In the present study Fourier Transform Infrared Spectroscopy was used to examine the effects of streptozotocin-induced diabetes mellitus on the structural components of slow- and fast-twitch rat skeletal muscles, at molecular level. Diabetes mellitus is a chronic disorder of carbohydrate, fat and protein metabolism, which is characterized by hyperglycemia caused by a defective or deficient insulin secretory response. The effect of diabetes is seen on a variety of tissues leading to important secondary complications such as kidney failure, liver dysfunction, cardiac disorders, etc. Skeletal muscle is one of the major tissues determining carbohydrate and lipid metabolism in the body / therefore, is one of the target tissues of diabetes. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. The FTIR spectra revealed a considerable decrease in lipid and protein content of diabetic skeletal muscles, indicating an increased lipolysis and protein breakdown or decreased protein synthesis. Moreover changes in protein structure and conformation were observed. In diabetes, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. Diabetes caused a decrease in the content of nucleic acids, especially RNA, and hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated slow-twitch muscle was more severely affected from diabetes. Thus, FTIR spectroscopy appears to be a useful method to evaluate the effect of diabetes on skeletal muscle tissues at molecular level.

京都大学 / 0048 / 新制・論文博士 / 博士(人間・環境学) / 乙第12883号 / 論人博第40号 / 新制||人||169(附属図書館) / 26||論人博||40(吉田南総合図書館) / 31601 / (主査)教授 石原 昭彦, 教授 船橋 新太郎, 教授 林 達也, 准教授 神﨑 素樹, 准教授 久代 恵介 / 学位規則第4条第2項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

In the present study Fourier Transform Infrared (FTIR) and Attenuated Total Reflectance FTIR (ATR-FTIR) Spectroscopy were used to examine the effects of simvastatin on structure, composition and function of macromolecules of three different rat skeletal muscles EDL (Extensor Digitorium Longus), DIA (Diaphragm) and SOL (Soleus) containing different amount of slow and fast twitch fibers, at molecular level. Simvastatin, a lipophilic statin, is widely used in the treatment of hypercholesterolemia and cardiovascular diseases due to its higher efficacy. However, long term usage of statins give rise to many adverse effects on different tissues and organs. Skeletal muscle accounts for around 45 % of the total body weight and has a high metabolic rate and blood flow. As a consequence, it is highly exposed to drugs within the circulation. Therefore, it is one of the target tissues of statins. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. EDL is a fast twitch muscle while SOL is slow twitch muscle. On the other hand, DIA shows intermediate properties between slow and fast twitch muscle. The results of ATR-FTIR and FTIR spectra revealed a considerable decrease in protein and lipid content of simvastatin treated skeletal muscles, indicating protein breakdown or decreased protein synthesis and increased lipolysis. Moreover changes in protein structure and conformation were observed. In simvastatin treatment, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. The drug treatment caused a decrease in the content of nucleic acids, especially RNA, and hydrogen and non-hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated EDL muscle was more severely affected from statin treatments while SOL was less affected from the drug treatments. Thus, FTIR and ATR-FTIR spectroscopy appear to be useful methods to evaluate the effects of statin on skeletal muscle tissues at molecular level.

Abstract Skeletal muscle cells (myofibers) are huge multinucleated cells responsible for muscle contraction and hence for the everyday movements of the joints. The structure of these voluminous cells differs greatly from that of the mononucleated cells – the characteristic features of the myofibers include dozens of peripherally located nuclei, tightly packed contractile apparatus and a sophisticatedly organized endomembrane system. The basic physiology involving myofibers is quite well known, but scarce data exist on the membrane biology of the myofibers. The purpose of this study was to examine the localization of mRNA and the site of protein synthesis in the myofibers. The characterization of the membrane dynamics in muscle cells was also performed. In this study we utilized a primary cell culture model obtained from the rat flexor digitorum brevis (FDB) muscle. Also frozen sections from the rat extensor digitorum longus muscle were used. The precursor cells of the myofibers – myoblasts and myotubes – were also utilized in some experiments. Furthermore, methods of immunohistochemistry and molecular biology were applied extensively in this study. We found that in FDB myofibers the mRNA lies just under the plasma membrane. Protein synthesis seemed to be concentrated in the vicinity of nuclei locating beneath the plasma membrane but also in interfibrillar dot-like structures. Protein products moved hundreds of micrometers away from the nuclei of origin. Moreover, there were no barriers for protein movement into the core regions of the myofibers. Movement of proteins was found to be rapid in the cytosol and in the endomembrane system, too. Interestingly, when examining exocytic trafficking we observed that ER-to-Golgi trafficking significantly differed from that of mononucleated cells. Finally, myofibers were found to be able to generate lipid bodies under stress conditions. The dynamics of lipid bodies seemed to deviate from the dynamics found in other cells types. Nowadays not much muscle research with primary myofibers is done worldwide, and therefore dilemmas involving myofibers such as insulin resistance and myotoxicity of statins are mostly unresolved. The knowledge gained from this study may be used in the future to solve clinical problems related to the cell biology of the myofibers. / Tiivistelmä Luurankolihassolut eli myofiiberit ovat jättimäisiä monitumaisia soluja, jotka vastaavat lihassupistuksen aikaansaamisesta ja siten mahdollistavat jokapäiväisen liikkumisemme. Näiden suurten solujen rakenne poikkeaa selkeästi yksitumaisten solujen rakenteesta: myofiiberien tunnusomaisia piirteitä ovat kymmenet solun reunoille sijoittuneet tumat, tiiviisti pakkautunut supistumiskoneisto ja monimutkaisesti järjestynyt solukalvostojärjestelmä. Vaikka myofiiberien perusfysiologia tunnetaankin hyvin, niin tiedetään itse myofiiberien kalvostobiologiasta sangen vähän. Kokonaisuutena tämän tutkimuksen tarkoituksena oli tarkastella mRNA:n ja proteiinisynteesin sijaintia myofiibereissä. Lisäksi selvitimme lihassolujen kalvostodynamiikkaa. Tässä tutkimuksessa käytimme rotan flexor digitorum brevis (FDB) -lihaksesta saatua primääristä soluviljelymallia. Lisäksi hyödynsimme rotan extensor digitorum longus -lihaksesta hankittuja jääleikkeitä. Joissakin kokeissa käytimme myös myofiiberien esiastesoluja (myoblasteja ja myotuubeja). Immunohistokemian ja molekyylibiologian menetelmiä sovellettiin tutkimuksessa laajasti. Havaitsimme, että FDB –myofiibereissä mRNA sijaitsee aivan solukalvon alla. Proteiinisynteesi vaikutti olevan keskittynyt solukalvon alla sijaitsevien tumien ympärille, mutta myös solusisäisiin pistemäisiin rakenteisiin. Proteiinituotteet ylsivät satojen mikrometrien päähän alkuperäisestä tumastaan. Lisäksi proteiineille ei ilmennyt leviämisestettä myofiiberin sisäosiin. Leviämisen havaittiin olevan nopeaa sekä solulimassa että solulimakalvostoissa. Tutkiessamme solun eritystoimintaa huomasimme, että kuljetus ER:stä Golgin laitteeseen eroaa huomattavasti yksitumaisten solujen vastaavasta kuljetuksesta. Lopuksi havaitsimme myofiiberien pystyvän muodostamaan rasvapisaroita rasitusolosuhteissa. Rasvapisaroiden käyttäytyminen näytti myös poikkeavan siitä, mitä muissa soluissa on havaittu. Nykyään lihastutkimusta primäärisoluilla ei juuri tehdä maailmalla, minkä vuoksi myofiibereihin liittyvät lääketieteelliset pulmat kuten insuliiniresistenssi ja statiinien lihashaitat ovat suurelta osin ratkaisematta. Tästä tutkimuksesta saatuja tuloksia voitaneen jatkossa käyttää myofiiberien solubiologiaan liittyvien kliinisten ongelmien selvittämiseen.

Abstract The prevalence of obesity is dramatically on the rise in the Western world. Obesity is associated with several chronic diseases, including diabetes and cardiovascular disease (CVD). Non-alcoholic fatty liver disease occurs when fat is ectopically stored in the liver. It is closely associated with serious metabolic abnormalities. Non-alcoholic fatty liver disease ranges from simple hepatic steatosis with no inflammation to hepatic steatosis with a necroinflammatory component, which may lead to cirrhosis and liver failure. Adiponectin is an adipokine that is solely secreted by adipocytes and has anti-inflammatory, antiatherogenic and insulin-sensitizing properties. Adipose tissue inflammation contributes to reduced plasma adiponectin levels in obesity leading to further metabolic complications. Adiponectin may be a mediator between obesity and fat accumulation in the liver and skeletal muscle. Fatty liver may play a role in the pathogenesis of CVD. Mortality data show that CVD as the cause of death accounts for almost half of all deaths in Finland. Traditional risk factors for CVD are age, gender, smoking, high low-density lipoprotein level, high blood pressure and diabetes. The aim of the thesis was to investigate the mediators of fat accumulation in the liver and skeletal muscle as well as the role of fatty liver in the future risk for CVD. If one considers the peptide hormones, then adiponectin turned out to be the strongest independent indicator of the brightness of the liver. In addition, an association between a low adiponectin concentration and large muscle fiber size was observed, and this was not dependent on the amount of total fatness. Furthermore, severe fatty liver increased the risk for cardiovascular events, predicted the risk for death from all causes and death from CVD in a long follow-up. Insulin sensitivity seemed to play a more dominant role in developing cardiovascular events. In conclusion, this study demonstrates that adiponectin may have an important effect on fat accumulation in the liver and skeletal muscle. Adiponectin could be a target when considering the treatment and prevention of ectopic fat accumulation. Fatty liver seems to play a significant role in developing cardiovascular event and mortality to CVD. / Tiivistelmä Lihavuus on kasvava ongelma länsimaissa. Lihavuudella on todettu olevan yhteyttä lukuisiin kroonisiin sairauksiin, kuten diabetekseen ja sydän- ja verisuonitautiin. Ei-alkoholiperäinen rasvamaksa aiheutuu rasvan kertymisestä maksaan. Tilan on todettu liittyvän läheisesti vaikeisiin aineenvaihdunnan häiriöihin. Ei-alkoholiperäinen rasvamaksa vaihtelee vakavuusasteeltaan poikkeavasta rasvan kertymisestä tulehdukseen, joka voi edelleen johtaa kirroosiin ja maksan toiminnan pettämiseen. Adiponektiini on pääasiassa rasvakudoksen erittämä hormoni, jolla on tulehdusta hillitseviä, ateroskleroosilta suojaavia ja insuliinia herkistäviä ominaisuuksia. Rasvakudoksen tulehdustila myötävaikuttaa alentuneeseen adiponektiinipitoisuuteen, joka voi johtaa vaikeutuneisiin aineenvaihdunnan häiriöihin. Adiponektiinin epäillään olevan välittäjäaine lihavuuden ja rasvamaksan ja lihaksensisäisen rasvan välillä. Rasvamaksan ja kardiovaskulaarisairauksien välillä saattaa olla yhteys. Sydän- ja verisuonisairaudet aiheuttavat lähes puolet kuolemista Suomessa. Perinteisiä kardiovaskulaaritaudin riskitekijöitä ovat ikä, sukupuoli, tupakointi, korkea LDL-kolesteroli, korkea verenpaine ja diabetes. Tutkimuksemme tavoitteena oli selvittää maksan ja lihaksen rasvan kertymiseen myötävaikuttavia tekijöitä sekä rasvamaksan vaikutusta riskiin sairastua sydän- ja verisuonisairauksiin. Tutkimuksessa havaittiin, että lihavuuteen liittyvistä hormoneista adiponektiini oli vahvin itsenäinen myötävaikuttaja rasvamaksan kehittymisessä. Plasman alentunut adiponektiinipitoisuus yhdistyi kasvaneeseen lihassolun kokoon riippumatta henkilöiden rasvakudoksen määrästä. Seurantatutkimuksen mukaan vaikeasti rasvoittunut maksa lisäsi riskiä sairastua kardiovaskulaaritautiin, ennusti yleistä kuolemanriskiä ja kuolemaa kardiovaskulaaritautiin. Insuliiniherkkyydellä näytti olevan merkittävä rooli sydän- ja verisuonitautitapahtumissa. Tutkimus osoittaa, että adiponektiinillä saattaa olla keskeinen rooli rasvan kertymisessä maksaan ja lihakseen. Adiponektiini voi olla keskeinen tutkimuskohde kehiteltäessä hoitomuotoja ja ehkäisymenetelmiä rasvakudoksen ulkopuolisen rasvan kertymiseen. Rasvamaksan rooli sairaalahoitoon tai kuolemaan johtavissa ateroskleroottisissa tapahtumissa on ilmeinen.

Introdução: A hipertensão arterial pulmonar (HAP) é uma doença progressiva extremamente grave, que evolui com insuficiência cardíaca direita e morte. Apesar do avanço do tratamento farmacológico, o prognóstico permanece reservado com taxa de sobrevida de 86%, 70% e 55% em 1, 3 e 5 anos, respectivamente. A dispnéia progressiva e a intolerância ao exercício são as principais manifestações clínicas e refletem a falência do ventrículo direito. O músculo esquelético periférico parece ser também um dos principais determinantes desta limitação funcional, visto que a redução da oferta de oxigênio e alterações na extração/utilização do oxigênio pelo músculo são diretamente relacionados com a tolerância ao exercício. Existem dois mecanismos potencialmente envolvidos na regulação da oferta de oxigênio, e portanto, na capacidade de exercício: mecanismos centrais (função do coração, pulmão e sistema nervoso autônomo) e mecanismos periféricos (associado ao fluxo sanguíneo periférico e a função do músculo esquelético). Os pacientes com HAP geralmente apresentam baixo débito cardíaco e estado adrenérgico exacerbado. A combinação destas alterações pode resultar em alterações estruturais e funcionais da musculatura estriada periférica. Porém, não existem informações sólidas que nos esclareçam se o acometimento muscular é preditor independente da limitação da capacidade de exercício. Objetivos: (1) Caracterizar o papel da musculatura periférica na limitação funcional em pacientes com HAP. (2) Avaliar o papel do sistema muscular periférico como um fator independente para a limitação ao exercício em HAP. Materiais e métodos: Dezesseis pacientes com HAP foram prospectivamente comparados com 10 indivíduos controle em termos de dados demográficos, qualidade de vida relacionada à saúde e limitação ao exercício, avaliada pelo teste de caminhada de seis minutos, teste cardiopulmonar, dinamometria isocinética e medições de pressão respiratória máxima. Pacientes com HAP também foram submetidos à biópsia do quadríceps, a fim de avaliar as mudanças estruturais. Resultados: Os pacientes com HAP apresentaram pior qualidade de vida (componente físico p<0,001), menor percentagem de massa magra (p=0,044), menor força muscular respiratória (p<0,001), menor resistência e força dos extensores de coxa (p=0,017 e p=0,012, respectivamente) e maior limitação funcional demonstrada pela distância percorrida no teste de caminhada de seis minutos (p<0,001) e pelo teste de exercício cardiopulmonar (p<0,001 para VO2/kg), em comparação ao grupo controle. Estes achados de redução de força e função muscular estão em acordo com os achados de redução da percentagem de fibras do Tipo I à biópsia muscular. O consumo de oxigênio, apresentou correlação com a função da musculatura respiratória e da musculatura extensora de coxa (resistência e força), e com a proporção de fibras oxidativas (Tipo I). O débito cardíaco também apresentou correlação com o VO2. o modelo de análise bivariada demonstrou que a função muscular é preditora independente do VO2 pico, mesmo com a correção para o perfil hemodinâmico. Conclusão: (1) Pacientes com HAP apresentam alteração estrutural e funcional da musculatura estriada periférica, e (2) estas alterações determinam limitação da capacidade global de exercício de forma independente do padrão hemodinâmico característico da HAP / Introduction: Pulmonary arterial hypertension (PAH) is a relentlessly progressive disease that leads to right heart failure and death. Despite advances in pharmacological treatment, prognosis is still poor with survival rates of 86%, 70% and 55% at 1, 3 and 5 years, respectively. Progressive dyspnea and exercise intolerance are the main clinical manifestations and reflect the impairment of right ventricular function. Peripheral skeletal muscle also seems to be a major determinant of functional limitation, as the reduction of oxygen supply and changes in extraction and utilization of oxygen by the muscle are directly associated to exercise tolerance. There are two potential mechanisms involved in the regulation of oxygen supply and therefore in exercise capacity: central (as a function of heart, lung and autonomic nervous system function) and peripheral (associated to peripheral blood flow and skeletal muscle function). Patients with PAH usually present low cardiac output and exacerbated adrenergic state. The combination of these features might result in changes of peripheral skeletal muscle and structure. However, there is no robust information that clearly clarifies whether the muscle involvement is an independent factor for exercise limitation. Objectives: (1) Characterize the role of the peripheral muscles in functional limitation in patients with PAH. (2) Address the role of the peripheral muscle system as an independent factor in exercise limitation in PAH. Materials and methods: Sixteen PAH patients were prospectively compared to 10 control individuals in terms of demographic data, health related quality of life and exercise limitation, assessed by six-minute walk test, cardiopulmonary test, isokinetic dynamometry and maximum respiratory pressure measurements. PAH patients also were submitted to vastus lateralis biopsy in order to assess structural changes. Results: PAH patients presented poorer quality of life (p <0.001), lower percentage of fat free mass (p = 0.044), lower respiratory muscle strength (p <0.001), lower resistance and strength of the extensor of the thigh (p = 0.017 and 0.012, respectively) and greater functional limitation demonstrated by the six-minute walk distance (p <0.001) and at the cardiopulmonary exercise test (p <0.001 for VO2max/kg), as compared to the control group. These findings of reduced muscle strength and function are in agreement with the findings of reduced percentage of Type I fibers at the muscle biopsy. The oxygen consumption correlated to the function of respiratory muscles and of extensor muscles of the thigh (endurance and strength) as well as to the proportion of oxidative fibers (Type I). The cardiac output also correlated with VO2. A bivariate model demonstrated that muscle function is an independent predictor of maximum oxygen consumption, even correcting for the hemodynamic profile. Conclusion: (1) PAH patients present functional and structural changes in peripheral skeletal muscles, and (2) these changes determine overall exercise capacity limitation, independently of the hemodynamic pattern

Introdução: A hipertensão arterial pulmonar (HAP) é uma doença progressiva extremamente grave, que evolui com insuficiência cardíaca direita e morte. Apesar do avanço do tratamento farmacológico, o prognóstico permanece reservado com taxa de sobrevida de 86%, 70% e 55% em 1, 3 e 5 anos, respectivamente. A dispnéia progressiva e a intolerância ao exercício são as principais manifestações clínicas e refletem a falência do ventrículo direito. O músculo esquelético periférico parece ser também um dos principais determinantes desta limitação funcional, visto que a redução da oferta de oxigênio e alterações na extração/utilização do oxigênio pelo músculo são diretamente relacionados com a tolerância ao exercício. Existem dois mecanismos potencialmente envolvidos na regulação da oferta de oxigênio, e portanto, na capacidade de exercício: mecanismos centrais (função do coração, pulmão e sistema nervoso autônomo) e mecanismos periféricos (associado ao fluxo sanguíneo periférico e a função do músculo esquelético). Os pacientes com HAP geralmente apresentam baixo débito cardíaco e estado adrenérgico exacerbado. A combinação destas alterações pode resultar em alterações estruturais e funcionais da musculatura estriada periférica. Porém, não existem informações sólidas que nos esclareçam se o acometimento muscular é preditor independente da limitação da capacidade de exercício. Objetivos: (1) Caracterizar o papel da musculatura periférica na limitação funcional em pacientes com HAP. (2) Avaliar o papel do sistema muscular periférico como um fator independente para a limitação ao exercício em HAP. Materiais e métodos: Dezesseis pacientes com HAP foram prospectivamente comparados com 10 indivíduos controle em termos de dados demográficos, qualidade de vida relacionada à saúde e limitação ao exercício, avaliada pelo teste de caminhada de seis minutos, teste cardiopulmonar, dinamometria isocinética e medições de pressão respiratória máxima. Pacientes com HAP também foram submetidos à biópsia do quadríceps, a fim de avaliar as mudanças estruturais. Resultados: Os pacientes com HAP apresentaram pior qualidade de vida (componente físico p<0,001), menor percentagem de massa magra (p=0,044), menor força muscular respiratória (p<0,001), menor resistência e força dos extensores de coxa (p=0,017 e p=0,012, respectivamente) e maior limitação funcional demonstrada pela distância percorrida no teste de caminhada de seis minutos (p<0,001) e pelo teste de exercício cardiopulmonar (p<0,001 para VO2/kg), em comparação ao grupo controle. Estes achados de redução de força e função muscular estão em acordo com os achados de redução da percentagem de fibras do Tipo I à biópsia muscular. O consumo de oxigênio, apresentou correlação com a função da musculatura respiratória e da musculatura extensora de coxa (resistência e força), e com a proporção de fibras oxidativas (Tipo I). O débito cardíaco também apresentou correlação com o VO2. o modelo de análise bivariada demonstrou que a função muscular é preditora independente do VO2 pico, mesmo com a correção para o perfil hemodinâmico. Conclusão: (1) Pacientes com HAP apresentam alteração estrutural e funcional da musculatura estriada periférica, e (2) estas alterações determinam limitação da capacidade global de exercício de forma independente do padrão hemodinâmico característico da HAP / Introduction: Pulmonary arterial hypertension (PAH) is a relentlessly progressive disease that leads to right heart failure and death. Despite advances in pharmacological treatment, prognosis is still poor with survival rates of 86%, 70% and 55% at 1, 3 and 5 years, respectively. Progressive dyspnea and exercise intolerance are the main clinical manifestations and reflect the impairment of right ventricular function. Peripheral skeletal muscle also seems to be a major determinant of functional limitation, as the reduction of oxygen supply and changes in extraction and utilization of oxygen by the muscle are directly associated to exercise tolerance. There are two potential mechanisms involved in the regulation of oxygen supply and therefore in exercise capacity: central (as a function of heart, lung and autonomic nervous system function) and peripheral (associated to peripheral blood flow and skeletal muscle function). Patients with PAH usually present low cardiac output and exacerbated adrenergic state. The combination of these features might result in changes of peripheral skeletal muscle and structure. However, there is no robust information that clearly clarifies whether the muscle involvement is an independent factor for exercise limitation. Objectives: (1) Characterize the role of the peripheral muscles in functional limitation in patients with PAH. (2) Address the role of the peripheral muscle system as an independent factor in exercise limitation in PAH. Materials and methods: Sixteen PAH patients were prospectively compared to 10 control individuals in terms of demographic data, health related quality of life and exercise limitation, assessed by six-minute walk test, cardiopulmonary test, isokinetic dynamometry and maximum respiratory pressure measurements. PAH patients also were submitted to vastus lateralis biopsy in order to assess structural changes. Results: PAH patients presented poorer quality of life (p <0.001), lower percentage of fat free mass (p = 0.044), lower respiratory muscle strength (p <0.001), lower resistance and strength of the extensor of the thigh (p = 0.017 and 0.012, respectively) and greater functional limitation demonstrated by the six-minute walk distance (p <0.001) and at the cardiopulmonary exercise test (p <0.001 for VO2max/kg), as compared to the control group. These findings of reduced muscle strength and function are in agreement with the findings of reduced percentage of Type I fibers at the muscle biopsy. The oxygen consumption correlated to the function of respiratory muscles and of extensor muscles of the thigh (endurance and strength) as well as to the proportion of oxidative fibers (Type I). The cardiac output also correlated with VO2. A bivariate model demonstrated that muscle function is an independent predictor of maximum oxygen consumption, even correcting for the hemodynamic profile. Conclusion: (1) PAH patients present functional and structural changes in peripheral skeletal muscles, and (2) these changes determine overall exercise capacity limitation, independently of the hemodynamic pattern