрозом при активации гликолиза. Остеопороз и остеопатии 2011; 1: 8—12.</p><p>Четина Е.В., Пул А.Р. Роль ростовых факторов в подавлении разрушения коллагена и дифференциации хондроцитов при остеоартрозе. Вестн. РАМН 2008; 5: 15—21.</p><p>Coggon D., Reading I., Croft P. et al. Knee osteoarthritis and obesity. Int J Obes Relat Metab Disord 2001; 25: 622—7.</p><p>Bliddal H., Leeds A.R., Stigsgaard L. et al. Weight loss as treatment for knee osteoarthritis symptoms in obese patients: 1-year results from a randomised controlled trial. Ann Rheum Dis 2011; 70: 1798—803.</p><p>Gwinn D.M., Shackelford D.B., Egan D.F. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 2008; 30: 214—26.</p><p>Ghosh S., Blumental H.J., Davidson E. et al. Glucosamine metabolism.V. Enzymatic synthesis of glucosamine 6-phosphate. J Biol Chem 1960; 235: 1265—73.</p><p>Johnston I.R., McGuire E.J., Jourdian G.W. et al. Incorporation of N-acetyl-D-glucosamine into glycoproteins. J Biol Chem 1966; 241: 5735—7.</p><p>Shikhman A.R., Brinson D.C., Valbracht J. et al. Differential metabolic effects of glucoseamine and N-acetylglucoseaminein human articular chondrocytes. Osteoarthr Cartilage 2009; 17: 1022—8.</p><p>Ryu J.H., Shin Y., Huh Y.H. et al. Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction. Cell Death Differ 2012; 19: 440—50.</p><p>Gouze J.N., Gouze E., Palmer G.D. et al. Adenovirus-mediated gene transfer of glutamine: fructose-6-phosphate amidotransferase antagonizes tha effect of interleukin-1beta on rat chondrocytes. Osteoarthr Cartilage 2004; 12: 217—24.</p><p>Tong K.M., Chen C.P., Huang K.C. et al. Adiponectin increases MMP-3 expression in human chondrocytes through AdipoR1 signaling pathway. J Cell Biochem 2011; 112: 1431—40.</p><p>Fermor B., Gurumurthy A., Diekman B.O. Hypoxia, RONS and energy metabolism in articular cartilage. Osteoarthr Cartilage 2010; 18: 1167—73.</p><p>Banhegyi G., Csala M., Benedetti A. Hexose-6-phosphate dehydrogenase: linking endocrinology and metabolism. J Mol Endocrinol 2009; 42: 283—9.</p><p>Senesi S., Marcolongo P., Manini I. et al. Constant expression of hexose-6-phosphate dehydrogenase during differentiation of human adipose-derived mesenchymal stem cells. J Mol Endocrinol 2008; 41: 125—33.</p><p>Bujaska I.J., Hewitt K.N., Hauton D. et al. Lack of hexose-6-phosphate dehydrogenase impairs lipid mobilization from mouse adipose tissue. Endocrinology 2008; 149: 2584—91.</p><p>Ford J.H. Saturated fatty acid metabolism is key link between cell division, cancer, and senescence in cellular and whole organism aging. Age 2010; 32: 231—7.</p><p>Rumberger J.M., Wu T., Hering M.A. et al. Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels. J Biol Chem 2003; 278: 28547—52.</p><p>McClain D.A., Hazel M., Parker G. et al. Adipocytes with increased hexosamine flux exhibit insulin resistance, increased glucose uptake, and increased synthesis and storage of lipid. Am J Physiol Endocrinol Metab 2005; 288: T973— T979.</p><p>Swagell C.D., Henly D.C., Morris C.P. Expression analysis of a human hepatic cell line in response to palmitate. Biochem Biophys Res Commun 2005; 328: 432—41.</p><p>Planey S.L., Keay S.K., Zhang C.O. et al. Palmitoylation of cytoskeleton associated protein 4 by DHHC2 regulates antiproliferative factor-mediated signaling. Mol Biol Cell 2009; 20: 1454—63.</p></div><br /><div><p>Henrotin Y., Lambert C., Couchourel D. et al. Nutraceuticals: do they represent a new era in the management of osteoarthritis? — А narrative review from the lessons taken with five products. Osteoarthr Cartilage 2011; 19: 1—21.</p><p>Marshall K.W., Zhang H., Nossova N. Chondrocyte genomics: implications for disease modification in osteoarthritis. Drug Discov Today 2006; 11: 825—32.</p><p>Четина Е.В. Сигнальные пути нутриентов и ревматические заболевания. Науч.-практич. ревматол. (в печати).</p><p>Raught B., Gingras A.C., Sonenberg N. The target of rapamycin (TOR) proteins. Proc Natl Аcad Sci USA 2005; 98: 7037—44.</p><p>Nicklin P., Bergman P., Zhang B. et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 2009; 136: 521—34.</p><p>Sofer A., Lei K., Johannessen C.M. et al. Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol Cell Biol 2005; 25: 5834—45.</p><p>Hara K., Yonezawa K., Weng Q.P. et al. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 1998; 273: 14484—94.</p><p>Мецлер Д. Биохимия. Химические реакции в живой клетке. М.: Мир, 1980.</p><p>Love D.C., Hanover J.A. The hexosamine signaling pathway: Deciphering the O-GlcNAc code. Sci. STKE 2005; 2005: re13.</p><p>Smith S., Witkowski A., Joshi A.K. Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res 2003; 42: 289—317.</p><p>Kahn B.B., Alquier T., Carling D. et al. AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 2005; 1: 15—25.</p><p>Carling D. The AMP-activated protein kinase cascade, а unifying system for energy control. Trends Biochem Sci 2004; 29: 18—24.</p><p>Четина Е.В., Братыгина Е.А., Зайцева Е.М. и др. Прогнозирование течения остеоартроза по экспрессии гена mTOR (mammalian target of rapamycin). Науч.-практич. ревматол. 2012; 1: 27—32.</p><p>Четина Е.В. Ингибирование активности расщепления коллагена в хряще больных остеоартрозом при активации гликолиза. Остеопороз и остеопатии 2011; 1: 8—12.</p><p>Четина Е.В., Пул А.Р. Роль ростовых факторов в подавлении разрушения коллагена и дифференциации хондроцитов при остеоартрозе. Вестн. РАМН 2008; 5: 15—21.</p><p>Coggon D., Reading I., Croft P. et al. Knee osteoarthritis and obesity. Int J Obes Relat Metab Disord 2001; 25: 622—7.</p><p>Bliddal H., Leeds A.R., Stigsgaard L. et al. Weight loss as treatment for knee osteoarthritis symptoms in obese patients: 1-year results from a randomised controlled trial. Ann Rheum Dis 2011; 70: 1798—803.</p><p>Gwinn D.M., Shackelford D.B., Egan D.F. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 2008; 30: 214—26.</p><p>Ghosh S., Blumental H.J., Davidson E. et al. Glucosamine metabolism.V. Enzymatic synthesis of glucosamine 6-phosphate. J Biol Chem 1960; 235: 1265—73.</p><p>Johnston I.R., McGuire E.J., Jourdian G.W. et al. Incorporation of N-acetyl-D-glucosamine into glycoproteins. J Biol Chem 1966; 241: 5735—7.</p><p>Shikhman A.R., Brinson D.C., Valbracht J. et al. Differential metabolic effects of glucoseamine and N-acetylglucoseaminein human articular chondrocytes. Osteoarthr Cartilage 2009; 17: 1022—8.</p><p>Ryu J.H., Shin Y., Huh Y.H. et al. Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction. Cell Death Differ 2012; 19: 440—50.</p><p>Gouze J.N., Gouze E., Palmer G.D. et al. Adenovirus-mediated gene transfer of glutamine: fructose-6-phosphate amidotransferase antagonizes tha effect of interleukin-1beta on rat chondrocytes. Osteoarthr Cartilage 2004; 12: 217—24.</p><p>Tong K.M., Chen C.P., Huang K.C. et al. Adiponectin increases MMP-3 expression in human chondrocytes through AdipoR1 signaling pathway. J Cell Biochem 2011; 112: 1431—40.</p><p>Fermor B., Gurumurthy A., Diekman B.O. Hypoxia, RONS and energy metabolism in articular cartilage. Osteoarthr Cartilage 2010; 18: 1167—73.</p><p>Banhegyi G., Csala M., Benedetti A. Hexose-6-phosphate dehydrogenase: linking endocrinology and metabolism. J Mol Endocrinol 2009; 42: 283—9.</p><p>Senesi S., Marcolongo P., Manini I. et al. Constant expression of hexose-6-phosphate dehydrogenase during differentiation of human adipose-derived mesenchymal stem cells. J Mol Endocrinol 2008; 41: 125—33.</p><p>Bujaska I.J., Hewitt K.N., Hauton D. et al. Lack of hexose-6-phosphate dehydrogenase impairs lipid mobilization from mouse adipose tissue. Endocrinology 2008; 149: 2584—91.</p><p>Ford J.H. Saturated fatty acid metabolism is key link between cell division, cancer, and senescence in cellular and whole organism aging. Age 2010; 32: 231—7.</p><p>Rumberger J.M., Wu T., Hering M.A. et al. Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels. J Biol Chem 2003; 278: 28547—52.</p><p>McClain D.A., Hazel M., Parker G. et al. Adipocytes with increased hexosamine flux exhibit insulin resistance, increased glucose uptake, and increased synthesis and storage of lipid. Am J Physiol Endocrinol Metab 2005; 288: T973— T979.</p><p>Swagell C.D., Henly D.C., Morris C.P. Expression analysis of a human hepatic cell line in response to palmitate. Biochem Biophys Res Commun 2005; 328: 432—41.</p><p>Planey S.L., Keay S.K., Zhang C.O. et al. Palmitoylation of cytoskeleton associated protein 4 by DHHC2 regulates antiproliferative factor-mediated signaling. Mol Biol Cell 2009; 20: 1454—63.</p></div><br />

The authors declare that there are no conflicts of interest present.