Cthrc1 controls adipose tissue formation, body composition, and physical activity.
ABSTRACT: This study investigated the effects of loss of Cthrc1 on adipogenesis, body composition, metabolism, physical activity, and muscle physiology.Complete metabolic and activity monitoring as well as grip strength measurements and muscle myography was performed in Cthrc1 null and wildtype mice.Compared to wildtypes, Cthrc1 null mice had similar body weights but significantly reduced energy expenditure, decreased lean mass, and increased fat mass, especially visceral fat. In vitro studies demonstrated that Cthrc1 inhibited adipocyte differentiation as well as PPAR and CREB reporter activity, while preadipocytes isolated from Cthrc1 null mice exhibited enhanced adipogenic differentiation. Voluntary physical activity in Cthrc1 null mice as assessed by wheel running was reduced to approximately half the distance covered by wildtypes. Reduced grip strength was observed in Cthrc1 null mice at the age of 15 weeks or older with reduced performance and mass of hyphenate muscle. In the brain, Cthrc1 expression was most prominent in neurons of thalamic and hypothalamic nuclei with evidence for secretion into the circulation in the median eminence.Our data indicate that Cthrc1 regulates body composition through inhibition of adipogenesis. In addition, central Cthrc1 may be a mediator of muscle function and physical activity.
Project description:Collagen triple helix repeat-containing1 (Cthrc1) has previously been implicated in osteogenic differentiation and positive regulation of bone mass, however, the underlying mechanisms remain unclear. Here we characterized the bone phenotype of a novel Cthrc1 null mouse strain using bone histomorphometry, ?CT analysis and functional readouts for bone strength. In male Cthrc1 null mice both trabecular bone as well as cortical bone formation was impaired, whereas in female Cthrc1 null mice only trabecular bone parameters were altered. Novel and highly specific monoclonal antibodies revealed that CTHRC1 is expressed by osteocytes and osteoblasts, but not osteoclasts. Furthermore, Cthrc1 null mice exhibited increased bone resorption with increased number of osteoclast and increased osteoclast activity together with enhanced expression of osteoclastogenic genes such as c-Fos, Rankl, Trap, and Nfatc1. Differentiation of bone marrow-derived monocytes isolated from Cthrc1 null mice differentiated into osteoclasts as effectively as those from wildtype mice. In the presence of CTHRC1 osteoclastogenic differentiation of bone marrow-derived monocytes was dramatically inhibited as was functional bone resorption by osteoclasts. This process was accompanied by downregulation of osteoclastogenic marker genes, indicating that extrinsically derived CTHRC1 is required for such activity. In vitro, CTHRC1 had no effect on osteogenic differentiation of bone marrow stromal cells, however, calvarial osteoblasts from Cthrc1 null mice exhibited reduced osteogenic differentiation compared to osteoblasts from wildtypes. In a collagen antibody-induced arthritis model Cthrc1 null mice suffered significantly more severe inflammation and joint destruction than wildtypes, suggesting that CTHRC1 expressed by the activated synoviocytes has anti-inflammatory effects. Mechanistically, we found that CTHRC1 inhibited NF?B activation by preventing I?B? degradation while also inhibiting ERK1/2 activation. Collectively our studies demonstrate that CTHRC1 secreted from osteocytes and osteoblasts functions as an inhibitor of osteoclast differentiation via inhibition of NF?B-dependent signaling. Furthermore, our data suggest that CTHRC1 has potent anti-inflammatory properties that limit arthritic joint destruction.
Project description:Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.
Project description:We recently reported expression of collagen triple helix repeat containing-1 (Cthrc1) in injured arteries and proteolytic cleavage of Cthrc1 in smooth muscle cells in vitro. The present study characterizes Cthrc1 processing and determines its biological significance.Domain-specific antibodies localized full-length Cthrc1 in the cytoplasm of vascular, gastrointestinal, and uterine smooth muscle as well as in some neurons. Unlike smooth muscle alpha-actin, Cthrc1 was not expressed in the embryonic myocardium. Intracellular localization of full-length Cthrc1 was sharply reduced in dedifferentiated smooth muscle of the developing neointima despite the previously shown increase in mRNA levels with accompanying extracellular Cthrc1 immunoreactivity. Immunoblotting suggested an apparent covalent association of monomeric full-length Cthrc1 with a cytoplasmic protein present in differentiated smooth muscle. Plasmin was identified as a protease that cleaved a putative propeptide generating an N-terminally truncated form of Cthrc1 with increased inhibitory activity of procollagen synthesis.Our data show that the differentiated smooth muscle cell phenotype is associated with the intracellular localization of noncleaved Cthrc1 despite the presence of a signal peptide. On arterial injury, increased Cthrc1 expression with apparent extracellular localization of N-terminally truncated Cthrc1 occurs. Removal of the propeptide correlated with increased activity of the molecule.
Project description:OBJECTIVE:Sarcopenia in older adults is closely related to vitamin D deficiency and reduced levels of physical activity, but little has been reported on the interaction between physical activity and the positive effects of vitamin D. The purpose of this study was to explore the interactive effect of vitamin D and physical activity on muscle mass and function through animal experiments and population surveys. METHODS:Male 4-week-old C57BL/6J mice were fed different purified diets: a vitamin D-deficient diet (with increased calcium and phosphorus to prevent the effects of abnormal mineral levels on muscle) or a 1,25-dihydroxyvitamin D3 (1,25D)-supplemented diet. After 24 weeks on the assigned diets, the mice were immobilized. The level of skeletal muscle atrophy in the mice was determined by grip strength, gastrocnemius (GA) muscle mass and muscle fiber cross-sectional area (CSA); additionally, the protein expression levels of FOXO3a and the E3 ubiquitin ligases MuRF1 and MAFbx were detected. A cross-sectional study included data from 4139 older adults (64.9% women, 67.9 ± 6.7 years) as part of a survey in Shenyang, Northeast China. The associations of serum 25(OH)D3 and physical activity with timed up and go test (TUG) performance, handgrip strength, calf circumference, and body muscle mass were assessed by a linear regression analysis that was adjusted for covariates. RESULTS:In activity-limited mice, vitamin D deficiency accelerated the decrease in GA muscle weight, muscle fiber CSA, and grip strength and increased the protein expression of MuRF1, MAFbx, and FOXO3a (all P < 0.05). In addition, 1,25D supplementation may inhibit the grip-strength reduction induced by limited activity (P = 0.069). Serum 25(OH)D3 and physical activity were linearly related to TUG time (P < 0.001) and handgrip strength (P < 0.05) after adjustment for sex, age, body mass index (BMI), education level, smoking status, and serum calcium level. Serum 25(OH)D3 and physical activity had interactive effects on TUG (P < 0.001) and handgrip strength (P < 0.05) but not calf circumference or body muscle mass in older adults. CONCLUSIONS:The effect of vitamin D on muscle strength and physical performance depends on physical activity level in the elderly. It is recommended that older adults strive to avoid both physical inactivity and vitamin D deficiency. Because physical inactivity and vitamin D deficiency may exacerbate muscle atrophy, the biological mechanism may involve synergistic effects of vitamin D and physical activity on the promotion of muscle protein ubiquitination and degradation.
Project description:Testosterone supplementation increases muscle mass in older men but has not been shown to consistently improve physical function and activity. It has been hypothesized that physical exercise is required to induce the adaptations necessary for translation of testosterone-induced muscle mass gain into functional improvements. However, the effects of testosterone plus low intensity physical exercise training (T/PT) on functional performance and bioenergetics are unknown. In this pilot study, we tested the hypothesis that combined administration of T/PT would improve functional performance and bioenergetics in male mice late in life more than low-intensity physical training alone. 28-month old male mice were randomized to receive T/PT or vehicle plus physical training (V/PT) for 2 months. Compare to V/PT control, administration of T/PT was associated with improvements in muscle mass, grip strength, spontaneous physical movements, and respiratory activity. These changes were correlated with increased mitochondrial DNA copy number and expression of markers for mitochondrial biogenesis. Mice receiving T/PT also displayed increased expression of key elements for mitochondrial quality control, including markers for mitochondrial fission-and-fusion and mitophagy. Concurrently, mice receiving T/PT also displayed increased expression of markers for reduced tissue oxidative damage and improved muscle quality.Testosterone administered with low-intensity physical training improves grip strength, spontaneous movements, and respiratory activity. These functional improvements were associated with increased muscle mitochondrial biogenesis and improved mitochondrial quality control.
Project description:Muscular dystrophy is characterized by progressive skeletal muscle weakness and dystrophic muscle exhibits degeneration and regeneration of muscle cells, inflammation and fibrosis. Skeletal muscle fibrosis is an excessive deposition of components of the extracellular matrix including an accumulation of Collagen VI. We hypothesized that a reduction of Collagen VI in a muscular dystrophy model that presents with fibrosis would result in reduced muscle pathology and improved muscle function. To test this hypothesis, we crossed ?-sarcoglycan-null mice, a model of limb-girdle muscular dystrophy type 2C, with a Col6a2-deficient mouse model. We found that the resulting ?-sarcoglycan-null/Col6a2?ex5 mice indeed exhibit reduced muscle pathology compared with ?-sarcoglycan-null mice. Specifically, fewer muscle fibers are degenerating, fiber size varies less, Evans blue dye uptake is reduced and serum creatine kinase levels are lower. Surprisingly, in spite of this reduction in muscle pathology, muscle function is not significantly improved. In fact, grip strength and maximum isometric tetanic force are even lower in ?-sarcoglycan-null/Col6a2?ex5 mice than in ?-sarcoglycan-null mice. In conclusion, our results reveal that Collagen VI-mediated fibrosis contributes to skeletal muscle pathology in ?-sarcoglycan-null mice. Importantly, however, our data also demonstrate that a reduction in skeletal muscle pathology does not necessarily lead to an improvement of skeletal muscle function, and this should be considered in future translational studies.
Project description:Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca (2+)) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca (2+) buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic episodes that resemble MH events in humans.We compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.About 25% of fibers in EDL muscles from CASQ1-null mice of 14 to 27 months of age exhibited large areas of structural disarray (named core-like regions), which were rarely observed in muscle from age-matched WT mice. To determine early events that may lead to the formation of cores, we analyzed EDL muscles from adult mice: at 4 to 6 months of age, CASQ1-null mice (compared to WT) displayed significantly reduced grip strength (40?±?1 vs. 86?±?1 mN/gr) and exhibited an increase in the percentage of damaged mitochondria (15.1% vs. 2.6%) and a decrease in average cross-sectional fiber area (approximately 37%) in EDL fibers. Finally, oxidative stress was also significantly increased (25% reduction in ratio between reduced and oxidized glutathione, or GSH/GSSG, and 35% increase in production of mitochondrial superoxide flashes). Providing ad libitum access to N-acetylcysteine in the drinking water for 2 months normalized GSH/GSSG ratio, reduced mitochondrial damage (down to 8.9%), and improved grip strength (from 46?±?3 to 59?±?2 mN/gr) in CASQ1-null mice.Our findings: 1) demonstrate that ablation of CASQ1 leads to enhanced oxidative stress, mitochondrial damage, and the formation of structural cores in skeletal muscle; 2) provide new insights in the pathogenic mechanisms that lead to damage/disappearance of mitochondria in cores; and 3) suggest that antioxidants may provide some therapeutic benefit in reducing mitochondrial damage, limiting the development of cores, and improving muscle function.
Project description:AIMS/HYPOTHESIS:The carcino-embryonic antigen-related cell adhesion molecule (CEACAM)2 is produced in many feeding control centres in the brain, but not in peripheral insulin-targeted tissues. Global Ceacam2 null mutation causes insulin resistance and obesity resulting from hyperphagia and hypometabolism in female Ceacam2 homozygous null mutant mice (Cc2 [also known as Ceacam2](-/-)) mice. Because male mice are not obese, the current study examined their metabolic phenotype. METHODS:The phenotype of male Cc2(-/-) mice was characterised by body fat composition, indirect calorimetry, hyperinsulinaemic-euglycaemic clamp analysis and direct recording of sympathetic nerve activity. RESULTS:Despite hyperphagia, total fat mass was reduced, owing to the hypermetabolic state in male Cc2(-/-) mice. In contrast to females, male mice also exhibited insulin sensitivity with elevated ?-oxidation in skeletal muscle, which is likely to offset the effects of increased food intake. Males and females had increased brown adipogenesis. However, only males had increased activation of sympathetic tone regulation of adipose tissue and increased spontaneous activity. The mechanisms underlying sexual dimorphism in energy balance with the loss of Ceacam2 remain unknown. CONCLUSIONS/INTERPRETATION:These studies identified a novel role for CEACAM2 in the regulation of metabolic rate and insulin sensitivity via effects on brown adipogenesis, sympathetic nervous outflow to brown adipose tissue, spontaneous activity and energy expenditure in skeletal muscle.
Project description:Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5-E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.
Project description:BACKGROUND:Late-middle age HIV patients are prone to fatigue despite effective viral control by antiretroviral therapies. Rodent models to recapitulate this phenotype are still not available. HYPOTHESIS:Drug treatment may compromise muscle strength and physical performance more in older individuals with pre-existing metabolic disorders than normal young ones. METHODS:Kaletra was given to overweight male mice at late-middle age and normal young adults; both on a rodent diet containing 30% fat calorie. Body composition and grip strength were measured at baseline and after drug treatment. Rota-rod running, insulin and glucose tolerance were measured at the end of the experiment. Drug effect on metabolic activity and spontaneous movements were assessed using the metabolic cage system. Representative muscle and fat tissue were analyzed for protein and mRNA expression. Selected findings were tested using murine C2C12 myotubes. RESULTS:Kaletra reduced grip strength in both young and older mice but impaired rotarod performance only in the old. Spontaneous movements were also reduced in Kaletra-treated old mice. Kaletra reduced IGF-1 expression in all muscle groups tested for the old and in cultured myotubes but to a less extent in the muscle of young animals. Reduced IGF-1 expression correlated with increased expression of muscle-specific atrogene MAFbx and MuRF1. Kaletra also increased abdominal fat mass markedly in the old animals and to a less extend in the young. CONCLUSION:Long-term Kaletra intake aggravated abdominal obesity and impaired muscle strength. This effect was worse in older animals than in normal young adults.