Effects of streptozotocin-induced diabetes and physical training on gene expression of titin-based stretch-sensing complexes in mouse striated muscle

In striated muscle, a sarcomeric noncontractile protein, titin, is proposed to form the backbone of the stress- and strain-sensing structures. We investigated the effects of diabetes, physical training, and their combination on the gene expression of proteins of putative titin stretch-sensing complexes in skeletal and cardiac muscle. Mice were divided into control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed for 1, 3, or 5 wk of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 h after the last training session. Gene expression of calf muscles (soleus, gastrocnemius, and plantaris) and cardiac muscle were analyzed using microarray and quantitative PCR. Diabetes induced changes in mRNA expression of the proteins of titin stretch-sensing complexes in Z-disc (MLP, myostatin), I-band (CARP, Ankrd2), and M-line (titin kinase signaling). Training alleviated diabetes-induced changes in most affected mRNA levels in skeletal muscle but only one change in cardiac muscle. In conclusion, we showed diabetes-induced changes in mRNA levels of several fiber-type-biased proteins (MLP, myostatin, Ankrd2) in skeletal muscle. These results are consistent with previous observations of diabetes-induced atrophy leading to slower fiber type composition. The ability of exercise to alleviate diabetes-induced changes may indicate slower transition of fiber type.     

Changes in the expression of small intestine extracellular matrix proteins in streptozotocin-induced diabetic rats

Diabetes mellitus is characterized by anatomical and functional alterations of the intestinal tract. However, the aetiology of these disturbances remains unclear. The aim of the present work was to investigate the effects of diabetes on the expression of laminin-1 and fibronectin in the small intestine of Streptozotocin (STZ)-induced diabetic rats. The Western immunoblotting of the extracts from the small intestine revealed that experimental diabetes resulted in a marked increase in the intensity of the bands corresponding to laminin-1 and fibronectin. Immunohistochemical studies demonstrated a strong labelling to these two extracellular matrix (ECM) proteins in the small intestine of diabetic rats, mainly localized in the smooth muscle layer. These results occur together with a thickening of the basement membrane (BM) of the smooth muscle cells, demonstrated by transmission electron microscopy (TEM). We propose that the accumulation of ECM proteins in the smooth muscle layer may be an effect mediated by hyperglycaemia, since insulin treatment of diabetic rats reversed this accumulation. These results could provide information on the potential role of the ECM in the intestine, an organ which is known to exhibit important alterations in diabetes.     

Effect of resistance exercise training on expression of Hsp70 and inflammatory cytokines in skeletal muscle and adipose tissue of STZ-induced diabetic rats

Impairment of adipose tissue and skeletal muscles accrued following type 1 diabetes is associated with protein misfolding and loss of adipose mass and skeletal muscle atrophy. Resistance training can maintain muscle mass by changing both inflammatory cytokines and stress factors in adipose tissue and skeletal muscle. The purpose of this study was to determine the effects of a 5-week ladder climbing resistance training program on the expression of Hsp70 and inflammatory cytokines in adipose tissue and fast-twitch flexor hallucis longus (FHL) and slow-twitch soleus muscles in healthy and streptozotocin-induced diabetic rats. Induction of diabetes reduced body mass, while resistance training preserved FHL muscle weight in diabetic rats without any changes in body mass. Diabetes increased Hsp70 protein content in skeletal muscles, adipose tissue, and serum. Hsp70 protein levels were decreased in normal and diabetic rats by resistance training in the FHL, but not soleus muscle. Furthermore, resistance training decreased inflammatory cytokines in FHL skeletal muscle. On the other hand, Hsp70 and inflammatory cytokine protein levels were increased by training in adipose tissue. Also, significant positive correlations between inflammatory cytokines in adipose tissue and skeletal muscles with Hsp70 protein levels were observed. In conclusion, we found that in diabetic rats, resistance training decreased inflammatory cytokines and Hsp70 protein levels in fast skeletal muscle, increased adipose tissue inflammatory cytokines and Hsp70, and preserved FHL muscle mass. These results suggest that resistance training can maintain skeletal muscle mass in diabetes by changing inflammatory cytokines and stress factors such as Hsp70 in skeletal muscle and adipose tissue.     

Physical training reverses defect in 3-ketoacid CoA-transferase activity in skeletal muscle of diabetic rats

To investigate one potential mechanism whereby physical training improves the plasma concentration of ketone bodies in experimental diabetes mellitus, we measured the activity of 3-ketoacid CoA-transferase, the key enzyme in the peripheral utilization of ketone bodies. Diabetes was induced with streptozotocin (50 mg/kg) and training carried out on a treadmill with a progressive 10-wk program. Diabetes resulted in an increase (P < 0.001) in plasma concentration of beta-hydroxybutyric acid in sedentary rats, which was partly reversed by training (P < 0.001). Diabetes was also associated with a decreased activity of 3-ketoacid CoA-transferase in gastrocnemius muscle. When expressed per total gastrocnemius, training increased the activity of 3-ketoacid CoA-transferase by 66% in nondiabetic rats (P < 0.001) and by 150% in diabetic rats (P < 0.001), the decrease present in diabetic rats being fully reversed by training. Simple linear regression between the log of 3-ketoacid CoA-transferase activity and the log of plasma beta-hydroxybutyric acid levels showed a statistically significant (r = 0.563, P < 0.001) negative correlation. The beneficial effects of training on plasma ketone bodies in diabetic rats are probably explained, at least in part, by an increase in ketone body utilization, mediated by an increase in skeletal muscle 3-ketoacid CoA-transferase activity.     

Effects of acute exercise, exercise training, and diabetes on the expression of lymphangiogenic growth factors and lymphatic vessels in skeletal muscle

Blood and lymphatic vessels together form the circulatory system, allowing the passage of fluids and molecules within the body. Recently we showed that lymphatic capillaries are also found in the capillary bed of skeletal muscle. Exercise is known to induce angiogenesis in skeletal muscle, but it is not known whether exercise has effects on lymphangiogenesis or lymphangiogenic growth factors. We studied lymphatic vessel density and expression of the main lymphangiogenic growth factors VEGF-C and VEGF-D and their receptor VEGFR-3 in response to acute running exercise and endurance exercise training in the skeletal muscle of healthy and diabetic mice. VEGF-C mRNA expression increased after the acute exercise bout (P < 0.05) in healthy muscles, but there was no change in diabetic muscles. VEGF-C levels were not changed either in healthy or in diabetic muscle after the exercise training. Neither acute exercise nor exercise training had an effect on the mRNA expression of VEGF-D or VEGFR-3 in healthy or diabetic muscles. Lymphatic vessel density was similar in sedentary and trained mice and was >10-fold smaller than blood capillary density. Diabetes increased the mRNA expression of VEGF-D (P < 0.01). Increased immunohistochemical staining of VEGF-D was found in degenerative muscle fibers in the diabetic mice. In conclusion, the results suggest that acute exercise or exercise training does not significantly affect lymphangiogenesis in skeletal muscle. Diabetes increased the expression of VEGF-D in skeletal muscle, and this increase may be related to muscle fiber damage.     

Effect of exercise training on glucose homeostasis in normal and insulin-deficient diabetic rats

The effect of 8-wk of treadmill training on plasma glucose, insulin, and lipid concentrations, oral glucose tolerance, and glucose uptake in the perfused hindquarter of normal and streptozocin-treated, diabetic Sprague-Dawley rats was studied. Diabetic rats with initial plasma glucose concentrations of 200-450 mg/dl and control rats were divided into trained and sedentary subgroups. Training resulted in lower plasma free fatty acid concentrations and increased triceps muscle citrate synthase activity in both the control and diabetic rats; triglyceride concentrations were lowered by training only in the diabetic animals. Oral glucose tolerance and both basal and insulin-stimulated glucose uptake in hindquarter skeletal muscle were impaired in the diabetic rats, and plasma glucose concentrations (measured weekly) gradually increased during the experiment. Training did not improve the hyperglycemia, impaired glucose tolerance, or decreased skeletal muscle glucose uptake in the diabetic rats, nor did it alter these parameters in the normal control animals. In considering our results and those of previous studies in diabetic rats, we propose that exercise training may improve glucose homeostasis in animals with milder degrees of diabetes but fails to cause improvement in the more severely insulin-deficient, diabetic rat.     

The ubiquitin-proteasome proteolytic pathway in heart vs skeletal muscle: effects of acute diabetes

The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P 相似文献   

Tocotrienol-rich fraction supplementation reduces hyperglycemia-induced skeletal muscle damage through regulation of insulin signaling and oxidative stress in type 2 diabetic mice

Chronic hyperglycemia induces impairment of muscle growth and development of diabetes mellitus (DM). Since skeletal muscle is the major site for disposal of ingested glucose, impaired glucose metabolism causes imbalance between protein synthesis and degradation which adversely affects physical mobility.In this study, we investigated the effect of tocotrienol-rich fraction (TRF) supplementation on skeletal muscle damage in diabetic mice. Diabetes was induced by a high-fat diet with streptozotocin (STZ) injection (100 mg/kg) in male C57BL/6J mice. After diabetes was induced (fasting blood glucose levels≥250 mg/dl), normal control (CON) and diabetic control (DMC) groups were administrated with olive oil, while TRF treatment groups were administrated with TRF (dissolved in olive oil) at low dose (100 mg/kg BW, LT) or high dose (300 mg/kg BW, HT) by oral gavage for 12 weeks.TRF supplementation ameliorated muscle atrophy, plasma insulin concentration and homeostatic model assessment estimated insulin resistance in diabetic mice. Moreover, TRF treatment up-regulated IRS-1 and Akt levels accompanied by increased translocation of GLUT4. Furthermore, TRF increased mitochondrial biogenesis by activating SIRT1, SIRT3 and AMPK in diabetic skeletal muscle. These changes were in part mechanistically explained by reduced levels of skeletal muscle proteins related to oxidative stress (4-hydroxynonenal, protein carbonyls, Nrf2 and HO-1), inflammation (NFkB, MCP-1, IL-6 and TNF-α), and apoptosis (Bax, Bcl₂ and caspase-3) in diabetic mice. Taken together, these results suggest that TRF might be useful as a beneficial nutraceutical to prevent skeletal muscle atrophy associated with diabetes by regulating insulin signaling via AMPK/SIRT1/PGC1α pathways in type 2 diabetic mice.     

The effects of neuropeptide Y on skeletal muscle contractile properties in streptozotocin diabetic rats

Diabetes induces changes in the structural, biochemical, electrical, and contractile properties of skeletal muscles. Neuropeptide Y (NPY) administered locally can induce angiogenesis in a rat ischemic limb model and restore the contractile function of the ischemic muscle. The effects of NPY on the contractile characteristics of limb skeletal muscles were examined in streptozotocin-induced diabetic rats. Rats were treated with sham pellets (control groups) or NPY-containing pellets (1 mg of NPY/pellet, 14 days releasing time) administered locally to the rat hind limb 2 months after induction of diabetes. Contractile properties and fatigability of the slow-twitch soleus and fast-twitch gastrocnemius medials muscle were compared in control (sham), control NPY, diabetic (sham), and diabetic NPY groups. In order to induce fatigue trains of repetitive tetanic stimulation were used (600 ms/1 s simulation-rest cycle per train, 112 trains at an 85-Hz fusion frequency). Two months of untreated diabetes significantly prolonged soleus contraction and slowed its relaxation, but had minimal effects on soleus tension. NPY ameliorated the diabetic effects on soleus speed-related contractile properties, restoring its contraction and relaxation times. Diabetes significantly reduced gastrocnemius medials tetanic tension, leaving its contractile characteristics mostly unaffected. NPY partially restored gastrocnemius tetanic tension production capacity. Diabetes significantly increased fatigability of both muscles, which was partially restored by NPY, as evidenced by restored endurance of soleus muscle. The results suggest that NPY administered locally tends to normalize muscle performance and improve fatigue resistance of skeletal muscles in streptozotocin diabetes. Further examination is needed to establish the mechanisms of local NPY action on muscle contractile properties in streptozotocin-induced diabetes.     

Effect of short- and long-term diabetes on carnitine and myo-inositol in rats.

1. The effect of short- (2 wk) and long-term (20 wk) streptozotocin diabetes was studied on urine, blood, liver, heart, brain, skeletal muscle, pancreas and kidney concentrations of acid-soluble carnitine and free myo-inositol. 2. Short-term diabetic rats excreted significantly higher concentrations of carnitine as well as myoinositol than normal rats. Blood carnitine and myo-inositol were not different between normal and diabetic rats. Diabetes caused a decrease in liver, brain and pancreatic carnitine, but not in heart, skeletal muscle and kidney. Myo-inositol concentration was decreased in liver, heart and kidney but not in brain, pancreas and skeletal muscle. 3. Long-term diabetic rats had higher urinary excretions of both carnitine and myo-inositol. Blood carnitine did not change; however, myo-inositol was higher in diabetic than in normal rats. Diabetes caused a significant increase in liver and a decrease in heart, brain, skeletal muscle and pancreatic content of carnitine; no difference in kidney carnitine was noted. Myo-inositol content was elevated only in liver of diabetic rats. 4. We suggest that carnitine and myo-inositol concentrations are influenced both by short- and long-term diabetes through changes in tissue metabolism.     

Defective collagen VI α6 chain expression in the skeletal muscle of patients with collagen VI-related myopathies

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the “classical” α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.     

有氧运动对2型糖尿病大鼠骨骼肌ERK1/2活性的影响

目的：观察有氧运动对2型糖尿病大鼠骨骼肌细胞外信号调节激酶（ERK1/2）活性的影响,探讨有氧运动对2型糖尿病的预防和调控机制。方法：将75只SD大鼠随机分为正常对照组（CON）、糖尿病对照1组（DC1）、糖尿病运动1组（DE1）、糖尿病对照2组（DC2）、糖尿病运动2组（DE2）5组（n=15）。正常对照组用普通饲料喂养,糖尿病组用高脂高糖配方饲料喂养。经过8周高脂高糖喂养后,糖尿病2组大鼠腹腔内注射链脲佐菌素（STZ）,诱发2型糖尿病;糖尿病运动1组游泳的最后1周初和糖尿病对照1组同时注射STZ,注射剂量为35 mg/kg,3 d后尾部取血测血糖≥ 16.7 mmol/L为造模成功。运动干预8周后,测定大鼠血清胰岛素、骨骼肌中ERK1/2蛋白表达等指标。结果：①与正常对照组比较,糖尿病各对照组血液中总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白（LDL-C）、游离脂肪酸（FFA）显著升高（P<0.05,P<0.01）,空腹血糖（FBG）、胰岛素（FIN）含量和胰岛素抵抗指数（HOMA-IR）显著升高（P<0.01）,ERK1/2磷酸化的蛋白表达显著下降（P<0.05）,糖尿病对照2组ERK1/2蛋白含量显著下降（P<0.05）;②8周游泳运动后,与糖尿病对照组比较,糖尿病运动组血液中TC、TG、FFA、LDL-C显著下降（P<0.05）,FBG、FIN、HOMA-IR显著下降（P<0.05,P<0.01）,ERK1/2磷酸化蛋白表达显著升高（P<0.05）。结论：长时间有氧运动,增加了骨骼肌ERK1/2磷酸化水平,改善了2型糖尿病大鼠胰岛素抵抗的状况,降低血糖。这可能是改善糖代谢紊乱,提高胰岛素敏感性的机制之一。     

Exercise-induced changes of MCT1 in cardiac and skeletal muscles of diabetic rats induced by high-fat diet and STZ

We hypothesized that a part of therapeutic effects of endurance training on insulin resistance is mediated by increase in cardiac and skeletal muscle mitochondrial lactate transporter, monocarboxylate transporter 1 (MCT1). Therefore, we examined the effect of 7 weeks endurance training on the mRNA and protein expression of MCT1 and MCT4 and their chaperon, CD147, on both sarcolemmal and mitochondrial membrane, separately, in healthy and type 2 diabetic rats. Diabetes was induced by injection of low dose of streptozotocin and feeding with high-fat diet. Insulin resistance was confirmed by homeostasis model assessment-estimated insulin resistance index and accuracy of two membranes separation was confirmed by negative control markers (glucose transporter 1 and cytochrome c oxidase. Real-time PCR and western blotting were used for mRNA and protein expression, respectively. Diabetes dramatically reduced MCT1 and MCT4 mRNA and their expression on sarcolemmal membrane whereas the reduction in MCT1 expression was less in mitochondrial membrane. Training increased the MCT1 mRNA and protein expression in both membranes and decreased insulin resistance as an adaptive consequence. In both tissues increase in CD147 mRNA was only parallel to MCT1 expression. The response of MCT1 on sarcolemmal and mitochondrial membranes was different between cardiac and skeletal muscles which indicate that intracellular lactate kinetic is tissue specific that allows a tissue to coordinate whole organism metabolism.     

超声微泡介导hVEGF165 基因靶向转染糖尿病鼠缺血骨骼肌

目的：探讨超声介导微泡破裂法促进血管内皮生长因子（VEGF）基因在糖尿病鼠缺血骨骼肌内转染的作用,评估其转染效 率和安全性。方法：建立糖尿病鼠缺血骨骼肌动物模型,以绿色荧光蛋白基因为报告基因, 观察接受超声及微泡治疗组hVEGF165 基因在糖尿病鼠缺血骨骼肌内表达,并与对照组相比。同时取糖尿病鼠缺血骨骼肌进行HE染色行组织学检查。结果：在超声介导 微泡破裂组内,hVEGF165 基因表达明显增强(42.87± 5.12),与单纯接受质粒治疗组(5.02± 1.21)和接受质粒和超声治疗组（8.16± 2.43）相比,差异具有统计学意义（P<0.001）,HE 切片未发现肌组织结构的改变。结论：超声介导微泡破裂法能有效促进外源基因 在糖尿病鼠缺血骨骼肌中表达, 为糖尿病周围血管疾病的基因治疗提供了实验依据。     

The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats

Glomerulosclerosis and tubulointerstitial fibrosis are the main structural changes found in the later stages of diabetic nephropathy, which is clinically characterized by proteinuria, and progressive renal insufficiency. Heat shock protein (HSP) 47, a collagen-binding stress protein, has a specific role in the intracellular processing of procollagen molecules during collagen synthesis. It is implicated in the pathogenesis of various fibrotic diseases. However, the expression and significance of HSP47 in acute and chronic phases of diabetic nephropathy is not yet known. In this study, we studied the expression of HSP47 in the kidneys obtained from streptozotocin-induced diabetic rats, in both short- and long-term diabetes. To determine the renal expression of HSP47, and collagens (type III and IV) in acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) diabetes, we have performed a time-course study using streptozotocin-induced diabetic rats. The expression pattern of alpha-smooth muscle actin (to identify mesangial cell damage), vimentin (to identify tubular epithelial cell damage), and desmin (to identify glomerular epithelial cell damage) was also determined in kidneys of these diabetic rats. Antibodies specific for HSP47, type III and type IV collagens, alpha-smooth muscle actin, vimentin, and desmin were used to assess the relative expression of their proteins in paraffin-embedded kidney sections by immunohistochemistry. Compared to control rat kidneys, no significant changes in the expression of HSP47 was found in the kidneys of acute diabetic rats. However a significant increase in the expression of HSP47 was noted in the kidneys of chronic diabetic rats; increased expression of HSP47 correlated with an increased renal deposition of types III and IV collagens. Similarly, compared to kidneys of control and acute diabetic rats, an increased expression of alpha-smooth muscle actin (in mesangial cells), vimentin (in tubular epithelial cells), and desmin (in glomerular epithelial cells) was detected in the kidneys of chronic diabetic rats; by dual immunostaining, these phenotypically-altered renal cells in kidneys of chronic diabetic rats were found to be HSP47-producing cells. Importantly, HSP47 up-regulation coincided with the initiation and progression of renal fibrosis, as determined by the expression and deposition of collagens. Our results strongly support a pathological role for HSP47 in the later stages (sclerotic phase) of streptozotocin-induced diabetic nephropathy, which is associated with glomerulosclerosis and tubulointerstitial fibrosis.