Adipocyte-derived exosomal miR-27a induces insulin resistance in skeletal muscle through repression of PPARγ

OBJECTIVE To explore increasingly exosomal serum miR-27 a derived from adipocytes could be taken up by skeletal muscle tissue and induce insulin resistance in skeletal muscle in obese state. METHODS The association between miR-27 a and insulin resistance in skeletal muscle was determined in obese children,high-fat diet-induced miR-27 a knockdown obese mice,db/db mice and C2C12 cells overexpressing miR-27 a.The crosstalk mediated by exosomal miR-27 a between adipose tissue and skeletal muscle was determined in C2C12 cel s incubated with conditioned medium prepared from palmitate-treated 3 T3-L1 adipocytes. RESULTS After knockdown miR-27 a in obese insulin resistance mice,impaired insulin resistance, glucose intolerance and insulin resistance of skeletal muscle were partly restored. In high-fat diet group, the expressions of IRS-1 and GLUT4 in glucose uptake signal pathway of skeletal muscle were significantly decreased, while the expression of IRS-1 and GLUT4 was restored after miR-27 a knockdown. The content of FABP4, a marker specific for exosomes from adipocytes, was detected in sera, skeletal muscle, supernatant of adipocytes and co-cultured C2C12 cells; furthermore,exosomal miR-27 a in serum and adipocyte supernatants were detect, and fluorescence co-localization experiments were conducted to detect whether the exosomal miR-27 a in serum is mainly derived from adipocyte; finally,we used the supernatant of adipose tissue to construct conditioned media to treat with C2C12 cells, and detected whether adipocytes derived exosomal miR-27 a could impaired glucose uptake signaling pathway of skeletal muscle. the expressions of PPARγ silencing high-fat diet induced C57 BL/6 J obese mouse model and adenovirus intervention miR-27 a knockdown model were examined,and a C2C12 cell model overexpressing miR-27 a in the absence or presence with rosiglitazone(PPARγ activator)were established to test glucose consumption, glucose uptake, and glucose uptake signaling pathways of skeletal muscle cells. CONCLUSION These results identify a novel crosstalk signaling pathway between adipose tissue and skeletal muscle in the development of insulin resistance, and indicate that adipose tissue-derived miR-27 a may play a key role in the development of obesity-triggered insulin resistance in skeletal muscle.     

Galectin-1 is a novel factor that regulates myotube growth in regenerating skeletal muscles

Adult skeletal muscles have a vigorous regenerative capacity in response to chemical, mechanical or physical injuries. Muscle satellite cells play a critical role in skeletal muscle regeneration. Activated satellite cells (myoblasts) proliferate and then differentiate. Differentiated myoblasts fuse with each other to form multinucleated myotubes, and the growth of myotubes is induced by both fusion with additional myoblasts and reinnervation of motor neurons. Cellular and molecular events underlying the regenerative processes are regulated by critical factors, which are produced by satellite cells, myoblasts, myotubes, extracellular matrix and inflammatory cells. Galectin-1 is abundantly synthesized in adult skeletal muscles, but its roles in muscle regeneration have not been fully elucidated. We reviewed previous studies on the function of galectin-1 regarding myogenesis in vivo and in vitro, and discussed the roles of this lectin in regenerating skeletal muscles based on our observations. In intact adult muscles, galectin-1 was associated with basement membranes of myofibers. After muscle injury, galectin-1 immunoreactivity was increased within the cytoplasm of activated satellite cells. Thereafter, differentiated myoblasts lost galectin-1 immunoreactivity, but galectin-1 expression associated with basement membranes was detected in myotubes. Administration of anti-galectin-1 antibody, which perturbs the function of galectin-1, decreased the size of myotubes. Furthermore, muscle injury induced abundant expression of galectin-1 in damaged intramuscular nerve axons. We conclude that galectin-1 is a novel factor that promotes both myoblast fusion and axonal growth following muscle injury, and consequently, regulates myotube growth in regenerating skeletal muscles.     

Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes

Aim:

To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice.

Methods:

Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses.

Results:

The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-β receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-β1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-β receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-β receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGFβ receptor/pSmad3 signaling.

Conclusion:

The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-β receptor signaling.     

Coaxing stem cells for skeletal muscle repair

Skeletal muscle has a tremendous ability to regenerate, attributed to a well-defined population of muscle stem cells called satellite cells. However, this ability to regenerate diminishes with age and can also be dramatically affected by multiple types of muscle diseases, or injury. Extrinsic and/or intrinsic defects in the regulation of satellite cells are considered to be major determinants for the diminished regenerative capacity. Maintenance and replenishment of the satellite cell pool is one focus for muscle regenerative medicine, which will be discussed. There are other sources of progenitor cells with myogenic capacity, which may also support skeletal muscle repair. However, all of these myogenic cell populations have inherent difficulties and challenges in maintaining or coaxing their derivation for therapeutic purpose. This review will highlight recent reported attributes of these cells and new bioengineering approaches to creating a supply of myogenic stem cells or implants applicable for acute and/or chronic muscle disorders.     

Selective D3 receptor agonist effects of (+)-PD 128907 on dialysate dopamine at low doses

An involvement of the D3 dopamine receptor in the modulation of extracellular dopamine concentrations is suggested by pharmacological studies. However, recent studies using D3 receptor knock out mice indicated that several functions previously attributed to the D3 receptor are mediated by other receptor types. In the present study, we used the no-net flux microdialysis technique to characterize: (i) basal dopamine dynamics in the ventral striatum of D3 knock out and wild type mice and (ii) the effects of the putative D3-receptor selective agonist (+)-PD 128907. Neither the extracellular dopamine concentration nor the in vivo extraction fraction, an indirect measure of basal dopamine uptake, differed between D3 knock out and wild type mice. Moreover, no differences in potassium (60 mM) or cocaine (5 or 20 mg/kg i.p.) evoked dopamine concentrations were detected between the two genotypes. However, intra-striatal or systemic administration of doses of (+)-PD 128907 that failed to modify dopamine concentrations in knock out mice significantly decreased dialysate dopamine concentrations in the wild type. Comparison of the concentration-response curve for (+)-PD 128907 revealed IC(25) values of 61 and 1327 nM in wild type and knock out mice, respectively, after intra-striatal infusions. Similar differences were obtained after systemic administration of the D3 preferring agonist (IC(25) 0.05 and 0.44 mg/kg i.p. in wild type and knock out mice, respectively). We conclude that the activation of the D3 receptor decreases extracellular dopamine levels and that, at sufficiently low doses, the effects of (+)-PD 128907 on extracellular dopamine are selectively mediated by the D3 receptor.     

Phosphatase and tensin homolog deleted on chromosome 10 suppression is an important process in peroxisome proliferator-activated receptor-gamma signaling in adipocytes and myotubes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) activation enhances insulin sensitivity in type 2 diabetes mellitus. However, downstream mediators of PPARgamma activation in adipocytes and myotubes, the most important cell types involved in glucose homeostasis, remained unclear. Here we show by using two synthetic PPARgamma agonists (rosiglitazone and KR-62776, a novel PPARgamma agonist) that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a key downstream mediator of PPARgamma signaling. The PPARgamma agonists down-regulated PTEN expression, resulting in glucose uptake increase in differentiated 3T3-L1 adipocytes and C2C12 skeletal muscle cells. In both cells, PTEN knockdown increased glucose uptake, whereas overexpression abolished the agonist-induced effects. The effects of PPARgamma agonists on PTEN expression and glucose uptake disappeared by pretreatment with a PPARgamma antagonist or by knockdown of PPARgamma expression. In vivo treatment of the agonists to C57BL/6J-ob/ob mice resulted in the reduction of PTEN level in both adipose and skeletal muscle tissues and decreased plasma glucose levels. Thus, these results suggest that PTEN suppression is a key mechanism of the PPARgamma-mediated glucose uptake stimulation in insulin-sensitive cells such as adipocytes and skeletal muscle cells, thereby restoring glucose homeostasis in type 2 diabetes.     

Isolation and characterization of primary skeletal muscle satellite cells from rats

The purpose of this study was to isolate and characterize skeletal muscle satellite cells from rats using tissue block culture method. Specific Pathogen Free (SPF) level Sprague-Dawley (SD) rats were used to isolate skeletal muscle satellite cells. Morphology, expression and distribution of α-actin and Desmin within the cytoplasm of skeletal muscle satellite cells were compared with those of C2C12 myoblasts. The results showed that tissue block culturing method achieved robust proliferation and excellent differentiation of skeletal muscle satellite cells. Immunofluorescence and immunohistochemistry results showed that α-actin and Desmin proteins were expressed in the cytoplasm of both skeletal muscle satellite cells and myoblasts. We concluded that tissue block culturing method can obtain highly purified skeletal muscle satellite cells with robust proliferation and excellent differentiation capabilities.     

Lack of functional D2 receptors prevents the effects of the D3-preferring agonist (+)-PD 128907 on dialysate dopamine levels

Substantial pharmacological evidence is consistent with an inhibitory effect of D3 receptor activation on dopamine (DA) release. Although receptor selectivity of the ligands employed in initial studies has been questioned, studies employing new, more selective, compounds continue to support an involvement of this receptor subtype in regulating extracellular dopamine levels in the dorsal striatum and nucleus accumbens. Consistent with this hypothesis, microdialysis studies have shown that the dose-effect curve for (+)-PD 128907, a moderately selective D3 agonist, is shifted to the right in D3 knock out mice. The present microdialysis studies sought to further examine the role of D2 vs. D3 receptors in mediating (+)-PD 128907-evoked alterations in basal and depolarization-evoked DA levels. Dialysate DA levels were determined in D2 knock out mice and wild type littermate controls following both systemic and local administration of (+)-PD 128907. In view of regional differences in D3 receptor localization, studies were conducted in the nucleus accumbens, a D3 receptor rich area, and in the dorsal striatum, a region with low D3 receptor abundance. Systemic or reverse dialysis of (+)-PD 128907 into the nucleus accumbens significantly decreased basal and depolarization-evoked DA levels in wild type mice. A similar effect was observed in the dorsal striatum. Regardless of the route of administration, (+)-PD 128907 was ineffective in modulating DA levels in either brain region of D2 knock out mice. These data contrast with previous results in D3 knock out mice and indicate that the D2 receptor is necessary for the inhibition of presynaptic DA neurotransmission produced by a preferential D3 agonist. Based on the documented physical interaction of D2 and D3 receptors in heterologous expression systems, we put forth a hypothesis that reconciles the seemingly paradoxical results of this and previous microdialysis studies.     

Satellite cell activity in muscle regeneration after contusion in rats

1. The role of satellite cells in muscle growth during development is well documented, but the involvement of these cells in muscle repair after contusion is less well known. In the present study, we investigated the time‐course of satellite cell activity (from 3 h to 7 days) after contusion of rat gastrocnemius muscle using specific molecular markers for immunofluorescence and real‐time polymerase chain reaction (PCR). 2. Inflammation of the injured muscle occurred within 6 h, followed by disintegration of the damaged myofibres within 12 h. Newly formed myofibres appeared by Day 7. 3. The number of MyoD‐positive nuclei (activated satellite cells) in the injured muscle was significantly increased by 6 h, reaching a maximum by 12 h after contusion. However, the number of MyoD‐positive nuclei decreased towards control levels by Day 7. Changes in the number of bromodeoxyuridine‐labelled nuclei (proliferating satellite cells) paralleled the changes seen in the number of MyoD‐positive nuclei. Conversely, expression of myogenin protein was not apparent until Day 3 and increased further by Day 7. Colabelling of MyoD and myogenin was seen in only a few cells. 4. The time‐course of MyoD mRNA expression corresponded with MyoD protein expression. However, there were two peaks in myogenin mRNA expression: 6 h and Day 7 after contusion. The second peak coincided with upregulation of myostatin mRNA levels. 5. The results of the present study suggest that contusion activates a homogeneous population of satellite cells to proliferate within 3 days, followed by differentiation to form new myofibres. The latter may be regulated, in part, by myostatin.     

Development of therapies against neuromuscular diseases causing muscle atrophy]

Skeletal muscles become atrophied by muscular disorders such as muscular dystrophy, wasting and even aging. In addition to muscle atrophy, progressive muscle damage, inflammation and replacement of muscle fibers with fibrous and fatty tissues are observed in muscular dystrophy. Neuronal innervation is required for skeletal muscle, and muscles become atrophic when motor neurons are affected by neurodegenerative disorders such as amyotrophic lateral sclerosis. Restoring muscle mass and function lost by diseases such as muscular dystrophy and neurodegenerative disorders is important. There are three rational therapies for muscular dystrophy and related diseases: gene therapy, cell therapy and drug therapy. Gene therapies to replace the defective genes have been tried with various degrees of effectiveness. Multiple myogenic stem cells including satellite cells, bone marrow cells, muscle side population cells, muscle-derived stem cells and mesoangioblast have been characterized. Cell therapies using these stem cells are one of the promising therapies for neuromuscular diseases causing muscle atrophy. As pharmacological drug therapies, increasing skeletal muscle mass by myostatin inhibition is quite promising and will be applied clinically in the near future.     

A novel coenzyme A:diacylglycerol acyltransferase 1 inhibitor stimulates lipid metabolism in muscle and lowers weight in animal models of obesity

Obesity is characterized by the accumulation of triacylglycerol in adipocytes. Coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final and only committed step in triacylglycerol synthesis. In this report, we describe the pharmacological effects of a novel selective DGAT1 inhibitor, Compound-A. This compound inhibited triacylglycerol synthesis in both adipocytes and skeletal myotubes, and increased fatty acid oxidation in skeletal myotubes at 1 μM. The repeated administration of Compound-A to diet-induced obese C57BL/6J and genetically obese KKA(y) mice (3-30 mg/kg for 3-4 weeks) significantly decreased the visceral fat pad weights and the hepatic lipid contents compared to controls without affecting food intake. In addition, fatty acid oxidation in skeletal muscle tissues was increased by the treatment of Compound-A in both mice strains. This is the first report demonstrating that a small synthetic DGAT1 inhibitor increases fatty acid oxidation in skeletal muscle in vitro and ex vivo. These results suggest that DGAT1 inhibition is a promising therapeutic approach for the treatment of obesity and lipid abnormalities such as hepatic steatosis.     

Stem cell ageing and apoptosis

Ageing has been defined as the process of deterioration of many body functions over the lifespan of an individual. In spite of the number of different theories about ageing, there is a general consensus in identifying ageing effects in a reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancer. In recent years the stem cell theory of ageing has gained much attention. Adult stem cells residing in mammalian tissues are essential for tissue homeostasis and repair throughout adult life. With advancing age, the highly regulated molecular signalling necessary to ensure proper cellular, tissue, and organ homeostasis loses coordination and leads, as a consequence, to a compromised potential of regeneration and repair of damaged cells and tissues. Although a complete comprehension of the molecular mechanisms involved in stem cell ageing and apoptosis is far to be reached, recent studies are beginning to unravel the processes involved in stem cell ageing, particularly in adult skeletal muscle stem cells, namely satellite cells. Thus, the focus of this review is to analyse the relationship between stem cell ageing and apoptosis with a peculiar attention to human satellite cells as compared to haematopoietic stem cells. Undoubtedly, the knowledge of age-related changes of stem cells will help in understanding the ageing process itself and will provide novel therapeutic challenges for improved tissue regeneration.     

Cultured myotubes from skeletal muscle of adult rats

Summary Mononucleated myogenic cells (satellite cells) were isolated from skeletal muscle of adult rats and grown in culture. These cells replicated and, beginning with the 6th day in culture, they fused and differentiated into multinucleated myotubes, which accumulated creatine kinase and developed cross striation and spontaneous contractions. The differentiation of the excitable membrane and the action of sea anemone toxin ATX II were investigated with microelectrode techniques. Mature myotubes reached a stable membrane potential of –47.3 mV (±6.5 mV) with the IIth day in culture. Action potentials could be generated in all myotubes. During maturation they became faster (increasing rate of rise) and shorter in duration. In spontaneously contracting myotubes spontaneous action potentials were recorded, which were often associated with subthreshold oscillations of membrane potential. ATX II reduced the membrane potential and prolonged the action potential duration with the lowest effective concentrations being 1 nmol/l and 0.5 nmol/l, respectively. Furthermore, ATX II induced electrical activity in quiescent myotubes. After fusion the development of the membrane electrical properties of satellite cell derived muscle cells followed essentially the same pattern as in primary cultures of embryonic myotubes. Electrophysiologically and with respect to their sensitivity to ATX II the mature myotubes resemble denervated muscle fibres. Send offprint requests to I. Tesseraux at the above address     

Ameliorating effect of hypothalamic brain-derived neurotrophic factor against impaired glucose metabolism after cerebral ischemic stress in mice

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has potent neuroprotective effects against brain injury. We recently reported that glucose intolerance/hyperglycemia could be induced by ischemic stress (i.e., post-ischemic glucose intolerance) following ischemic neuronal damage. Therefore, the aim of this study was to determine the effects of BDNF on the development of post-ischemic glucose intolerance and ischemic neuronal damage. Male ddY mice were subjected to middle cerebral artery occlusion (MCAO) for 2 h. On day 1, the expression levels of BDNF were significantly decreased in the cortex, hypothalamus, liver, skeletal muscle, and pancreas. The expression levels of tyrosine kinase B receptor, a BDNF receptor, decreased in the hypothalamus and liver and increased in the skeletal muscle and pancreas, but remained unchanged in the cortex. Intrahypothalamic administration of BDNF (50 ng/mouse) suppressed the development of post-ischemic glucose intolerance on day 1 and neuronal damage on day 3 after MCAO. In the liver and skeletal muscle, the expression levels of insulin receptors decreased, while gluconeogenic enzyme levels increased on day 1 after MCAO. These changes completely recovered to normal levels in the presence of BDNF. These results indicate that regulation of post-ischemic glucose intolerance by BDNF may suppress ischemic neuronal damage.     

2型糖尿病小鼠骨骼肌细胞受体后胰岛素信号转导蛋白的下降调节

目的研究2型糖尿病小鼠骨骼肌细胞受体后胰岛素信号转导蛋白:胰岛素受体底物-1(IRS-1)、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(PKB,Akt)的蛋白表达和磷酸化情况。方法雌性C57BL/6J小鼠予高脂、高糖膳食,制成2型糖尿病动物模型,提取完整的骨骼肌用胰岛素刺激2、15和30min。Westernblot技术检测IRS-1、PI3-Kp85α、PKB的蛋白水平,免疫沉淀技术检测IRS-1酪氨酸磷酸化水平。结果对照组、2型糖尿病模型组骨骼肌细胞的信号转导蛋白IRS-1、PKB未发现数量上的不同,2型糖尿病模型组的PI3Kp85的蛋白比对照组明显减少(P<0·05),在基础状态下,对照组和2型糖尿病模型组的IRS-1、PKB磷酸化水平相似,但2型糖尿病模型组胰岛素刺激后的这些蛋白磷酸化反应曲线上升幅度较之对照组明显降低。结论2型糖尿病小鼠骨骼肌细胞存在受体后胰岛素信号传导蛋白的下降调节。     

Reduction of RhoC is involved in the decreased migration of neural stem/precursor cells

OBJECTIVE Alzheimer′s disease(AD) is mainly characterized by a progressive loss of neurons and the deposition of beta-amyloid peptides(Aβ).It was demonstrated that transplanted and endogenous neural stem cells or neural precursor cells can survive,migrate,and differentiate into neurons.Previously we found that Aβ42 could disturb the migratory ability of neural stem/precursor cells.We therefore hypothesized that Aβ42 may affect some important proteins through Rho pathway and result in the decreased migration of neural stem/precursor cells.METHODS AND RESULTS We applied siRNA technology to knock down the expression of RhoC in neural stem/precursor cells and found that the expression of RhoC was down-regulated;Trans well assay was used to measure the migratory ability of the neural stem/precursor cells and the result showed that the migratory ability was disturbed when the expression of RhoC was down-regulated;WRW4 was used for inhibiting the effects of the Aβ42 through the FPRL1.CONCLUSION The reduction of RhoC was involved in the decreased migration of neural stem cells.