Function Of MTOR In Skleatal Muscle And Adipose Tissue Development Revealed By Conditional Knockout Mice

The skeletal muscle constitutes ~40% of the animal body mass and plays an important role in locomotion and metabolism. As such, proper muscle growth and homeostasis is a critical determinant of farm animal meat production and human motor performance. Conversely, muscle wasting due to genetic factors(muscular dystrophy), aging(sarcopenia), disuse(atrophy) or cancer(cachexia) severely compromises the life quality of humans. There are mainly two kinds of adipose tissue: white adipose tissue and brown adipose tissue. Beige adipose tissue was recently found in the white adipose tissue of mice. The excess energy is stored in white adipose tissue in the form of triglycerides and finally leads to obesity, diabetes and heart disease. Whereas brown and beige adipose tissue could fight with obesity by burning excess energy to heat. Mammalian(or mechanistic) target of rapamycin(m TOR) is a serine/threonine kinase that serves as the catalytic subunit of two protein complexes: the m TOR complex 1(m TORC1) and m TOR complex 2(m TORC2). The m TOR-mediated signaling pathway has been shown to regulate a wide range of cellular processes including protein translation, cell proliferation, apoptosis and autophagy. Knock out of the m TOR associated protein Raptor or Rictor in skeletal muscle led to severe myopathy, including impaired oxidative metabolism, altered mitochondrial regulation and glycogen accumulation. Deletion of Raptor in adipose tissue results in less white adipose tissue and up regulate metabolism weight. In contrast, deletion of Rictor increased mice body weight without affecting adipose tissue. Whereas these results has shed light on the relative role of m TOR in skeletal muscle and adipose tissue development, however the exactrole of m TOR remains unknown. With the developing of Cre-Lox P system. it is possible to knockout target gene in specific tissue. Based on this method, we established skeletal muscle and adipose tissue specific knockout of m TOR to investigate the role of m TOR.The main results as follows:1 The role of m TOR for proper satellite cell activity and skeletal muscle regenerationWe established a satellite cell specific m TOR conditional knockout(c KO) mouse model by crossing Pax7-Cre ER and m TOR flox/flox mice. Skeletal muscle regeneration after injury was severely compromised in the absence of m TOR, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the m TOR c KO mice compared to wild type(WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The m TOR c KO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the m RNA and protein level, the m TOR c KO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, and Myog than did the WT myoblasts. These results suggest that m TOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes.2 The role of m TOR for cardiomyocyte activity and heart functionWe established a muscle-specific m TOR conditional knockout mouse model(m TOR-m KO) by crossing Mck-Cre and m TOR flox/flox mice. Although the m TOR-m KO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of m TOR-m KO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of m TOR-m KO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the m TOR-m KO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of m TOR dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of m TORC1 and m TORC2 mediating the normal function of m TOR. These results demonstrate that m TOR is essential for cardiomyocyte survival and cardiac muscle function.3 The function of m TOR in adipose tissue growth and developmentWe established an adipose tissue-specific m TOR conditional knockout mouse model(Ad-m TOR) by crossing Adiponectin-Cre and m TOR flox/flox mice. Notably,all of the BAT,anterior subcutaneous WAT(as WAT),inguinal white adipose tissue(ing WAT),(visceral white adipose tissue,v WAT) from the Ad-m TOR mice decreased to less than 50% of the WT. In addition, the size of the BAT and WAT adipocytes were smaller in Ad-m TOR mice. Of note, multilocular adipocytes were found in some regions of Ad-m TOR WAT. The BAT marker genes, such as UCP1 and Prdm16 elevated to twice of the WT. Adipogenesis related gene Pparγ increased to three times of the WT. In contrast to this, another adipogenesis related gene C/EBPα decreased significantly. In WAT, the adipogenesis related genes such as Adiponectin,Leptin,Pparγand Srebp1 c were found decreased in m TOR knockout mice compared to WT, whereas fatty acid metabolism genes, such as Ucp1,Pgc1α and Pparα, increased significantly. Taken together, these results suggested knockout of m TOR may inhibit adipose tissue development and elevated energy metabolism. Since the expression levels of beige marker genes were not change significantly., beige adipose tissue may not form, Next, we found the knockout of m TOR inhibited the differentiation of SVF cells by isolation and culture of primary SVF cells from WAT and BAT. Consistent with the defection of differentiation, the expression levels of aipogenesis genes were also down regulated in the m TOR deleted cells. Though BAT metabolism is important to keep the body temperature in cold environment, we did not found difference in the body temperature between m TOR knockout mice and WT. Consistent with this, the metabolic chambers result showed no difference in the heat production between m TOR deleted and WT mice. This may be explained by that the metabolism levels were increased in BAT. Next, we induced mice obesity by feeding with high fat diet for 12 weeks. The weight of BAT and WAT adipose tissue were significantly lower in the Ad-m TOR mice compares to WT. These results provide compelling evidence that knockout of m TOR inhibit adipose tissue development and growth. The metabolism levels were elevated in m TOR deleted mice, thus resistant to high fat diet induced obesity.