Testosterone and trenbolone enanthate increase mature myostatin protein expression despite increasing skeletal muscle hypertrophy and satellite cell number in rodent muscle.

The androgen-induced alterations in adult rodent skeletal muscle fibre cross-sectional area (fCSA), satellite cell content and myostatin (Mstn) were examined in 10-month-old Fisher 344 rats (n = 41) assigned to Sham surgery, orchiectomy (ORX), ORX + testosterone (TEST; 7.0 mg week-1 ) or ORX + trenbolone (TREN; 1.0 mg week-1 ). After 29 days, animals were euthanised and the levator ani/bulbocavernosus (LABC) muscle complex was harvested for analyses. LABC muscle fCSA was 102% and 94% higher in ORX + TEST and ORX + TREN compared to ORX (p < .001). ORX + TEST and ORX + TREN increased satellite cell numbers by 181% and 178% compared to ORX, respectively (p < .01), with no differences between conditions for myonuclear number per muscle fibre (p = .948). Mstn protein was increased 159% and 169% in the ORX + TEST and ORX + TREN compared to ORX (p < .01). pan-SMAD2/3 protein was ~30-50% greater in ORX compared to SHAM (p = .006), ORX + TEST (p = .037) and ORX + TREN (p = .043), although there were no between-treatment effects regarding phosphorylated SMAD2/3. Mstn, ActrIIb and Mighty mRNAs were lower in ORX, ORX + TEST and ORX + TREN compared to SHAM (p < .05). Testosterone and trenbolone administration increased muscle fCSA and satellite cell number without increasing myonuclei number, and increased Mstn protein levels. Several genes and signalling proteins related to myostatin signalling were differentially regulated by ORX or androgen therapy.

Effects of testosterone treatment on markers of skeletal muscle ribosome biogenesis.

The effects of testosterone (TEST) treatment on markers of skeletal muscle ribosome biogenesis in vitro and in vivo were examined. C2 C12 myotubes were treated with 100 nm TEST for short-term (24-h) and longer-term (96-h) treatments. Moreover, male 10-month-old Fischer 344 rats were housed for 4 weeks, and the following groups were included in this study: (i) Sham-operated (Sham) rats, (ii) orchiectomised rats (ORX) and (iii) ORX+TEST-treated rats (7.0 mg week-1 ). For in vitro data, TEST treatment increased c-Myc mRNA expression by 38% (P = 0.004) after 96 h, but did not affect total RNA, 47S pre-rRNA, Raptor mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA at 24 h or 96 h following the treatment. For in vivo data, ORX decreased levator ani/bulbocavernosus (LABC) myofibril protein versus Sham (P = 0.006), whereas ORX+TEST (P = 0.015) rescued this atrophic effect. ORX also decreased muscle ribosome content (total RNA) compared to Sham (P = 0.046), whereas ORX+TEST tended to rescue this effect (P = 0.057). However, other markers of ribosome biogenesis including c-Myc mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA decreased with ORX independently of TEST treatments (P < 0.05). Finally, lower phospho-(Ser235/236)-to-total rps6 protein and lower rpl5 protein levels existed in ORX+TEST rats versus other treatments, suggesting that chronic TEST treatment may lower translational capacity.

Bone loss in a new rodent model combining spinal cord injury and cast immobilization.

OBJECTIVES: Characterize bone loss in our newly developed severe contusion spinal cord injury (SCI) plus hindlimb immobilization (IMM) model and determine the influence of muscle contractility on skeletal integrity after SCI. METHODS: Female Sprague-Dawley rats were randomized to: (a) intact controls, (b) severe contusion SCI euthanized at Day 7 (SCI-7) or (c) Day 21 (SCI-21), (d) 14 days IMM-alone, (e) SCI+IMM, or (f) SCI+IMM plus 14 days body weight supported treadmill exercise (SCI+IMM+TM). RESULTS: SCI-7 and SCI-21 exhibited a >20% reduction in cancellous volumetric bone mineral density (vBMD) in the hindlimbs (p⋜0.01), characterized by reductions in cancellous bone volume (cBV/TV%), trabecular number (Tb.N), and trabecular thickness. IMM-alone induced no observable bone loss. SCI+IMM exacerbated cancellous vBMD deficits with values being >45% below Controls (p⋜0.01) resulting from reduced cBV/TV% and Tb.N. SCI+IMM also produced the greatest cortical bone loss with distal femoral cortical area and cortical thickness being 14-28% below Controls (p⋜0.01) and bone strength being 37% below Controls (p⋜0.01). SCI+IMM+TM partially alleviated bone deficits, but values remained below Controls. CONCLUSIONS: Residual and/or facilitated muscle contractility ameliorate bone decrements after severe SCI. Our novel SCI+IMM model represents a clinically-relevant means of assessing strategies to prevent SCI-induced skeletal deficits.

Myogenic and proteolytic mRNA expression following blood flow restricted exercise.

AIM: Resistance exercise performed at low loads (20-30% of maximal strength) with blood flow restriction (BFR) acutely increases protein synthesis and induces hypertrophy when performed chronically. We investigated myogenic and proteolytic mRNA expression 8 h following an acute bout of knee extension exercise. METHODS: Fifteen subjects (22.8 ± 3.7 years, eight men and seven women) were randomized to two exercise conditions: BFR or control exercise. All participants performed four sets of exercise (30, 15, 15 and 15 repetitions) at 20% of maximal strength. Persons in the BFR group had a cuff placed on the upper thigh inflated to 1.5 times brachial systolic blood pressure (cuff pressure range: 135-186 mmHg). Muscle biopsies from the vastus lateralis were excised 24 h before and 8 h following the exercise. RESULTS: RT-PCR analysis demonstrated no change in myogenic gene expression (insulin-like growth factor-1, MyoD, myogenin, myostatin - a negative regulator) with either exercise condition (P > 0.123). However, BFR exercise downregulated mRNA expression in transcripts associated with proteolytic pathways (FOXO3A, Atrogin-1 and MuRF-1) with no change in the control exercise condition. Specifically, median mRNA expression of FOXO3A decreased by 1.92-fold (P = 0.01), Atrogin-1 by 2.10-fold (P = 0.01) and MuRF-1 by 2.44-fold (P = 0.01). CONCLUSION: These data are consistent with the downregulation of proteolytic transcripts observed following high-load resistance exercise. In summary, myogenic genes are unchanged and proteolytic genes associated with muscle remodelling are reduced 8 h following low-load BFR exercise.

Testosterone administration induces protection against global myocardial ischemia.

We tested the hypothesis that chronic testosterone treatment would promote a cardioprotective phenotype against ischemia/reperfusion (I/R) injury. For this study, 3-month-old F344 male rats underwent sham-surgery, orchiectomy (ORX), or ORX plus 21 days testosterone treatment (1.0 mg testosterone/day). At sacrifice, cardiac performance was assessed in a working heart model of I/R (25 min of global ischemia and 45 min of reperfusion). ORX reduced serum testosterone by approximately 98% and testosterone administration elevated serum testosterone to a concentration of 4.6-fold over that of Sham-operated controls (p<0.05). ORX did not significantly impair recovery of cardiac performance following I/R, but did increase cardiac release of lactate dehydrogenase (LDH) during pre- and post-ischemia (p<0.05). Testosterone administration prevented the ORX-induced increase in LDH during both pre- and post-ischemia and increased post-ischemic recovery of aortic flow, cardiac output, cardiac work, left ventricular developed pressure, and contractility (p<0.05) during reperfusion. Testosterone administration also increased left ventricular expression of catalase, but did not affect the expression of manganese superoxide dismutase, glutathione peroxidase, or sarcolemmal K (ATP) channel protein Kir6.2. Neither circulating nor cardiac concentrations of estradiol were altered by either treatment. We conclude that administration of high-dose testosterone confers cardioprotection through yet to be identified androgen-dependent mechanism(s).

beta-3 adrenergic agonist restores skeletal muscle insulin responsiveness in Sprague-Dawley rats.

Between 7 and 14 weeks of age, male Sprague-Dawley rats develop a greater than 50% loss in insulin-stimulated glucose transport in skeletal muscle. We treated rats aged 14 weeks with the beta-3 adrenergic agonist CL316,243 (1 mg/kg/day by minipump for 14 days). Treatment resulted in a 56% reduction in visceral fat (P < 0.05). Muscle mass and body weight were unchanged. In strips of soleus muscle isolated from rats treated with CL316,243, basal transport of [(3)H]-2-deoxyglucose (2-DOG) was unchanged (105.8 +/- 7.5 nmol/g/min for vehicle vs 122.0 +/- 8.7 for CL316,243). However, in rats treated with CL316,243, the increase in 2-DOG transport in response to a maximal concentration of insulin was substantially increased (55.5 +/- 13.1 nmol/g/min for vehicle vs 102.4 +/- 13.5 for CL316,243, P < 0.03). CL 316,243 caused no significant changes in fasting glucose, insulin, or free fatty acids. Treatment of soleus muscle strips in vitro with CL316,243 (either 0.1 nM or 1.0 nM for 120 min at 37 degrees C) had no effect either on basal 2-DOG transport or on insulin-stimulated transport. We conclude that the CL316,243 causes a reduction in visceral fat and a reversal of muscle insulin resistance. The effect CL 316,243 on muscle insulin responses appears to be indirect, as it did not occur in vitro.

Metformin, but not exercise training, increases insulin responsiveness in skeletal muscle of Sprague-Dawley rats.

We assessed the effects of combined metformin treatment and exercise training on body composition, on insulin concentration following glucose loading, on insulin-stimulated glucose transport in skeletal muscle, and on muscle glycogen content. Male Sprague-Dawley rats were treated for 35 days with or without metformin (320 mg/kg/day) and/or treadmill exercise training (20 min at 20 m/min, 5 days/wk). Because metformin reduces food intake, pair-fed controls were included. Metformin, training, and pair-feeding all decreased food intake, body weight, and insulin concentration following glucose loading. Metformin and training reduced intra-abdominal fat, but pair feeding did not. In isolated strips derived from soleus, epitrochlearis and extensor carpi ulnaris muscles, metformin increased insulin-stimulated transport of [3H]-2-deoxyglucose by 90%, 89% and 125%, respectively (P < 0.02) and training increased [3H]-2-deoxyglucose transport in the extensor carpi ulnaris muscle only (66%, P < 0.05). Pair-feeding did not alter [3H]-2-deoxyglucose transport. Training increased gastrocnemius muscle glycogen by 100% (P < 0.001). Metformin and pair-feeding did not alter muscle glycogen. We conclude that metformin reverses the maturation-induced impairment of insulin responsiveness in Sprague-Dawley rats by increasing insulin-stimulated glucose transport in skeletal muscle and that this effect is not secondary to reduced food intake. We also conclude that metformin and exercise training may increase insulin sensitivity by different mechanisms, with training causing increased glucose transport only in some muscles and also causing increased muscle glycogen storage.

Effects of resistance training on insulin-like growth factor-I and IGF binding proteins.

PURPOSE: Our goal was to determine the effects resistance training on circulating IGF-I and on two of its major binding proteins, IGFBP-1 and IGFBP-3. Additional goals were to compare the time course of hormonal changes with the time course of strength changes and to determine the effect of training volume on the extent of hormonal changes. METHODS: Thirty-one men and women (mean age = 37 +/- 7 yr) completed a 25-wk, 3 d x wk(-1) program in which they performed single-set resistance training (1-SET, N = 11), multiple-set resistance training (3-SET, N = 11), or no exercise (Control, N = 9). Before training, and after 13 and 25 wk of training, blood hormones were analyzed and strength was assessed as the sum of one-repetition maximum (1-RM) for leg extension and chest press exercises. RESULTS: During the first 13 wk of resistance training, circulating IGF-I increased by approximately 20% in both the 1-SET and 3-SET groups (P = 0.041). No further increases occurred between 13 and 25 wk. In the 3-SET group, IGFBP-3 decreased 20% between 13 and 25 wk (P = 0.008). Training did not alter IGFBP-1. Increases in 1-RM strength occurred mainly during the first 13 wk of training and were significantly higher with 3-SET training compared to 1-SET. CONCLUSIONS: These findings indicate that increased circulating IGF-I may, at least in part, mediate increases in strength that result from resistance training.

Metformin treatment enhances insulin-stimulated glucose transport in skeletal muscle of Sprague-Dawley rats.

Although the glucose-lowering properties of metformin are well-established, its effects on glucose metabolism in skeletal muscle have not been clearly defined. We tested the effects of metformin in young adult male Sprague-Dawley rats, which have a documented reduced response to insulin in skeletal muscle. Rats were treated with metformin for 20 days (320 mg/kg/day) in the drinking water. During this period, metformin completely prevented the increase in food intake and decreased adiposity by 30%. Metformin also reduced insulin secretion by 37% following an intra-peritoneal injection of glucose. Finally, metformin enhanced transport of [3H]-2-deoxyglucose in isolated strips of soleus muscle. Metformin substantially increased insulin-stimulated transport, while having no effect on basal transport. In control rats, a maximal concentration of insulin stimulated transport 77% above basal. In metformin-treated rats, insulin stimulated transport 206% above basal. We conclude that in the Sprague-Dawley rat model, metformin causes a significant increase in insulin-responsiveness.

Altered IGF-I and IGFBPs in senescent male and female rats.

The effects of senescence on muscle characteristics and the insulin-like growth factor I (IGF-I) pathway were assessed in male and female BN/F344 rats. The mass and total ATPase activity of gastrocnemius and plantaris muscles were reduced with age and to a greater extent in males than in females. The mass and total ATPase activity of soleus muscle were not significantly altered with age. Circulating IGF-I was also significantly reduced with age, 60% in females and 21% in males. Circulating IGF-binding protein 3 (IGFBP-3) was reduced with age. In liver and gastrocnemius muscle, mRNAs for IGF-1, IGFBP-2, and IGFBP-3 were analyzed in young and aged males of two strains, BN/F344 and Sprague-Dawley. In BN/F344 rats, liver mRNAs were unchanged with age. Also in BN/F344 rats, muscle mRNAs for IGFBP-2, and IGFBP-3 displayed nonsignificant trends toward increase with age. In aged Sprague-Dawley males, liver mRNA for IGF-I was increased 15% and muscle mRNA for IGFBP-2 was increased 110%. Thus, different age-related changes in the growth hormone (GH)/IGF pathway occur in males and females between the sexes and strains. These changes may play a role in the muscle atrophy associated with senescence.

Role of IGF-I in muscular atrophy of aging.

Loss of muscle mass and strength are well-known consequences of aging. The growth hormone/IGF-I pathway is both impaired with aging and essential for growth and maintenance of skeletal muscle. Despite this, growth hormone (GH) replacement has not been effective in increasing muscle mass and strength in the elderly. Possible explanations for this failure include 1. The presence of other age-related impairments in muscle that prevent the actions of GH and IGF-I and 2. Complications arising from a failure to deliver GH and IGF-I in the physiologically correct manner. This article summarizes the results of clinical trials of GH and IGF-I in the elderly and discusses the current status of strategies for safely and effectively stimulating the GH/IGF-I pathway in this population.

Alpha-adrenergic receptor-mediated thermogenesis in brown adipose tissue of rat.

1. The present studies were undertaken to characterize the thermogenic response to the alpha 1-adrenergic agonist phenylephrine, to compare this response with the beta-adrenergic response and to assess the role of brown adipose tissue (BAT). 2. Phenylephrine and the beta 3-adrenergic agonist CGP 12177A each caused similar increases in O2 consumption. No synergism was observed when combining the two drugs. Phenylephrine stimulated O2 consumption via an alpha-adrenergic mechanism, as indicated by effective blockade with phenoxybenzamine, but not propranolol. 3. Further evidence for the alpha-adrenergic mechanism of phenylephrine action was seen in studies with BAT membranes. Phenylephrine did not stimulate adenylyl cyclase and did not potentiate beta-adrenergic stimulation of adenylyl cyclase. 4. Skeletal muscle was not a major site of phenylephrine-stimulated O2 consumption, as the response was not inhibited by a concentration of dantrolene which inhibited cold-induced muscle shivering. 5. Phenylephrine caused an increase in the density of available 3H-GDP binding sites in BAT mitochondria, indicating an activation of BAT thermogenesis in vivo. This increase was equal in magnitude to what we have reported previously for CGP-12177A. No changes were observed in the affinity for 3H-GDP. 6. We concluded that phenylephrine stimulates O2 consumption by an alpha-adrenergic mechanism that involves activation of BAT thermogenesis. It remains to be determined whether the activation of BAT occurs by a direct or indirect mechanism.

Alpha 1-adrenergic and arginine vasopressin stimulation of inositide hydrolysis in rat hepatocytes is unaltered in senescence.

Alpha 1-adrenergic receptors in the liver support glucose counterregulation by stimulating glycogenolysis and gluconeogenesis. We have recently reported that senescence is accompanied by reductions both in the density of alpha 1 receptors in liver homogenates and in the affinity of these receptors for agonists (Borst and Scarpace, 1990a). The present studies were undertaken to determine what consequences these reductions may have for agonist-stimulated second messenger production. To this end we measured the density of alpha 1-adrenergic receptors and agonist-stimulated phosphoinositide (PI) hydrolysis in short-term hepatocyte cultures derived from young and senescent rats. Receptor density in hepatocytes was unchanged with age. PI hydrolysis response to epinephrine was alpha 1 in nature, as it was fully blocked by prazosin. The epinephrine response was unchanged with age, both in maximum response and in sensitivity. Similarly, the response to arginine vasopressin (AVP) was also unchanged with age.

The association of E. coli peritonitis with an impaired and delayed fever response in senescent rats.

Infection is one of the leading causes of death in elderly humans, and the importance of the early diagnosis of severe infection is undisputed. In the elderly a delay in diagnosis is often due to a reduced or absent fever. To understand more fully the pathogenesis of fever in senescence, we assessed the febrile response to E. coli peritonitis in 3-, 12-, and 24-month-old rats. Baseline temperatures were unchanged with age. Following infection with 1 x 10(8) CFU E. coli, a fever was evident in 2.8 h in the young, 3.9 h in the 12-month-old rats, and delayed until 5.8 h in the senescent rats. The magnitude of the fever was quantitatively less in the older rats compared with the two younger age groups throughout the time course of the fever. Because beta-adrenergic-mediated thermogenesis in brown adipose tissue has been implicated in the genesis of fever, we also assessed adenylate cyclase activity in this tissue. There was a progressive age-related decrease in both receptor- and postreceptor-stimulated adenylate cyclase activity. Our findings indicate there is both a delay in the onset of the fever and a reduced febrile response in the senescent rats following E. coli infection.

Impaired febrile response with age: role of thermogenesis in brown adipose tissue.

We demonstrated previously that in Escherichia coli-infected rats, the heat necessary for the febrile response is a result of thermogenesis in brown adipose tissue (BAT). To investigate whether senescent rats have an impaired febrile response to infection and whether such an impairment is a result of attenuated sympathetically activated thermogenesis in BAT, we assessed body temperature and the increase in mitochondrial guanosine 5'-diphosphate (GDP) binding sites in interscapular BAT in response to E. coli administration in young and senescent male F-344 rats. There was a significant delay of 2 hr in the onset of fever in the older animals. In addition, in senescent rats, the peak fever (1.0 +/- 0.1 delta degrees C vs 2.2 +/- 0.1) and the cumulative fever (383 +/- 43 delta degrees C.min vs 775 +/- 69) were significantly less than in the young rats (P less than 0.005). Baseline levels of GDP binding were the same in young and old rats. In young rats, during the rising phase of the fever, E. coli infection resulted in a 50% increase in the density of GDP binding sites in BAT mitochondria. In contrast, there was no increase in GDP binding in the older rats following infection. The failure to increase GDP binding may be a result of a reduced ability to unmask reserve GDP binding sites. Alternatively, there may be fewer total GDP binding sites (masked and unmasked) in senescent rats and these sites may already be unmasked. Collectively, these data suggest that the impaired febrile response with age is due to reduced thermogenesis in BAT.

Beta-adrenergic responsiveness in cultured aorta smooth muscle cells. Effects of subculture and aging.

beta-Adrenoceptor-mediated vasorelaxation is diminished in vessels from a variety of aged species including humans. This phenomenon was studied for the first time in cultured aorta smooth muscle cells (ASMC) from young (4- to 6-month) and old (24- to 26-month) F-344 rats. Cyclic AMP (cAMP) accumulation was assessed following isoproterenol and forskolin stimulations in primary cultures and after 1-4 passages of aorta smooth muscle cells. Isoproterenol and forskolin increased cAMP accumulation 6- and 10-fold, respectively, in primary cultures from young rats. Isoproterenol stimulation was reduced markedly in passaged cells. Forskolin stimulation was unaffected, indicating passage-related phenotypic changes in receptor-mediated stimulation, but not in post-receptor adenylate cyclase activation. The response to isoproterenol was diminished in old animals, but that to forskolin was unaltered. Thus, cultured ASMC from F-344 rats are highly responsive to beta-adrenoceptor stimulation and demonstrate age-related changes, but undergo phenotypic modulation during passage.

Thermogenesis and mitochondrial GDP binding with age in response to the novel agonist CGP-12177A.

The ability to regulate body temperature diminishes with age in both humans and rodents. To investigate whether attenuation of sympathetically activated thermogenesis in brown adipose tissue (BAT) may account for the loss of thermoregulation with age, we assessed O2 consumption and body temperature in response to norepinephrine and the specific BAT beta-adrenergic agonist CGP-12177A in 6-, 18-, and 24-mo-old rats. In addition, the effects of this agonist on interscapular BAT mitochondrial GDP binding in young and senescent rats were determined. CGP-12177A rapidly induced an elevation in O2 consumption, which peaked at 25 min, followed by a decline over 4 h. The peak increase in O2 consumption over baseline and the cumulative 4-h response were decreased with age [P less than 0.02, analysis of variance (ANOVA)]. CGP-12177A induced an increase in body temperature that paralleled but appropriately lagged behind the increase in O2 consumption and that was decreased with age (P less than 0.02, ANOVA). The norepinephrine-induced increase in O2 consumption was also reduced with age but was not paralleled by a change in body temperature and was associated with a four- to fivefold increase in physical activity. In young rats CGP-12177A increased the number of available BAT mitochondrial GDP binding sites at 20 and 60 min post-injection, but in senescent rats GCP-12177A was unable to increase GDP binding. These data indicate that CGP-12177A is a novel agonist for BAT thermogenesis. With age there is a reduced capacity for thermogenesis that involves a failure to increase GDP binding, either due to a diminished amount of uncoupling protein with age or a failure to unmask reserve GDP binding sites.