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.

Two types of rat pituitary somatotrophs secrete growth hormone with different biological and immunological profiles.

OBJECTIVE: Two stable subpopulations of somatotrophs reside in the rat pituitary gland. We tested the hypothesis that one produced growth hormone (GH) with greater activity when tested in the tibial line bioassay (BGH) than the other, while differences in the activities between the two groups would be less dramatic when measured by immunoassay (IGH). DESIGN: A series of studies using hypophysectomized rats, hollow fibers, treatments and culture models were used to differentiate differences in Type I and Type II anterior pituitary somatotrophs in both function and production of immunoactive and bioactive growth hormone. RESULTS: We found that dense, Type II somatotrophs (>1.070g·cm-3) differed markedly in their secretion patterns of IGH vs BGH in different In vitro and in vivo tests. In culture, Type II cells secreted five times as much BGH, and three fourths as much IGH as the less dense Type I cells. Production (storage and secretion) of BGH was 7-fold greater by Type II cells whereas IGH production was identical for the two cell types. Implantation of Type II cells into hypophysectomized rats significantly increased body weight, epiphyseal cartilage thickness, and muscle weight of the recipients; in contrast, Type I cells elicited only a small increase in body weight. Type I somatotrophs isolated from rats which had been previously fasted or insulin-treated subsequently showed only small, inconsistent changes in release relative to that from cells in the unfractionated cell population. However, release of BGH from the Type II cells was markedly decreased. CONCLUSION: Both IGH and BGH should be considered in the elucidation of GH physiology.

Systemic administration of IGF-I enhances healing in collagenous extracellular matrices: evaluation of loaded and unloaded ligaments.

BACKGROUND: Insulin-like growth factor-I (IGF-I) plays a crucial role in wound healing and tissue repair. We tested the hypotheses that systemic administration of IGF-I, or growth hormone (GH), or both (GH+IGF-I) would improve healing in collagenous connective tissue, such as ligament. These hypotheses were examined in rats that were allowed unrestricted activity after injury and in animals that were subjected to hindlimb disuse. Male rats were assigned to three groups: ambulatory sham-control, ambulatory-healing, and hindlimb unloaded-healing. Ambulatory and hindlimb unloaded animals underwent surgical disruption of their knee medial collateral ligaments (MCLs), while sham surgeries were performed on control animals. Healing animals subcutaneously received systemic doses of either saline, GH, IGF-I, or GH+IGF-I. After 3 weeks, mechanical properties, cell and matrix morphology, and biochemical composition were examined in control and healing ligaments. RESULTS: Tissues from ambulatory animals receiving only saline had significantly greater strength than tissue from saline receiving hindlimb unloaded animals. Addition of IGF-I significantly improved maximum force and ultimate stress in tissues from both ambulatory and hindlimb unloaded animals with significant increases in matrix organization and type-I collagen expression. Addition of GH alone did not have a significant effect on either group, while addition of GH+IGF-I significantly improved force, stress, and modulus values in MCLs from hindlimb unloaded animals. Force, stress, and modulus values in tissues from hindlimb unloaded animals receiving IGF-I or GH+IGF-I exceeded (or were equivalent to) values in tissues from ambulatory animals receiving only saline with greatly improved structural organization and significantly increased type-I collagen expression. Furthermore, levels of IGF-receptor were significantly increased in tissues from hindlimb unloaded animals treated with IGF-I. CONCLUSION: These results support two of our hypotheses that systemic administration of IGF-I or GH+IGF-I improve healing in collagenous tissue. Systemic administration of IGF-I improves healing in collagenous extracellular matrices from loaded and unloaded tissues. Growth hormone alone did not result in any significant improvement contrary to our hypothesis, while GH + IGF-I produced remarkable improvement in hindlimb unloaded animals.

Insulin-independent pathways mediating glucose uptake in hindlimb-suspended skeletal muscle.

Insulin resistance accompanies atrophy in slow-twitch skeletal muscles such as the soleus. Using a rat hindlimb suspension model of atrophy, we have previously shown that an upregulation of JNK occurs in atrophic muscles and correlates with the degradation of insulin receptor substrate-1 (IRS-1) (Hilder TL, Tou JC, Grindeland RF, Wade CE, and Graves LM. FEBS Lett 553: 63-67, 2003), suggesting that insulin-dependent glucose uptake may be impaired. However, during atrophy, these muscles preferentially use carbohydrates as a fuel source. To investigate this apparent dichotomy, we examined insulin-independent pathways involved in glucose uptake following a 2- to 13-wk hindlimb suspension regimen. JNK activity was elevated throughout the time course, and IRS-1 was degraded as early as 2 wk. AMP-activated protein kinase (AMPK) activity was significantly higher in atrophic soleus muscle, as were the activities of the ERK1/2 and p38 MAPKs. As a comparison, we examined the kinase activity in solei of rats exposed to hypergravity conditions (2 G). IRS-1 phosphorylation, protein, and AMPK activity were not affected by 2 G, demonstrating that these changes were only observed in soleus muscle from hindlimb-suspended animals. To further examine the effect of AMPK activation on glucose uptake, C2C12 myotubes were treated with the AMPK activator metformin and then challenged with the JNK activator anisomycin. While anisomycin reduced insulin-stimulated glucose uptake to control levels, metformin significantly increased glucose uptake in the presence of anisomycin and was independent of insulin. Taken together, these results suggest that AMPK may be an important mediator of insulin-independent glucose uptake in soleus during skeletal muscle atrophy.

International collaboration on Russian spacecraft and the case for free flyer biosatellites.

Animal research has been critical to the initiation and progress of space exploration. Animals were the original explorers of "space" two centuries ago and have played a crucial role by demonstrating that the space environment, with precautions, is compatible with human survival. Studies of mammals have yielded much of our knowledge of space physiology. As spaceflights to other planets are anticipated, animal research will continue to be essential to further reveal space physiology and to enable the longer missions. Much of the physiology data collected from space was obtained from the Cosmos (Bion) spaceflights, a series of Russian (Soviet)-International collaborative flights, over a 22 year period, which employed unmanned, free flyer biosatellites. Begun as a Soviet-only program, after the second flight the Russians invited American and other foreign scientists to participate. This program filled the 10 year hiatus between the last US biosatellite and the first animal experiments on the shuttles. Of the 11 flights in the Cosmos program nine of them were international; the flights continued over the years regardless of political differences between the Soviet Union and the Western world. The science evolved from sharing tissues to joint international planning and development, and from rat postmortem tissue analysis to in vivo measurements of a host of monkey physiological parameters during flight. Many types of biological specimens were carried on the modified Vostok spacecraft, but only the mammalian studies are discussed herein. The types of studies done encompass the full range of physiology and have begun to answer "critical" questions of space physiology posed by various ad hoc committees. The studies have not only yielded a prodigious and significant body of data, they have also introduced some new perspectives in physiology. A number of the physiological insights gained are relevant to physiology on Earth. The Cosmos flights also added significantly to flight-related technology, some of which also has application on our planet. In summary, the Cosmos biosatellite flights were extremely productive and of low cost. The Bion vehicles are versatile in that they can be placed into a variety of orbits and altitudes, and can carry radiation sources or other hazardous material which cannot be carried on manned vehicles. With recent advances in sensor, robotic, and data processing technology, future free flyers will be even more productive, and will largely preclude the need to fly animal experiments on manned vehicles. Currently, mammalian researchers do not have access to space for an unknown time, seriously impeding the advancement and understanding of space physiology during long duration missions. Initiation of a new, international program of free flyer biosatellites is critical to our further understanding of space physiology, and essential to continued human exploration of space.

Effect of hind limb muscle unloading on liver metabolism of rats.

In response to decreased use, skeletal muscle undergoes an adaptive reductive remodeling. There is a shift in fiber types from slow twitch to fast twitch fiber types. Associated with muscle unloading is an increased reliance on carbohydrate metabolism for energy. The hind limb suspended (HLS) rat model was used as the experimental model to determine whether skeletal muscle unloading had any impact on the liver. We used a combination of actual enzyme assays and microarray mRNA expression to address this question. The GenMAPP program was used to identify altered metabolic pathways. We found that the major changes in the liver with HLS were increases in the expression of genes involved in the generation of energy fuels for export, specifically gluconeogenesis and lipogenesis. The expression of mRNA was increased (P<0.05) for three of the four enzymes involved in the regulation of gluconeogenesis pathway (pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G-6-Pase). Actual assay of enzymatic activity, in micromol . min(-1) . mg protein(-1) showed G-6-Pase (0.14+0.01 vs 0.17+0.01 P<0.05), fructose 1,6, bisphophosphatase (0.048+0.002 vs 0.054+0.002, P<0.07), and PEPCK (0.031+0.002 vs 0.038+0.012 (P<0.05) to be increased. We conclude that 1) atrophied muscle is not the only tissue to be affected by HLS, as there is also a response by the liver; and 2) the major changes in liver substrate metabolism induced by HLS appear to be limited to glucose and triglyceride production. The increase in glycolytic capacity in disused muscle is paralleled by an increase in glucogenic capacity by the liver.

Effects of diet and exposure to hindlimb suspension on estrous cycling in Sprague-Dawley rats.

Various factors can disrupt the female reproductive cycle resulting in subfertility. The primary objective of this study was to determine whether physiological changes associated with exposure to hypogravity disrupt reproductive cycles. The hindlimb suspension (HLS) model was used to simulate the major physiological effects of hypogravity in female Sprague-Dawley rats. Also, to determine whether diet may influence reproductive results, rats were fed purified American Institute of Nutrition (AIN)-93G or chow diet. Rats (n = 9-11/group) subjected to HLS had lengthened estrous cycles due to prolonged diestrus, indicating hypoestrogenism. Interestingly, HLS rats fed AIN-93G but not chow diet had significantly reduced time spent in estrus and decreased plasma estradiol. Attenuation of hypoestrogenism in the chow-fed rats suggested that diet provided an exogenous source of estrogen. The mechanism involved in the disruption of estrous cycling remains to be determined. HLS increased urinary corticosterone (CORT) levels during the initial 4 days of HLS, suggesting that physiological responses to acute stress may be a potential mechanism in the disruption of estrous cycles. Higher basal urinary CORT was observed in rats fed chow vs. AIN-93G diet. HLS resulted in increased urinary CORT. However, two-way ANOVA indicated a significant HLS effect (P < 0.001) but no effect of HLS x diet effect on urinary CORT levels, suggesting that estrogenic activity associated with the chow diet did not enhance the stress response. The results of this study indicate that HLS, diet, and the combination of HLS and diet influence estrous cycling. This has important implications for future reproductive success in the hypogravity environment of space.

Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle.

c-Jun NH(2)-terminal kinase (JNK) has been shown to negatively regulate insulin signaling through serine phosphorylation of residue 307 within the insulin receptor substrate-1 (IRS-1) in adipose and liver tissue. Using a rat hindlimb suspension model for muscle disuse atrophy, we found that JNK activity was significantly elevated in atrophic soleus muscle and that IRS-1 was phosphorylated on Ser(307) prior to the degradation of the IRS-1 protein. Moreover, we observed a corresponding reduction in Akt activity, providing biochemical evidence for the development of insulin resistance in atrophic skeletal muscle.

Hindlimb unloading alters ligament healing.

We investigated the hypothesis that hindlimb unloading inhibits healing in fibrous connective tissue such as ligament. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing, and hindlimb-suspended healing. Ambulatory and suspended animals underwent surgical rupture of their medial collateral ligaments, whereas sham surgeries were performed on control animals. After 3 or 7 wk, mechanical and/or morphological properties were measured in ligament, muscle, and bone. During mechanical testing, most suspended ligaments failed in the scar region, indicating the greatest impairment was to ligament and not to bone-ligament insertion. Ligament testing revealed significant reductions in maximum force, ultimate stress, elastic modulus, and low-load properties in suspended animals. In addition, femoral mineral density, femoral strength, gastrocnemius mass, and tibialis anterior mass were significantly reduced. Microscopy revealed abnormal scar formation and cell distribution in suspended ligaments with extracellular matrix discontinuities and voids between misaligned, but well-formed, collagen fiber bundles. Hence, stress levels from ambulation appear unnecessary for formation of fiber bundles yet required for collagen to form structurally competent continuous fibers. Results support our hypothesis that hindlimb unloading impairs healing of fibrous connective tissue. In addition, this study provides compelling morphological evidence explaining the altered structure-function relationship in load-deprived healing connective tissue.

The calcium endocrine system of adolescent rhesus monkeys and controls before and after spaceflight.

The calcium endocrine system of nonhuman primates can be influenced by chairing for safety and the weightless environment of spaceflight. The serum of two rhesus monkeys flown on the Bion 11 mission was assayed pre- and postflight for vitamin D metabolites, parathyroid hormone, calcitonin, parameters of calcium homeostasis, cortisol, and indexes of renal function. Results were compared with the same measures from five monkeys before and after chairing for a flight simulation study. Concentrations of 1,25-dihydroxyvitamin D were 72% lower after the flight than before, and more than after chairing on the ground (57%, P < 0.05). Decreases in parathyroid hormone did not reach significance. Calcitonin showed modest decreases postflight (P < 0.02). Overall, effects of spaceflight on the calcium endocrine system were similar to the effects of chairing on the ground, but were more pronounced. Reduced intestinal calcium absorption, losses in body weight, increases in cortisol, and higher postflight blood urea nitrogen were the changes in flight monkeys that distinguished them from the flight simulation study animals.