京都大学 / 新制・課程博士 / 博士(医学) / 甲第23412号 / 医博第4757号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)特定拠点教授 妻木 範行, 教授 戸口田 淳也, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Obesity and type 2 diabetes mellitus (T2DM) are both complex diseases with multifactorial etiologies. Together they affect over 640 million people worldwide and have a significant impact on the global healthcare system incurring costs of over 800 billion dollars. The overall goal of my doctoral research has been to elucidate metabolic predictors and underlying mechanisms in obesity and T2DM. Specifically, I have examined mechanisms contributing to disordered oxidative metabolism in skeletal muscle. My research included participants who were recruited from the Ottawa Hospital Weight Management Clinic in which they completed a clinically supervised meal-replacement and lifestyle intervention program. More so, my doctoral studies evaluated characteristics of muscle mitochondrial function in obesity and T2DM and revealed impaired mitochondrial respiration and electron transport chain supercomplex assembly in muscle from patients with T2DM. The first aim was to study the impact of T2DM on weight loss ability in a large population of obese patients participating in a standardized meal replacement and lifestyle modification program. As there is considerable variability in weight loss propensity, it was found that T2DM significantly deters weight loss although the effect is not large. Since skeletal muscle energetics are central in the development and progression of obesity and T2DM, the second and third aims were to study mitochondrial function in this tissue with the idea of uncovering molecular etiologies. The second aim found deficiencies in mitochondrial respiration in individuals with obesity and T2DM compared to individuals with obesity alone. Reductions in mitochondrial respiration were correlated with increasing levels of HbA1C and attributed to paucity in supercomplex formation in the mitochondrial inner membrane (MIM) of the electron transport chain (ETC). The third aim was to delineate differential fuel oxidation mechanisms and circulating protein biomarkers in obese diet-sensitive (ODS) and obese diet-resistant (ODR) participants following a high fat meal (HFM) challenge. Whole-body analyses were conducted in addition to measures in blood, adipose tissue, skeletal muscle and primary cells. Remarkable increases in oxidative capacity were measured post-HFM. In addition, impaired mitochondrial function was found in the ODR group despite lack of differences in mitochondrial content or the assembly of supercomplexes. Differences were also found in circulating acylcarnitines as well as expression of several proteins including Heat shock 70 kDa protein 1A/1B, Tyrosine-protein kinase Fgr, and Peptidyl-prolyl cis-trans isomerase D. Ultimately, a better understanding of mechanisms involved could lead to significant improvements in personalized medical approaches in obesity and T2DM.

Le coactivateur de facteurs de transcription PGC-1a (PPARC1A) est connu pour jouer un rôle clé dans la thermogénèse adaptative ainsi que dans l’homéostasie et la croissance musculaire chez l’homme et la souris. Le gène codant pour PGC-1 est contrôlé par deux promoteurs et est soumis à un épissage alternatif, il en résulte de multiples protéines. Chez le bovin, malgré son implication dans la croissance et dans les caractéristiques du lait relevée par des études de SNP, le gène et les transcrits de PGC-1a restent peu étudiés. Ainsi, notre objectif a été de mettre en évidence la structure et l’expression des transcrits de PGC-1a chez le bovin. Nous avons montré que deux formes longues PGC-1a-a et PGC-1a-b étaient exprimées chez le bovin de même que deux formes tronquées NT-PGC-1a-a et NT-PGC-1a-b (aussi appelé PGC-14). En conditions basales, les formes tronquées sont plus exprimées que les formes longues dans le muscle squelettique. De plus, les transcrits dérivants du promoteur proximal sont prédominants, ce qui suggère que NT-PGC-1a serait la forme prédominante dans le muscle squelettique bovin. Nous avons également créé des lignées cellulaires sur-exprimant indépendamment les formes longues ou tronquées et montré que la sur-expression des isoformes bovins entrainait une différenciation accrue des myoblastes associée à une augmentation de l’expression d’IGF-1 et une sousexpression de la myostatine. La multitude d’isoformes codée par le gène PGC-1a ainsi que leur implication dans la myogenèse positionne PGC-1a en gène d’intérêt dans l’étude de la variabilité phénotypique retrouvé chez certaines races bovines. De plus, les transcrits de PGC-1a semblent être de puissants modulateurs de la masse musculaire. PGC-1a pourrait donc être un gène de plus à étudier lors de la sélection des animaux domestiques présentant une plus grande musculature. / The transcriptional co-activator PGC-1α (PPARGC1A) has been reported to play a key role in adaptive thermogenesis and to influence muscle homeostasis and growth in mouse and human. PGC-1α has a complex structure with multiple protein domains whose gene is controlled by two promoters and is subject to alternative splicing events. In cattle, very little is currently known about PGC-1α, despite its implication in growth and milk characteristics revealrd by SNP study. So, the aim of our study was to investigate the presence and the structure of bovine PGC-1α alternative transcripts. We found different transcripts, two full-length isoforms named PGC-1α-a and PGC-1α-b, and two truncated forms, NT-PGC-1α and PGC-1α4. In basal conditions, our results showed that the truncated forms are the most expressed in bovine muscle. In addition, the transcripts derived from the proximal promoter are predominant, suggesting that NT-PGC-1 would be the main form. Finally, we showed that the overexpression of either fulllength or truncated isoforms of bovine PGC-1 enhances myoblasts differentiation. The multiplicity of isoforms resulting from PGC-1α as well as their implication in myogenesismakes PGC-1α as a gene of interest for the study of the muscular phenotypic variability found in different cattle breeds. In addition, PGC-1 transctipts appear to be a strong modulators of muscle mass. So the bovine PGC-1a isoforms could be used to engineer future breeds with higher muscularity.

abstract: Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential source of autologous stem cells for regenerative medicine. This work summarizes efforts to further understanding of satellite cell biology, using novel model organisms, bioengineering, and molecular and cellular approaches. Lizards are evolutionarily the closest vertebrates to humans that regenerate entire appendages. An analysis of lizard myoprogenitor cell transcriptome determined they were most transcriptionally similar to mammalian satellite cells. Further examination showed that among genes with the highest level of expression in lizard satellite cells were an increased number of regulators of chondrogenesis. In micromass culture, lizard satellite cells formed nodules that expressed chondrogenic regulatory genes, thus demonstrating increased musculoskeletal plasticity. However, to exploit satellite cells for therapeutics, development of an ex vivo culture is necessary. This work investigates whether substrates composed of extracellular matrix (ECM) proteins, as either coatings or hydrogels, can support expansion of this population whilst maintaining their myogenic potency. Stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, while the ECM composition was not significantly important. Additionally, satellite cells on hydrogels entered a quiescent state that could be reversed when the cells were subsequently cultured on Matrigel. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for satellite cells. To further understand how different signaling pathways control satellite cell behavior, an investigation of the Notch inhibitor protein Numb was carried out. Numb deficient satellite cells fail to activate, proliferate and participate in muscle repair. Examination of Numb isoform expression in satellite cells and embryonic tissues revealed that while developing limb bud, neural tube, and heart express the long and short isoforms of NUMB, satellite cells predominantly express the short isoforms. A preliminary immunoprecipitation- proteomics experiment suggested that the roles of NUMB in satellite cells are related to cell cycle modulation, cytoskeleton dynamics, and regulation of transcription factors necessary for satellite cell function. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020

Skeletal muscle regeneration is hindered in severe injuries and degenerative diseases, including volumetric muscle loss (VML), due to the failure of current treatments to induce functional tissue growth. Various biological functions in skeletal muscle are supported by the extracellular matrix (ECM), a collection of proteins and glycosaminoglycans. <i>In vivo</i> studies on murine plantaris muscle hypertrophy indicate that ECM remodeling facilitates muscle growth, but a global analysis of ECM protein dynamics during skeletal muscle hypertrophy and repair are unknown. Understanding this influence of the ECM can establish instructive cues for regenerative therapies. Here, we define global proteomic changes throughout stages of plantaris muscle hypertrophy, with an emphasis on characterizing ECM proteins. Synergistic ablation of the gastrocnemius and soleus muscles induced a compensatory hypertrophic effect causing a 40% mass increase in the plantaris muscle 28 days post injury. Liquid chromatography-tandem mass spectrometry revealed the differential abundance of 1233 proteins, including 99 ECM proteins, across five time points. After two days of injury, a significant increase of ECM glycoproteins was observed although the overall collagen abundance decreased. Throughout the duration of injury, the relative abundance of type I collagen decreased while there was an increase of proteins associated with type I collagen fibrillogenesis (types III and V) and basement membrane (types IV and VI). Collectively, these results provide a better understanding of ECM dynamics throughout skeletal muscle hypertrophy. Future studies will evaluate protein synthesis by using non-canonical amino acids to identify newly synthesized proteins. Temporal analysis of protein dynamics symbolic to injury and tissue growth will provide tissue engineers with precise information to develop successful regenerative therapies to restore functional muscle in VML.

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated when cellular energy is low and causes muscle and other cells to increase glucose uptake and fat oxidation, diminish lipid synthesis, and alter expression of various genes. AMPK activity is diminished in animals with the metabolic syndrome, though the mechanisms causing this reduction are unknown. To examine nutrient-induced changes in AMPK activity over time and factors that may regulate it, we compared rat muscle incubated with high glucose (HG) (30min-2h) and muscle of glucose-infused rats (3-8h) with appropriate controls. In addition to diminished AMPK activity (measured by the SAMS peptide assay) and phosphorylation of its activation loop at Thr172, we observed increased muscle glycogen, phosphorylation of AMPK’s α1/α2 subunit at Ser485/491, and PP2A activity, and decreased SIRT1 expression, all of which have been shown to diminish AMPK activity. Dysregulation of one or more of these factors could contribute to pathophysiological changes leading to metabolic syndrome associated disorders. Since recent studies suggest phosphorylation at Ser485/491 may play an important role in AMPK inhibition, we sought to determine how phosphorylation of this site is regulated. We investigated whether insulin or diacylglycerol (DAG) signaling pathways may be involved, since both are increased in at least one of the HG models. Akt and Protein Kinase (PK)D1 phosphorylated AMPK at Ser485/491 and diminished its activity in C2C12 myotubes, downstream of insulin and the DAG-mimetic PMA, respectively. Additionally, p-AMPK Ser485/491 was increased in muscle and liver of fed versus fasted mice and liver of diabetic mice. Our results suggest that Akt- and PKD1-mediated inhibition of AMPK via Ser485/491 phosphorylation may inhibit energy-metabolizing processes, while favoring energy-storing processes. Our results highlight the fact that phosphorylation of Ser485/491 can inhibit AMPK activity independent of changes in p-AMPK Thr172, a measure which is often used as a readout of AMPK activity. We hypothesize that Akt-mediated inhibition of AMPK is an acute, physiological response to insulin, whereas PKD1-mediated inhibition may be associated with more chronic pathophysiological changes. Thus, PKD1 inhibition or prevention of Ser485/491 phosphorylation may represent new strategies for therapeutic AMPK activation as treatment for the metabolic syndrome.

Eukaryotic DNA is packaged into highly ordered structures knows as chromatin that further packs the DNA into higher-order structures, limiting the accessibility of the underlying genetic information for the processes like transcription, replication, and repair. However, eukaryotic cells have evolved proteins called chromatin regulators that regulate the accessibility of the genetic information when needed. This dissertation aims to characterize the role of two such proteins, JARID2 and the polycomb repressive complex-2 (PRC2), during skeletal muscle proliferation and differentiation.JARID2 is an inactive yet evolutionarily conserved histone demethylase that is shown to be a sub-stoichiometric component of the PRC2 complex. The PRC2 complex represses gene expression through the trimethylation of lysine 27 of histone 3 (H3K27me) tails. H3K27 methylation leads to chromatin compaction. JARID2 helps in targeting of the PRC2 complex to its target loci. JARID2 is shown to be required for the normal development of mice, as loss of Jarid2 leads to lethality in utero. We, for the first time, show that JARID2 is required for the normal skeletal muscle differentiation. We show that the JARID2 regulates the expression of myogenic regulatory factor, Myod1, both through direct repression and activation through the modulation of canonical Wnt signaling pathway. JARID2, in association with the PRC2 complex, represses Wnt antagonist Sfrp1 to modulate the activity of the canonical Wnt signaling pathway. The translocation of Wnt effector protein, b-catenin, from the cytoplasm to the nucleus modulates the activity of the canonical Wnt signaling pathway during activation. We also show that b-catenin directly regulates the expression of Myod1 gene through its direct binding in the distal regulatory region.We further extend the role of JARID2 during skeletal muscle proliferation. We show that JARID2 also plays an essential role in restraining the skeletal muscle proliferation through its direct repression of positive cell cycle regulators cyclin D1 (Ccnd1) and cyclin E1 (Ccne1). Furthermore, we show that retinoblastoma protein 1 (Rb1), a negative regulator of cell proliferation that promotes cell cycle exit and differentiation, is also directly regulated by JARID2 in PRC2 dependent manner. Together, we show that JARID2 precisely controls cell proliferation and differentiation during skeletal muscle differentiation.Further, we show that the regulation of cell proliferation by JARID2 is PRC2 complex dependent. When the PRC2 complex was depleted or inhibited to a modest level, the cells have an increased cell proliferation ability compared to severe loss or inhibition of EZH2, the catalytic subunit of the PRC2 complex, that leads to the apoptosis of the cells. It is also marked by increased expression of known PRC2 targets genes. We show that the increased proliferation upon modest inhibition or depletion of EZH2 is through direct de-repression of positive cell cycle genes, Ccnd1, and Ccne1. It is the first work that shows a context-dependent role of the PRC2 complex during skeletal muscle proliferation and differentiation.My dissertation also makes an extraordinary discovery as to why myogenin is required for the proper function of MyoD during skeletal muscle differentiation, even though both proteins share a large set of overlapping target genes. We show that myogenin is required for the nucleosome disassembly and reassembly at the target genes through recruitment of the FACT complex, a histone chaperone. We also show that myogenin is required for the assembly of the basic transcription machinery and RNA polymerase II to the target muscle genes during differentiation. Surprisingly, we also show that myogenin reinforces its own expression through the activation of Myod1 expression during skeletal muscle differentiation. Myogenin is a known target of MyoD.Taken together, this dissertation provides a molecular mechanism for the crosstalk between a signaling pathway with chromatin regulatory proteins, JARID2, and the PRC2 complex in regulating skeletal muscle differentiation. It also extends the role of JARID2 and the PRC2 complex - known oncogenes, in precise, context-dependent control of cell proliferation and differentiation in skeletal muscle.

Skeletal muscle is a highly adaptable tissue that comprises approximately 40% of total body weight while accounting for up to 90% of whole-body oxygen consumption and energy expenditure during exercise. The loss of skeletal muscle protein and subsequent decrease in muscle mass (atrophy) that accompanies disuse results primarily from a decrease in intracellular protein synthesis combined with an increase in proteolytic activity. Interestingly, these processes of skeletal muscle atrophy are amplified by changes in mitochondrial capacity, with evidence suggesting that the maintenance of mitochondria during periods of disuse protects skeletal muscle against atrophy. Remarkably, rodents with denervated muscle are protected against muscle atrophy following whole-body heat stress. The mechanism of protection appears to be tied to the observed increases in heat shock protein (HSP) and PGC-1α, which accompany the heat stress. Without any published observations as to whether such heat-induced protection against muscle atrophy would translate to human muscle, the aim of this project was to determine the extent to which deep tissue heating (via pulsed shortwave diathermy) might provide protection against skeletal muscle atrophy.

Purpose: To investigate the effects of eccentric exercise on lower body skeletal muscle mass during rapid body mass loss induced by bariatric surgery. Methods: All participants began 6 to 8 weeks after undergoing Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Skeletal muscle mass (SMM) in the lower body was measured via magnetic resonance imaging (MRI); additional exercise measurements included muscular strength and functional capacity. Quality of life was measured using Short Form 36 (SF-36). Nineteen females (age = 37.6 ± 9.8 yr, height = 164.4 ± 7.2 cm, mass = 106.9 ± 15.6 kg) were randomly assigned to 1 of 3 groups: eccentric exercise (EEX; n = 6), concentric exercise (CEX; n = 7), or standard-of-care control (CON; n = 6). Exercise groups performed 30-minute lower-body exercise sessions 3 times per week for 16 weeks. Each month the exercise tests were evaluated. At the end of 16 weeks, all participants performed the final exercise tests, received a final MRI scan, and completed the SF-36 questionnaire. Results: Thirteen individuals completed the study. All groups lost mass: CON: 21.4 ± 3.7 kg (p < 0.001), CEX: 19.9 ± 4.0 kg (p = 0.001), and EEX: 21.8 ± 3.3 kg (p < 0.001). SMM decreased in all groups: CON: 0.77 ± 0.5 kg (p = 0.18), CEX: 1.19 ± 0.6 kg (p = 0.06), and EEX: 0.90 ± 0.5 kg (p = 0.09). The skeletal muscle loss in percent of total mass loss was 3.7 ± 4.1%. All measures of muscular strength showed no difference, except for a small decrease in dynamic (60°·sec-1) strength in the eccentric group. Functional capacity and physical quality of life increased significantly in all groups (p < 0.05). Conclusion: SMM loss still occurred in the lower body regardless of resistance training, but the loss was less than what was previously documented. Improved postsurgical functional capacity and physical quality of life may be due to a reduction in fat mass and maintenance of muscular strength during the period of rapid mass loss.