Effects of testosterone on skeletal muscle architecture in intermediate-frail and frail elderly men.

BACKGROUND: Testosterone increases lean mass and may help to counter the changes in muscle architecture associated with sarcopenia. This study was designed to investigate the effects of testosterone replacement therapy on skeletal muscle architecture in intermediate-frail and frail elderly men. METHODS: A subgroup of 30 intermediate-frail and frail elderly men (65-89 years) with low to borderline-low testosterone levels were enrolled from a single-center randomized, double-blind placebo-controlled trial. Participants received either a transdermal testosterone (50 mg) or placebo gel daily for 6 months. Architecture (muscle thickness, fascicle length, and pennation angle) of the gastrocnemius medialis muscle was assessed by ultrasound imaging at baseline and after 6 months of treatment. RESULTS: Serum testosterone increased from 11.6 ± 3.5 to 18.0 ± 8.1 nmol/L by 10 days after randomization in the active group (but not the placebo group) and was maintained throughout the treatment period. Testosterone treatment resulted in a preservation of muscle thickness at 6 months while it decreased in the placebo group (effect size 1.4 [95% confidence interval = 0.3-2.5; p = .015]). There was no significant effect of treatment on fascicle length (effect size 1.9 mm [95% confidence interval = -1.2 to 5.0 mm; p = .22]) or pennation angle (effect size 1.2° [95% confidence interval = -1.3 to 3.7°; p = .32]). CONCLUSIONS: Testosterone replacement in intermediate-frail and frail elderly men is associated with preservation of muscle thickness. The results suggest that testosterone mitigates sarcopenia by improving muscle tissue to maintain a state of normality in aging men.

Fatty acid-induced defects in insulin signalling, in myotubes derived from children, are related to ceramide production from palmitate rather than the accumulation of intramyocellular lipid.

The elevation of free fatty acids (FFAs), observed in childhood obesity results in intramyocellular lipid (IMCL) accumulation with consequent insulin resistance. Using in vitro differentiated myotubes from normal weight pre-pubertal children (n = 8), we examined the effects of saturated (palmitate) and unsaturated (oleate) FFAs on insulin-stimulated AKT phosphorylation (pAKT) and IMCL accumulation. Palmitate decreased pAKT (Mean [SEM] % change pAKT with palmitate 750 microM vs. control; pThr308 site -50.5% [28.7] and pSer473 site -38.7% [11.7]; P < 0.001) with no effect on IMCL formation. Equimolar bromopalmitate did not effect pAKT and blocking ceramide production abolished the palmitate-induced reduction in signalling, suggesting that ceramide synthesis is critical for palmitate's actions. Oleate did not effect pAKT (1,000 microM oleate; pSer473 site -3.4% [11.4]; P = NS) but increased IMCL accumulation (+32.3% [7.1%]; P < 0.001). Co-administration of oleate diminished the reduction in pAKT seen with palmitate (+36.4% [23.6] vs. -13.3% [13.6]; P = 0.28), with similar IMCL levels to oleate alone. Co-administration also caused a significant reduction in 14C-ceramide synthesis from 14C-palmitate (101.6 [21.6] vs. 371.5 [122.4] DPM/mg protein; P < 0.001). In summary, palmitate appears to cause insulin resistance in children's myotubes via its metabolism to ceramide, and this process appears unrelated to IMCL formation and is ameliorated by oleate.

Waste management - cytokines, growth factors and cachexia.

Muscle damage with a lack of regeneration, manifests itself in several life-threatening diseases, including cancer cachexia, congestive heart failure, AIDS and sepsis. Often misdiagnosed as a condition simply of weight loss, cachexia is actually a highly complex metabolic disorder involving features of anorexia, anaemia, lipolysis and insulin resistance. A significant loss of lean body mass arises from such conditions, resulting in wasting of skeletal muscle. Unlike starvation, the weight loss seen in chronic illnesses arises equally from loss of muscle and of fat. The cachectic state is particularly problematic in cancer, typifying poor prognosis and often lowering responses to chemotherapy and radiation treatment. More than half of cancer patients suffer from cachexia, and strikingly, nearly one-third of cancer deaths are related to cachexia rather than the tumour burden. In considering this disorder, we are faced with a conundrum; how is it possible for uncontrolled growth to prevail in the tumour, in the face of unrestrained tissue loss in our muscles? Consistently, the catabolic state has been associated with a shift in the homeostatic balance between muscle synthesis and degradation mediated by the actions of growth factors and cytokines. Indeed, tumour necrosis factor-alpha (TNF-alpha) levels are raised in several animal models of cachectic muscle wasting, whereas the insulin-like growth factor (IGF) system acts potently to regulate muscle development, hypertrophy and maintenance. This concept of skeletal muscle homeostasis, often viewed as the net balance between two separate processes of protein synthesis and degradation has however changed. More recently, the view is that these two biochemical processes are not occurring independently of each other but in fact are finely co-ordinated by a web of intricate signalling networks. This review, therefore, aims to discuss data currently available regarding the mechanisms of degeneration and regeneration with specific emphasis on the potential and controversial cross-talk which may exist between anabolic growth factors (e.g. IGF-I) and catabolic cytokines (e.g. TNF-alpha). Also importantly, the potential impact at a cellular level of exercise, diet and age will be addressed. Finally, the ability to 'hi-jack' signalling pathways traditionally believed to be for growth and survival or death will be reviewed. It is anticipated that such a review will highlight significant gaps in our knowledge of the cachectic state as well as provide caution with regards to therapeutics suggesting total block on inflammatory processes such as that associated with TNF-alpha action.

The effects of retinoic acid on the insulin-like growth factor axis in primary tissue culture from hyperparathyroidism.

BACKGROUND: The importance of the IGF system in HPT has been previously demonstrated. Additionally, the role of vitamin A in HPT has been reported. Retinoic acid (RA), a derivative of vitamin A, is a ligand for the IGF II receptor (IGF2R). We have evaluated the interactions of RA with the IGF system in a primary parathyroid cell culture model. MATERIALS AND METHODS: Primary cell cultures were prepared from nine patients. Following adhesion, the cells were transferred to serum-free medium and dosed once with growth factors +/- RA for 96 hours. Proliferation was assessed by measuring tritiated thymidine incorporation. RESULTS: Compared with the control group (100%), both IGF I and II increased DNA synthesis significantly. Retinoic acid significantly reduced the basal DNA synthesis to 82.2% +/- 4.2% compared with control (P < 0.05). Retinoic acid x10(-5) M completely abrogated the proliferative actions of IGF II (70.2% +/- 9.7%, P < 0.05) but had no significant effect on the IGF I response (P > 0.05). To evaluate the role of IGF2R or IGFBPs in mediating the actions of RA, the IGF II analogs [Leu27]IGF II (10-20-fold reduced IGF I receptor affinity) and des(1-6) IGF II (lower IGFBP binding affinity) were used. The IGF II inhibitory effect of RA was enhanced in the presence of analogs [Leu27]IGF II (P = 0.052) but not with des(1-6)IGF II (P > 0.05), compared with wild-type IGF II. CONCLUSIONS: These data implicate a novel antiproliferative role for RA in enhancing the pericellular clearance of IGF II via the IGF2R preventing ligand activation of the IGF I receptor. This may have broader implications for RA effects in other tumors.

Mature subcutaneous and visceral adipocyte concentrations of adiponectin are highly correlated in prepubertal children and inversely related to body mass index standard deviation score.

CONTEXT: Adiponectin is an adipocyte-specific protein with insulin-sensitizing properties. Several studies have examined the expression of adiponectin mRNA or tissue/secreted protein levels in fat obtained from adults, but none has assessed tissue levels in childhood. PATIENTS: Paired subcutaneous (Sc) and visceral (V) fat samples were obtained from 12 normal-weight children. MAIN OUTCOME MEASURES: Mature adipocytes were isolated and total adiponectin levels determined by ELISA. Insulin sensitivity and lipid parameters were assessed in fasting blood samples taken at the time of biopsy collection. RESULTS: A positive correlation was seen between the adiponectin concentration within the Sc and V mature adipocytes derived from each child (r = 0.924; P < 0.001). After logarithmic transformation of the Sc and V adiponectin concentrations (log-Sc and log-V) to render the data Gaussian, both log-Sc and log-V were found to be lower in those children with higher body mass index sd score (r = -0.621 and r = -0.357 respectively), although this reached statistical significance only in the Sc adipocytes (P = 0.03). Age was not related to either log-Sc or log-V adiponectin levels, although a significant negative association was seen with serum adiponectin (r = -0.589; P = 0.04). Log-Sc or log-V did not correlate with serum adiponectin concentrations, markers of insulin sensitivity, or circulating lipid levels. CONCLUSIONS: These data indicate a relationship between total adiponectin levels in different tissue compartments, suggesting either some form of interaction or coregulation by systemic factors, possibly related to body size/fat mass. Serum concentrations of total adiponectin were inversely related to age but showed no relationship with either tissue levels or body mass index sd score.

Adult stem cells: the therapeutic potential of skeletal muscle.

Embryonic stem cells have revolutionised our understanding of normal and deregulated growth and development. The potential to produce cells and tissues as needed offers enormous therapeutic potential. The use of these cells, however, is accompanied by ongoing ethical, religious and biomedical issues. The expansion potential and plasticity of adult stem cells have therefore received much interest. Adult skeletal muscle is highly adaptable, responding to both the hypertrophic and degenerative stresses placed upon it. This extreme plasticity is in part regulated by resident stem cells. In addition to regenerating muscle, if exposed to osteogenic or adipogenic inducers, these cells spontaneously form osteoblasts or adipocytes. The potential for and heterogeneity of muscle stem cells is underscored by the observation that CD45+ muscle side population cells are capable of reconstituting bone marrow in lethally irradiated mice and of contributing to neo-vascularisation of regenerating muscle. Finally, first attempts to replace infarcted myocardium relied on injection of skeletal myoblasts into the heart. Cells successfully engrafted and cardiac function was improved. Harnessing their differentiation/trans-differentiation capacity provides enormous potential for adult stem cells. In this review, current understanding of the different stem cells within muscle will be discussed as will their potential utility for regenerative medicine.

Differential signalling mechanisms predisposing primary human skeletal muscle cells to altered proliferation and differentiation: roles of IGF-I and TNFalpha.

To gain a clearer insight into the mechanisms of skeletal muscle cell growth, differentiation and maintenance, we have developed a primary adult human skeletal muscle cell model. Cells were cultured from biopsies of rectus muscle from the anterior abdominal wall of patients undergoing elective surgery. Under differentiating conditions, all cultures formed myotubes, irrespective of initial myoblast number. Stimulation with both IGF-I and tumour necrosis factor alpha (TNFalpha) increased cellular proliferation but while IGF-I subsequently increased myoblast differentiation, via both hyperplasia and hypertrophy, TNFalpha inhibited the initiation of differentiation, but did not induce apoptosis. Addition of IGF-I stimulated both the MAP kinase and the phosphatidylinositide 3-kinase (PI 3-kinase) signalling pathways while treatment with TNFalpha preferentially led to MAP kinase activation although with a very different profile of activation compared to IGF-I. Data using the MEK inhibitor UO126 showed MAP kinase activity is not only needed for cellular proliferation but is also necessary for both the initiation and the progression of primary human myoblast differentiation. The PI 3-kinase pathway is also involved in differentiation, but activation of this pathway could not relieve inhibition of differentiation by TNFalpha or UO126. Our results show that the controlled temporal and amplitude of activation of multiple signalling pathways is needed for successful myoblast differentiation.

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts.

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.

Selective estrogen receptor modulators inhibit the effects of insulin-like growth factors in hyperparathyroidism.

BACKGROUND: Primary hyperparathyroidism (HPT) predominantly affects perimenopausal women, leading to speculations that an estrogen imbalance may be liable. We have previously demonstrated the importance of the insulin-like growth factor (IGF) axis in HPT. Because the antiestrogen tamoxifen has been shown to modulate the IGF axis, we examined the interactions of selective estrogen receptor modulators (SERMs) and IGF in HPT. METHODS; Estrogen receptors were evaluated by Western immunoligand blotting. Sixteen parathyroid glands from 19 patients were included. After adhesion, the cells were treated with IGF (I or II) +/- estrogen +/- SERMs (tamoxifen, ICI 182,780) for 96 hours in serum-free media. Proliferation was assessed by measuring tritiated thymidine incorporation. RESULTS: Both primary and secondary HPT express estrogen receptors alpha and beta. Primary and secondary HPT had comparable responses to SERMs, they were analyzed together. Compared with control (100%), IGFs (I and II) induced a significant increase in DNA synthesis. Estradiol at 10(-8) and 10(-7) mol/L (physiologic range) had no significant effects on IGF (I and II, P >.05). Both tamoxifen and ICI 182,780 inhibited basal DNA synthesis (P <.05) and abolished the effects of both IGF I and II (P <.05). CONCLUSIONS: SERMs are capable of reducing basal and IGF-stimulated DNA synthesis. This reduction in proliferation has implications for cancer biology and therapeutic potential for SERMs in HPT.