Increased workload fully activates the blunted IRS-1/PI3-kinase/Akt signaling pathway in atrophied uremic muscle

Muscle wasting in chronic renal failure is associated with increased morbidity and mortality; however, resistance exercise is effective at increasing muscle mass while improving muscle strength and function. To study the mechanism by which this occurs, we compared uremic and control rats where work overload was surgically induced unilaterally in the plantaris muscle. We found that work overload increases muscle insulin-like growth factor-1 and mechano-growth factor expression. This in addition to direct mechanical activation of signaling was likely the cause of the observed increased in the protein levels and phosphorylation of the mediators of these growth factors, the insulin receptor substrate-1/phosphoinositide 3-kinase/Akt pathway. The mechanical enhancement of signal transduction appeared to be mediated in part by increased signal protein levels and decreased SOCS2 mRNA expression (suppressor of cytokine signaling-2). Despite impaired basal signaling, the work-induced signaling response was similar to that observed in nonuremic rats and produced changes consistent with decreased muscle protein degradation, increased protein synthetic capacity, and an increased number of multinucleated muscle cells. Our studies suggest that these work-induced changes account for the improved uremic muscle mass reaching levels comparable to those seen in normal rats.     

Modulation of melanocortin signaling ameliorates uremic cachexia

Insulin-like growth factor (IGF)-I increases muscle mass while myostatin inhibits its development. Muscle wasting is common in patients with uremic cachexia and may be due to imbalance of this regulation. We had proposed a central mechanism involving leptin and melanocortin signaling in the pathogenesis of uremic cachexia since agouti-related peptide (AgRP), a melanocortin-4 receptor antagonist, reduced uremic cachexia. Here we found that injection of AgRP into the cerebral ventricles resulted in a gain of body mass and improved metabolic rate regulation in a mouse model of uremic cachexia. These salutary effects occurred independent of increased protein and calorie intake. Myostatin mRNA and protein concentrations were increased while those of IGF-I were decreased in the skeletal muscle of uremic mice. AgRP treatment partially corrected these uremia-induced changes. Suppressor of cytokine signaling-2 gene expression (SOCS2) was significantly increased in uremic animals and AgRP reduced this expression. We suggest that AgRP improves uremic cachexia and muscle wasting by a peripheral mechanism involving the balance between myostatin and IGF-I.     

Apoptosis and myostatin mRNA are upregulated in the skeletal muscle of patients with chronic kidney disease

Apoptosis and myostatin are major mediators of muscle atrophy and might therefore be involved in the wasting of uremia. To examine whether they are expressed in the skeletal muscle of patients with chronic kidney disease (CKD), we measured muscle apoptosis and myostatin mRNA and their related intracellular signal pathways in rectus abdominis biopsies obtained from 22 consecutive patients with stage 5 CKD scheduled for peritoneal dialysis. Apoptotic loss of myonuclei, determined by anti-single-stranded DNA antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays, was significantly increased three to fivefold, respectively. Additionally, myostatin and interleukin (IL)-6 gene expressions were significantly upregulated, whereas insulin-like growth factor-I mRNA was significantly lower than in controls. Phosphorylated JNK (c-Jun amino-terminal kinase) and its downstream effector, phospho-c-Jun, were significantly upregulated, whereas phospho-Akt was markedly downregulated. Multivariate analysis models showed that phospho-Akt and IL-6 contributed individually and significantly to the prediction of apoptosis and myostatin gene expression, respectively. Thus, our study found activation of multiple pathways that promote muscle atrophy in the skeletal muscle of patients with CKD. These pathways appear to be associated with different intracellular signals, and are likely differently regulated in patients with CKD.     

Effects of growth hormone treatment on the pituitary expression of GHRH receptor mRNA in uremic rats

BACKGROUND: A decreased ability of pituitary cells to secrete growth hormone (GH) in response to growth hormone releasing hormone (GHRH) stimulation has been shown in young uremic rats. The aim of the current study was to examine the effect of uremia and GH treatment on pituitary GHRH receptor expression. METHODS: Pituitary GHRH receptor mRNA levels were analyzed by RNase protection assay in young female rats made uremic by subtotal nephrectomy, either untreated (UREM) or treated with 10 IU/kg/day of GH (UREM-GH), and normal renal function animals fed ad libitum (SAL) or pair-fed with the UREM group (SPF). Rats were sacrificed 14 days after the second stage nephrectomy. RESULTS: Renal failure was confirmed by concentrations (X +/- SEM) of serum urea nitrogen (mmol/L) and creatinine (micromol/L) in UREM (20 +/- 1 and 89.4 +/- 4.5) and UREM-GH (16 +/- 1 and 91.4 +/- 6.9) that were much higher (P < 0.001) than those of sham animals (SAL, 3 +/- 0 and 26.5 +/- 2.2; SPF, 4 +/- 0 and 26.5 +/- 2.1). UREM rats became growth retarded as shown by a daily longitudinal tibia growth rate below (P < 0.05) that observed in SAL animals (156 +/- 3 vs. 220 +/- 5 microm/day). GH treatment resulted in significant growth rate acceleration (213 +/- 6 microm/day). GHRH receptor mRNA levels were no different among the SAL (0.43 +/- 0.03), SPF (0.43 +/- 0.08) and UREM (0.44 +/- 0.04) groups, whereas UREM-GH rats had significantly higher values (0.72 +/- 0.07). CONCLUSIONS: The status of pituitary GHRH receptor is not modified by nutritional deficit or by severe uremia causing growth retardation. By contrast, the growth promoting effect of GH administration is associated with stimulated GHRH receptor gene expression.     

Effect of exercise on mRNA levels for growth factors in skeletal muscle of hemodialysis patients.

OBJECTIVES: Muscle mass and muscle mRNA levels for certain growth factors are reduced in maintenance hemodialysis (MHD) patients. This study tested the hypothesis that in MHD patients endurance exercise training (EET) increases mRNA levels for insulin-like growth factors and reduces myostatin mRNA. DESIGN: Biopsies of the right vastus lateralis muscle were performed before and at the end of 8.9 +/- 0.9 (SEM) weeks of EET in MHD patients. Muscle tissue was analyzed histologically by electron microscopy and for fiber cross-sectional area, and, in 8 pairs of biopsies, muscle was examined for mRNA levels for the following proteins: myostatin, insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF binding proteins (IGFBPs)-1, -2, -3, -4, and -5, and IGF-binding protein-related protein-1 (IGFBP-rP1). SETTING: Outpatient MHD centers. PATIENTS: This was a pilot study conducted in sedentary clinically stable MHD patients undergoing EET with no control group. INTERVENTION: EET that was carefully supervised by exercise trainers. MAIN OUTCOME MEASURE: Skeletal muscle mRNA levels, especially myostatin mRNA. RESULTS: With EET, skeletal muscle myostatin mRNA decreased by 51%, mRNA levels increased significantly for IGF-IR (by 41%), IGFBP-2, -4, and -5, and IGFBP-rP1. IGF-I mRNA increased by 35%; this change was not significant. IGFBP-3 mRNA did not change, and IGFBP-1 mRNA was undetectable. There were mild to moderate alterations in skeletal muscle ultrastructure that did not change significantly with EET. Muscle fiber size, measured in 5 patients, did not change. CONCLUSION: In MHD patients who undergo approximately 9 weeks of EET, skeletal muscle mRNA for myostatin decreases and mRNA for IGF-IR, IGFBPs -2, -4, and -5 and IGFBP-rP1 increases. These changes may indicate mechanisms by which EET improves muscle exercise capacity in MHD patients.     

慢性压迫性脊髓损伤后大鼠运动功能及周围神经和骨骼肌中胰岛素样生长因子-1表达的变化

目的 观察慢性压迫性脊髓损伤后大鼠运动功能变化及周围神经和骨骼肌中胰岛素样生长因子-1(IGF-1)表达变化.方法 将50只Wistar雌性大鼠随机分为正常组、假手术组和慢性压迫组.慢性压迫组置入平头塑料螺钉对大鼠脊髓进行后路渐进性压迫,于2个月后分别压迫至20%、40%、60%左右程度.行神经功能观察;处死大鼠后取腓肠肌作为标本,分别进行IGF-1的免疫组织化学和原位杂交染色.结果 大鼠后肢瘫痪程度随压迫程度加重而加重,各组坐骨神经和骨骼肌中IGF-1蛋白和mRNA表达分别为:20%组(236.9±3.2)、(231.5±2.9)、(245.6±3.4)、(246.6±2.7);40%组(205.3±2.7)、(202.2±3.4)、(209.4±2.6)、(214.6±2.5);60%组(215.4±3.5)、(219.3±4.1)、(231.9±2.3)、(238.5±2.7).各压迫组坐骨神经及骨骼肌中IGF-1 mRNA和蛋白表达增加,与正常组比较差异有统计学意义(P<0.05).结论脊髓压迫性损伤可引起周围神经及骨骼肌中IGF-1表达增加,提示机体调动保护因素以减轻脊髓损伤并促进其再生.

Abstract：

Objective To observe the changes in motor function and expression of insulin-like growth factor-1 (IGF-1) of peripheral nerve and skeletal muscle in rats after chronic spinal cord compression. Methods A total of 50 Wistar rats were randomly divided into normal group (n=10), sham operation group (group A,n=10) and chronic compressive group (goup B,n=30). The rats in group B were given gradual compression on the posterior spinal cord using blunt plastics screw. Compression degree reached 20% (n=10), 40% (n=10) and 60% (n=10) respectively after two months. The rats were killed, and gastrocnemius muscle cells were removed. The expression levels of IGF-1 protein and mRNA in peripheral nerve and skeletal muscle were detected by immunohistochemistry and hybridization respectively after chronic compressive spinal cord injury. Results The rat hind limb paralysis was exacerbated with the increase of the compression. In the sciatic nerve and skeletal muscle, the expression levels of IGF-1 protein and mRNA were: (236.9±3.2), (231.5±2.9), (245.6±3.4), (246.6±2.7) in 20% group; (205.3±2.7), (202.2±3.4), (209.4±2.6), (214.6±2.5) in 40% group; (215.4±3.5), (219.3±4.1), (231.9±2.3), (238.5±2.7) in 60% group. The expression levels of IGF-1 protein and mRNA in peripheral nerve and skeletal muscle were significantly up-regulated after compression (P<0.05). Conclusion The results indicate that body transfers the protective factor to relieve injury of CNS.     

Effect of prolonged uremia on insulin metabolism by isolated liver and muscle.

As the prolonged metabolic clearance rate of insulin in chronic uremia cannot be entirely explained by impaired removal and degradation of insulin by the kidney, we set out to determine whether prolonged uremia depresses other major sites of insulin degradation. The study was conducted with livers and skeletal muscle obtained from normal control rats and uremic rats 4 weeks after 80% nephrectomy. Despite a significant difference between renal function in the control and uremic rats (BUN, 18 vs. 46 mg/dl), there was no significant difference in the clearance of insulin by isolated uremic or control livers perfused with a bloodless medium. Similarly, the 125I-insulin degrading activity of liver homogenates was not depressed by uremia. In contrast, binding and degradation by uremic liver cell membranes was significantly reduced to 58% and 85% of the controls, respectively. Degradation by homogenates of skeletal muscle and by intact epitrochlaris muscle was significantly less in uremics than in controls. These results indicate that chronic uremia depresses skeletal muscle insulin degradation but not hepatic insulin removal or degradation despite a decrease in insulin binding and degradation by liver plasma membranes. It thus appears that depression of insulin degradation by muscle may contribute to the prolonged insulin metabolic clearance rate seen in chronic uremia. Furthermore, it is possible that the impaired binding of insulin to liver membranes may play a role in the insulin resistance of uremia.     

Exercise training increases glucose transporter content in skeletal muscles more efficiently from aged obese rats than young lean rats.

Glucose uptake in rat skeletal muscles decreases with age and obesity, but increases with chronic exercise training. The purpose of our study was to determine whether the GLUT4 content in several skeletal muscles from 1-mo-old young, lean rats and 12-mo-old aged, obese rats alters with exercise training. For exercise, a treadmill run of approximately 1 km/day was made for 4 wk by both groups of rats. The concentration of GLUT4 per protein in membrane fraction from several skeletal muscles was measured by immunoblotting. The amount of GLUT4 in the gastrocnemius and white quadriceps from aged rats slightly but significantly decreased to 73% and 78% of that from young rats, respectively. However, no significant difference in GLUT4 amount in the soleus, plantaris, and red quadriceps was observed between young and aged rats. The exercise training resulted in a larger increase in the amount of GLUT4 in each muscle from aged rats than in muscles from young rats. In aged rats, GLUT4 amount increased significantly with exercise training by 30, 33, 41, and 27% in the soleus, plantaris, gastrocnemius, and red quadriceps, respectively, compared with the sedentary controls. However, in young rats, exercise-induced increase of GLUT4 amount was significant only in the plantaris, and the increase was 17%. In exercised aged, obese rats, decreases of body weight, plasma triglyceride levels, and plasma free fatty acid were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Therapies for Musculoskeletal Disease: Can we Treat Two Birds with One Stone?

Musculoskeletal diseases are highly prevalent with staggering annual health care costs across the globe. The combined wasting of muscle (sarcopenia) and bone (osteoporosis)—both in normal aging and pathologic states—can lead to vastly compounded risk for fracture in patients. Until now, our therapeutic approach to the prevention of such fractures has focused solely on bone, but our increasing understanding of the interconnected biology of muscle and bone has begun to shift our treatment paradigm for musculoskeletal disease. Targeting pathways that centrally regulate both bone and muscle (eg, GH/IGF-1, sex steroids, etc.) and newly emerging pathways that might facilitate communication between these 2 tissues (eg, activin/myostatin) might allow a greater therapeutic benefit and/or previously unanticipated means by which to treat these frail patients and prevent fracture. In this review, we will discuss a number of therapies currently under development that aim to treat musculoskeletal disease in precisely such a holistic fashion.     

Catabolic effects of ethanol in chronically uremic rats

Both ethanol consumption and uremia are considered to be associated with wasting, malnutrition and debilitation. The present study was designed to investigate as to whether ethanol exerts a stimulatory effect on the catabolic state of renal failure. Rats underwent 5/6-nephrectomy and were fed either with or without ethanol. The degree of uremia was comparable in both groups. Ethanol-fed uremic rats, however, displayed higher serum levels of urea (+ 103%) and glucose (+29%), as compared to uremic animals without alcohol. Subsequently, the urea N appearance was enhanced (+60%) in uremic rats with alcohol as compared to uremic animals without alcohol. In sham rats urea N appearance was also increased (+39%) following ethanol administration in comparison to sham-operated rats without alcohol, albeit to a lesser degree. Urinary Nt-methylhistidine excretion, an indicator of myofibrillar protein breakdown, was enhanced throughout the experiment in uremic rats receiving ethanol. Finally, ethanol caused higher urinary excretion rates of corticosterone in uremic animals as compared to uremic rats without ethanol. There was a significant correlation between urinary corticosterone excretion and both urea N appearance and urinary Nt-methylhistidine excretion. We conclude that ethanol consumption further aggravates the catabolic state of uremia and that this is mediated by an increment in glucocorticoid production.     

Growth hormone and growth hormone-related mRNA in uremic rats: effect of a growth hormone secretagogue

In experimental animals, the decreased growth during mild uremia is not accompanied by a loss in the capacity of the pituitary gland to secrete growth hormone (GH). With the development of orally administered GH "secretagogues" (GHS), it might be possible to stimulate growth during uremia without injections. This study was designed to determine the effects of the GHS, L-163,255. Uremia was induced by 5/6 nephrectomy (NX). GHS was given orally, 3 mg/kg, twice a week. Four groups of animals included: (1) sham-operated, (2) sham-operated, pair-fed, (3) uremic (NX), and (4) uremic, GHS-treated (NX+GHS). Blood sampling was conducted via intra-atrial catheters, and GH was quantitated. Pituitary GH mRNA was measured by Northern blot, and liver GH receptor and insulin-like growth factor-I mRNAs by RNAase protection. Untreated NX animals had a specific decrease in the "mass" of the GH pulses. A burst of GH was induced by GHS, but the pulsatile pattern of GH secretion over 6 h was not affected. An increase or a return to non-uremic levels of GH-related mRNAs occurred after GHS. Thus, GHS stimulated an acute burst of GH secretion and increased specific mRNAs encoding GH-related proteins in uremic animals.     

不同配方肠外营养对肝硬化大鼠生长激素/胰岛素样生长因子-1轴的影响

目的探讨不同配方肠外营养对肝硬化大鼠肝部分切除术后生长激素/胰岛素样生长因子-1轴的影响。方法正常大鼠作为对照组,肝硬化大鼠随机分为肝硬化术前组,肝硬化肝部分切除术后1 d组,术后行Novamin肠外营养5 d组,术后行Hepa肠外营养5 d组,各组n=6。测大鼠肝功能、血糖及血清GHI、GF-1I、GFBP-3水平,用RT-PCR法检测肝ALBmRNAI、GF-1 mRNA、IG-FBP-3mRNA的表达,肝组织行Ki67免疫组化染色。结果Hepa组肠外营养5 d后血清ALT、ALP、GH分别为(103±23)IU/L(、571±92)IU/L、(1.55±0.12)ng/ml,均比Novamin组明显降低,而血清IGF-1I、GFPB-3分别为(966.4±54.7)ng/ml(、6.9±0.2)ng/ml,均明显升高,肝ALBmRNA、IGF-1mRNAI、GFBP-3mRNA表达水平分别为(1.24±0.06)、(0.85±0.00)、(0.69±0.02),也明显升高,但肝Ki67指数(4.8%±0.3%vs 4.4%±0.4%)却无显著性差异。血清IGF-1I、GFPB-3与血清AST、ALT、ALP水平呈负相关,与血清ALB呈正相关。结论肝硬化大鼠不同配方肠外营养均可反映在生长激素/胰岛素样生长因子-1轴的变化,检测血清IGF-1,IGFBP-3水平有助于营养素的选择。     

Effects of exercise training on insulin-regulatable glucose-transporter protein levels in rat skeletal muscle

Exercise training has been shown to enhance the ability of insulin to stimulate glucose uptake in responsive tissues. The purpose of this study was to determine the effects of exercise training on the levels of the insulin-regulatable glucose transporter (IRGT) in rat skeletal muscle. After 6 wk of voluntary running in exercise-wheel cages, male Sprague-Dawley rats were rested for approximately 27 h and fasted overnight before removal of plantaris and soleus muscles. The concentration of glucose transporters per unit of muscle protein or DNA was quantitated by immunoblotting with an anti-IRGT polyclonal antibody raised against a synthetic peptide. The IRGT protein was increased by 60% (141 +/- 14 vs. 229 +/- 24 counts/min [cpm]/25 micrograms protein, P less than 0.01) in plantaris muscle from exercise-trained rats compared with controls. Total protein yield, DNA content, and 5'-nucleotidase activity were not different in plantaris muscle from control and exercise-trained rats. In contrast, there was no significant increase in the IRGT protein in soleus muscle after training when data were expressed per unit of muscle protein (292 +/- 22 vs. 346 +/- 16 cpm/25 micrograms protein). These data indicate that the increase in the IRGT in plantaris muscle is a selective response to exercise training that does not reflect an overall increase in muscle protein. The changes in IRGT for these muscles with exercise training parallel changes observed in insulin-mediated glucose uptake. We propose that this increase in the total number of glucose transporters may be a major component of the increase in insulin-mediated glucose uptake that is observed with exercise training.     

Persistent downregulation of calcium-sensing receptor mRNA in rat parathyroids when severe secondary hyperparathyroidism is reversed by an isogenic kidney transplantation

Experimental severe secondary hyperparathyroidism (HPT) is reversed within 1 wk after reversal of uremia by an isogenic kidney transplantation (KT) in the uremic rats. Abnormal parathyroid hormone (PTH) secretion in uremia is related to downregulation of CaR and vitamin D receptor (VDR) in the parathyroid glands (PG). The aim of this investigation was to examine the expression of CaR and VDR genes after reversal of uremia and HPT in KT rats. 5/6 nephrectomized rats were kept on a normal or high-phosphorus (hP) diet for 8 wk to induce severe HPT (n = 8 in each group). In another group of seven uremic hP rats, uremia was reversed by an isogenic KT and PG were harvested within 1 wk posttransplant. Plasma urea, creatinine, total calcium, phosphorus, and PTH levels were measured. Parathyroid CaR and VDR mRNA were measured by quantitative PCR. Uremic hP rats had significantly elevated levels of creatinine, urea, and phosphorus (P < 0.001) and developed significant hypocalcemia (plasma calcium 1.83 +/- 0.2 mmol/L; P < 0.001) compared with normal control rats. After KT, the levels were normalized from day 3 to 7: creatinine from 0.117 +/- 0.016 to 0.050 +/- 0.002 mmol/L; urea from 23 +/- 4 to 7 +/- 0.3 mmol/L; phosphorus from 3.9 +/- 0.6 to 1.5 +/- 0.06 mmol/L; calcium from 1.8 +/- 0.2 to 2.5 +/- 0.02 mmol/L. Plasma PTH levels fell from 849 +/- 224 to a normal level of 38 +/- 9 pg/ml (P < 0.01). In uremic rats on a standard diet, CaR mRNA was similar to that of normal control rats, whereas VDR mRNA was significantly decreased. In uremic rats kept on hP diet, CaR mRNA was significantly decreased to 26 +/- 7% of control rats (P = 0.01) and VDR mRNA reduced to 36 +/- 11% (P < 0.01). In KT, previously hP uremic rats, both CaR mRNA and VDR mRNA remained severely reduced (CaR, 39 +/- 7%; VDR, 9 +/- 3%; P < 0.01) compared with normal rats. In conclusion, circulating plasma PTH levels normalized rapidly after KT, despite persisting downregulation of CaR and VDR gene expression. This indicates that upregulation of CaR mRNA and VDR mRNA is not necessary to induce the rapid normalization of PTH secretion from hyperplastic parathyroid glands.     

Reduction of urea generation and muscle protein degradation by adrenalectomy in acutely uremic rats

The effect of adrenalectomy on the enhanced protein degradation in acute uremia was investigated. Therefore, serum urea nitrogen, urea N appearance and Nt-methylhistidine were followed in bilaterally nephrectomized rats. At 48 h after induction of uremia the animals displayed serum urea nitrogen levels of 223 +/- 9.5 mg/dl as compared to 26.0 +/- 1.0 mg/dl in sham-treated rats. This increment was significantly attenuated in acutely uremic, adrenalectomized animals (176 +/- 6.0 mg/dl). When these rats were substituted with corticosterone (5 mg/kg body weight), serum urea nitrogen readily increased to levels of acutely uremic animals with intact adrenal glands (225 +/- 6.0 mg/dl). The net generation of urea, as determined by the urea N appearance, was significantly increased during acute uremia (370 +/- 26 mg/48 h) as compared to SHAM animals (220 +/- 15 mg/48 h). This increment of urea formation could almost be completely reversed by simultaneous adrenalectomy (238 +/- 20 mg/48 h). When these rats were substituted with corticosterone, the urea N appearance rebounded to values quite comparable to acutely uremic rats with intact adrenal glands (363 +/- 30 mg/48 h). To determine whether skeletal muscle proteins might serve as a source for the enhanced protein degradation in acute uremia, plasma levels of Nt-methylhistidine were measured. Bilaterally nephrectomized rats had Nt-methylhistidine values of 9.6 +/- 1.0 micrograms/ml. In acutely uremic rats without adrenal glands, Nt-methylhistidine levels were found to be significantly decreased (6.0 +/- 0.4 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)