Bioactive growth hormone in humans: Controversies, complexities and concepts.

OBJECTIVE: To revisit a finding, first described in 1978, which documented existence of a pituitary growth factor that escaped detection by immunoassay, but which was active in the established rat tibia GH bioassay. METHODS: We present a narrative review of the evolution of growth hormone complexity, and its bio-detectability, from a historical perspective. RESULTS: In humans under the age of 60, physical training (i.e. aerobic endurance and resistance training) are stressors which preferentially stimulate release of bioactive GH (bGH) into the blood. Neuroanatomical studies indicate a) that nerve fibers directly innervate the human anterior pituitary and b) that hind limb muscle afferents, in both humans and rats, also modulate plasma bGH. In the pituitary gland itself, molecular variants of GH, somatotroph heterogeneity and cell plasticity all appear to play a role in regulation of this growth factor. CONCLUSION: This review considers more recent findings on this often forgotten/neglected subject. Comparison testing of a) human plasma samples, b) sub-populations of separated rat pituitary somatotrophs or c) purified human pituitary peptides by GH bioassay vs immunoassay consistently yield conflicting results.

Muscle-Specific Sensitivity to Voluntary Physical Activity and Detraining.

Aerobic physical activity triggers adaptations in skeletal muscle including a fast-to-slow shift in myosin heavy chain (MHC) isoforms, an enhanced capillary network, and mitochondrial biogenesis to meet increased demands placed upon this tissue. Although the magnitude of these responses appears to be dependent on muscle phenotype as well as training volume and/or intensity, the whole-muscle response to detraining remains mostly unexplored. Here, we hypothesized that the shifts toward slower MHC phentotype and the increased capillarity and mitochondrial oxidative markers induced with training would return toward sedentary (SED) control levels sooner in the fast plantaris than in the slow soleus muscle as a result of detraining. Soleus and plantaris muscles from 8-week (TR 8wk) voluntarily running adult female Sprague-Dawley rats were compared to muscles from SED and detrained rats (DETR) (4 weeks voluntary running followed by 4 weeks of reduced activity), which were subdivided into low- (DETR Lo) and high-running-distance (DETR Hi) groups. We show that maintaining the fast-to-slow MHC isoform shift required consistent aerobic training in the soleus and plantaris muscles: detraining clearly abolished any fast-to-slow gains in the plantaris, whereas the training volume in DETR Hi rats appeared to influence the MHC return to basal levels in the soleus. Total capillary number (per mm2) in the plantaris increased in all groups compared to SED levels, but, in the soleus, this enhancement was observed only in the TR 8wk rats. Generally, increased mitochondrial markers for aerobicitiy were observed in TR 8wk plantaris, but not soleus, muscles. In a second experiment, we show that the muscle-specific adaptations were similar after 4 weeks of voluntary exercise (TR 4wk) as in 4 weeks (TR 8wk). Taken together, our findings suggest that the plantaris muscle is more sensitive to voluntary physical activity and detraining than the soleus muscle; these results also demonstrate that the soleus muscle requires a greater aerobic challenge (i.e., intensity, duration) to trigger phenotypic, angiogenic, or aerobic enzyme adaptations. Our findings generally suggest that muscular aerobic fitness to voluntary running, or its loss during detraining, manifests as changes occurring primarily within fast, rather than slow, muscle phenotypes.

Acute vascular endothelial growth factor expression during hypertrophy is muscle phenotype specific and localizes as a striated pattern within fibres.

Skeletal muscle hypertrophy requires the co-ordinated expression of locally acting growth factors that promote myofibre growth and concurrent adaptive changes in the microvasculature. These studies tested the hypothesis that vascular endothelial growth factor (VEGF) and heparin-binding epidermal growth factor (HB-EGF) expression are upregulated during the early stages of compensatory muscle growth induced by chronic functional overload (FO). Bilateral FO of the plantaris and soleus muscles was induced for 3 or 7 days in the hindlimbs of adult female Sprague-Dawley rats (n = 5 per group) and compared with control (non-FO) rats. Relative muscle mass (in mg (kg body weight)(-1)) increased by 18 and 24% after 3 days and by 20 and 33% after 7 days in the plantaris and soleus muscles, respectively. No differences in HB-EGF mRNA or protein were observed in either muscle of FO rats relative to control muscles. The VEGF mRNA was similar in the soleus muscles of FO and control rats, whereas a significant elevation occurred at 3 and 7 days of FO in the plantaris muscle. However, VEGF protein expression after 3 days of FO exhibited a differential response; expression in the soleus muscle decreased 1.6-fold, whereas that in the plantaris muscle increased 1.8-fold compared with the control muscle. After 7 days of FO, VEGF protein remained elevated within the plantaris muscle, but returned to basal levels in the soleus. Robust basal HB-EGF and VEGF protein expression was consistently seen in control muscles. In all groups, immunohistochemistry for VEGF protein displayed a distinct striated expression pattern within myofibres, with considerably less labelling in extracellular spaces. Constitutive expression of HB-EGF and VEGF in control myofibres is consistent with housekeeping roles for these growth factors in skeletal muscle tissue. However, the specific patterns of VEGF expression in these muscles during FO may reflect the chronic changes in neural recruitment between muscles and the co-ordination of angiogenic and/or other hypertrophic responses.

Acute daily psychological stress causes increased atrophic gene expression and myostatin-dependent muscle atrophy.

Psychological stress is known to attenuate body size and lean body mass. We tested the effects of 1, 3, or 7 days of two different models of psychological stress, 1 h of daily restraint stress (RS) or daily cage-switching stress (CS), on skeletal muscle size and atrophy-associated gene expression in mice. Thymus weights decreased in both RS and CS mice compared with unstressed controls, suggesting that both models activated the hypothalamic-pituitary-adrenal axis. Body mass was significantly decreased at all time points for both models of stress but was greater for RS than CS. Mass of the tibialis anterior (TA) and soleus (SOL) muscles was significantly decreased after 3 and 7 days of RS, but CS only significantly decreased SOL mass after 7 days. TA mRNA levels of the atrophy-associated genes myostatin (MSTN), atrogin-1, and the phosphatidylinositol 3-kinase inhibitory subunit p85alpha were all significantly increased relative to unstressed mice after 1 and 3 days of RS, and expression of MSTN and p85alpha mRNA remained elevated after 7 days of RS. Expression of muscle ring finger 1 was increased after 1 day of RS but returned to baseline at 3 and 7 days of RS. MSTN, atrogin-1, and p85alpha mRNA levels also significantly increased after 1 and 3 days of CS but atrogen-1 mRNA levels had resolved back to normal levels by 3 days and p85alpha with 7 days of CS. p21CIP mRNA levels were significantly decreased by 3 days of CS or RS. Finally, body mass was minimally affected, and muscle mass was completely unaffected by 3 days of RS in mice null for the MSTN gene, and MSTN inactivation attenuated the increase in atrogin-1 mRNA levels with 4 days of RS compared with wild-type mice. Together these data suggest that acute daily psychological stress induces atrophic gene expression and loss of muscle mass that appears to be MSTN dependent.

Transcriptional regulation of the myosin heavy chain IIb gene in inactive rat soleus.

The myosin heavy chain (MHC) isoform composition of skeletal muscle is dependent, in part, on the functional demands of the muscle. The rat soleus muscle primarily expresses the slow-contracting type I MHC; however, chronic inactivity increases expression of the faster-contracting type II MHC isoforms. The purpose of this study was to identify the type IIb MHC promoter region(s) that regulate de novo transcription during chronic inactivity of the soleus induced by spinal cord isolation (SI; complete mid-thoracic and high sacral spinal cord transections plus deafferentation). Seven days after SI, transcription of IIb MHC was evidenced by increases in IIb pre-mRNA and mRNA. The activity of an approximately 2.2-kb IIb promoter-firefly luciferase reporter plasmid increased in SI soleus over control as compared to that of a promoterless plasmid. Deletion analyses indicated that the regions encompassing -2237 to -1431, -1048 to -461, and -192 to -161 basepairs (bp) each contributed to the increase in transcriptional activity. Moreover, deletions or mutations of AT-rich regions in the proximal -192 bp region abolished the increased promoter activity. These results provide important insights related to how proximal IIb MHC promoter elements regulate the increased expression of the IIb MHC gene in response to inactivity of a predominantly slow postural muscle as it undergoes a remodeling of its phenotype and functional characteristics.

PAX3/7 expression coincides with MyoD during chronic skeletal muscle overload.

Paired box (Pax) proteins 3 and 7 are key determinants for embryonic skeletal muscle development by initiating myogenic regulatory factor (MRF) gene expression. We show that Pax3 and 7 participate in adult skeletal muscle plasticity during the initial responses to chronic overload (< or =7 days) and appear to coordinate MyoD expression, a member of the MRF family of genes. Pax3 and 7 mRNA were higher than control within 12 h after initiation of overload, preceded the increase in MyoD mRNA on day 1, and peaked on day 2. On days 3 and 7, Pax7 mRNA remained higher than control, suggesting that satellite cell self-renewal was occurring. Pax3 and 7 and MyoD protein levels were higher than control on days 2 and 3. These data indicate that Pax3 and 7 coordinate the recapitulation of developmental-like regulatory mechanisms in response to growth-inducing stimuli in adult skeletal muscle, presumably through activation of satellite cells.

Modulation of HSP25 and TNF-alpha during the early stages of functional overload of a rat slow and fast muscle.

Early events in response to abrupt increases in activation and loading with muscle functional overload (FO) are associated with increased damage and inflammation. Heat shock protein 25 (HSP25) may protect against these stressors, and its expression can be regulated by muscle loading and activation. The purpose of this study was to investigate the responses of HSP25, phosphorylated HSP25 (pHSP25), and tumor necrosis factor-alpha (TNF-alpha) during FO of the slow soleus and fast plantaris. We compared the HSP25 mRNA, HSP25 protein, pHSP25, and TNF-alpha responses in the soleus and plantaris after 0.5, 1, 2, 3, and 7 days of FO. HSP25 and pHSP25 were quantified in soluble and insoluble fractions. HSP25 mRNA increased immediately in both muscles and decreased with continued FO. However, HSP25 mRNA levels were consistently higher in the muscles of FO than control rats. In the soluble fraction, HSP25 increased in the plantaris after 2-7 days of FO with the greatest response at 3 and 7 days. The pHSP25 response to FO was greater in the plantaris than soleus at all points in the soluble fraction and at 0.5 days in the insoluble fraction. TNF-alpha levels in the plantaris, but not soleus, were higher than control at 0.5-2 days of FO. This may have contributed to the greater FO response in pHSP25 in the plantaris than soleus as TNF-alpha increased pHSP25 in C2C12 myotubes. These results suggest that the initial responses of pHSP25 and TNF-alpha to mechanical stress and inflammation associated with FO are greater in a fast than slow extensor muscle.