R. E. Grindeland

Energy metabolism pathways in rat muscle under conditions of simulated microgravity

Evidence from rats flown in space suggests that there is a decrease in the ability of the soleus muscle to oxidize long chain fatty acids during space flight. The observation suggests that a shift in the pathways involved in muscle fuel utilization in the absence of load on the muscle has occurred. It is also possible that the reduction is part of a general down-sizing of metabolic capacity since energy needs of inactive muscle are necessarily less. The rodent hind limb suspension model has proved to be a useful ground based model for studying the musculo-skeletal systems changes that occur with space flight. Microarray technology permits the screening of a large number of the enzymes of the relevant pathways thereby permitting a distinction to be made between a shift fuel utilization pattern or a general decrease in metabolic activity. The soleus muscle was isolated from 5 control and 5 hindlimb suspended rats (21 days) and the Affymetrix system for assessing gene expression used to determine the impact of hindlimb unloading on fuel pathways within the muscle of each animal. RESULTS: Suspended rats failed to gain weight at the same rate as the controls (337 +/- 5 g vs 318 +/- 6 g, p < 0.05) and muscle mass from the soleus was reduced (135 +/- 3 mg vs 48 +/- 4 mg, p < 0.05). There was a consistent decrease (p < 0.05) in gene expression of proteins involved in fatty acid oxidation in the suspended group whereas glycolytic activity was increased (p < 0.05). Gene expressions of individual key regulatory enzymes reflected these changes. Carnitine palmitoyltransferase I and II were decreased (p < 0.05) whereas expression of hexokinase, phosphofructokinase and pyruvate kinase were increased (p < 0.05). CONCLUSION: Disuse atrophy is associated with a change in mRNA levels of enzymes involved in fuel metabolism indicative of a shift in substrate utilization away from fat towards glucose.

Cosmos 1887 mission overview: effects of microgravity on rat body and adrenal weights and plasma constituents.

The Cosmos 1887 biosatellite carried 10 male rats and 2 rhesus monkeys on its 12.5‐day mission. Upon reentry the Vostok vehicle overshot the designated landing site, which resulted in fasting of the animals for 42 h, exposure to cage temperatures of 12‐15°C, and 2 days delay in death of the rats. No overt untoward effects of the delayed recovery were apparent. Tissues from the rats were harvested by Soviet scientists, appropriately preserved, and provided to U.S. investigators. Flight rats grew more slowly and had larger adrenal glands than earth gravity controls. Analysis of plasma revealed increased concentrations of hepatic alkaline phosphatase, glucose, urea nitrogen, and creatinine in flight rats. In contrast, electrolytes, total protein, albumin, corticosterone, prolactin, and immunoreactive growth hormone levels were unchanged. However, testosterone concentration was marginally decreased after flight and thyroid hormone levels were suggestive of reduced thyroid function. Due to the possible effects of reentry and the delay in recovery of the animals, it is not clear what relationship postflight levels of plasma constituents bear to their concentrations in flight.—Grindeland, R. E.; Popova, I. A.; Vasques, M.; Arnaud, S. B. Cosmos 1887 mission overview: effects of microgravity on rat body and adrenal weights and plasma constituents. FASEB J. 4: 105‐109; 1990.

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

c‐Jun NH2‐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 Ser307 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.

A specific radioimmunoassay for osteocalcin with advantageous species crossreactivity

The specificities of immunoassays to rat and bovine osteocalcin are examined. Extracts of noncalcified tissues and tissue fractions are unreactive to the antibody, with the exception of the kidney, in which the reactive component appears to be identical with osteocalcin by gel filtration and dose dilution analysis. The assays, developed against protein isolated from bone, are also demonstrated to be reactive to the native protein (bone in situ) and to osteocalcin in serum. The assays are sensitive to less than 50 pg osteocalcin. Intra- and interassay coefficients of variation are less than 6.8%. The bovine antibody crossreacts with human, horse, monkey, baboon, and cat osteocalcin, while the rat antibody crossreacts with dog and mouse.

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

BackgroundInsulin-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.ResultsTissues 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.ConclusionThese 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.

Models to Study Gravitational Biology of Mammalian Reproduction

Abstract Mammalian reproduction evolved within Earths 1-g gravitational field. As we move closer to the reality of space habitation, there is growing scientific interest in how different gravitational states influence reproduction in mammals. Habitation of space and extended spaceflight missions require prolonged exposure to decreased gravity (hypogravity, i.e., weightlessness). Lift-off and re-entry of the spacecraft are associated with exposure to increased gravity (hypergravity). Existing data suggest that spaceflight is associated with a constellation of changes in reproductive physiology and function. However, limited spaceflight opportunities and confounding effects of various nongravitational factors associated with spaceflight (i.e., radiation, stress) have led to the development of ground-based models for studying the effects of altered gravity on biological systems. Human bed rest and rodent hindlimb unloading paradigms are used to study exposure to hypogravity. Centrifugation is used to study hypergravity. Here, we review the results of spaceflight and ground-based models of altered gravity on reproductive physiology. Studies utilizing ground-based models that simulate hyper- and hypogravity have produced reproductive results similar to those obtained from spaceflight and are contributing new information on biological responses across the gravity continuum, thereby confirming the appropriateness of these models for studying reproductive responses to altered gravity and the underlying mechanisms of these responses. Together, these unique tools are yielding new insights into the gravitational biology of reproduction in mammals.

Muscle Afferent–pituitary Axis: A Novel Pathway for Modulating the Secretion of a Pituitary Growth Factor

McCALL, G.E., K.L. GOSSELINK, A.J. BIGBEE, R.R. ROY, R.E. GRINDELAND, and V.R. EDGERTON. Muscle afferent–pituitary axis: A novel pathway for modulating the secretion of a pituitary growth factor. Exerc. Sport Sci. Rev., Vol. 29, No. 4, pp 164–169, 2001. Evidence is presented for a novel muscle-neuro-endocrine pathway for modulating the secretion of an unidentified pituitary growth factor by proprioceptive input from skeletal muscle afferents. Exercise stimulates the release of this growth factor, whereas chronic unloading associated with bed rest or spaceflight abrogates the normal exercise stimulus.

Synergistic ablation does not affect atrophy or altered myosin heavy chain expression in the non-weight bearing soleus muscle.

The purpose of this study was to investigate whether the soleus muscle undergoes atrophy and alterations in myosin heavy chain (MHC) composition during non-weight bearing in the absence of synergists. Thirty-two female rats were randomly assigned to four groups: control (C), synergistic ablation (ABL) of the gastrocnemius and plantaris muscles to overload the soleus muscle, hindlimb suspension (HLS), or a combination of synergistic ablation and hindlimb suspension (HLS-ABL). After 28 days of hindlimb suspension, soleus atrophy was more pronounced in HLS (58%) than in HLS-ABL (43%) rats. Compared to C rats, non-weight bearing decreased mixed and myofibrillar protein contents and Type I MHC 49%, 45%, and 7%, respectively, in HLS animals. In addition, de novo expression of fast Type IIx and Type IIb MHC (5% and 2%, respectively) was observed in HLS animals. Similarly, when compared to C rats, mixed and myofibrillar protein contents and Type I MHC decreased 43%, 46%, and 4%, respectively, in HLS-ABL animals. Also, de novo expression of Type IIx (4%) and IIb (1%) MHC was observed. Collectively, these data indicate that the loss of muscle protein and Type I MHC, and the de novo expression of Type IIx and Type IIb MHC in the rat soleus occur independently of the presence of synergists during non-weight bearing. Furthermore, these results confirm the contention that soleus mass and MHC expression are highly sensitive to alterations in mechanical load.

Growth Hormone Plus Resistance Exercise Attenuate Structural Changes in Rat Myotendinous Junctions Resulting from Chronic Unloading

Myotendinous junctions (MTJs) are specialized sites on the muscle surface where forces generated by myofibrils are transmitted across the sarcolemma to the extracellular matrix. At the ultrastructural level, the interface between the sarcolemma and extracellular matrix is highly folded and interdigitated at these junctions. In this study, the effect of exercise and growth hormone (GH) treatments on the changes in MTJ structure that occur during muscle unloading, has been analyzed. Twenty hypophysectomized rats were assigned randomly to one of five groups: ambulatory control, hindlimb unloaded, hindlimb unloaded plus exercise (3 daily bouts of 10 climbs up a ladder with 50% body wt attached to the tail), hindlimb unloaded plus GH (2 daily injections of 1 mg/kg body wt, i.p.), and hindlimb unloaded plus exercise plus GH. MTJs of the plantaris muscle were analyzed by electron microscopy and the contact between muscle and tendon was evaluated using an IL/B ratio, where B is the base and IL is the interface length of MTJ’s digit-like processes. After 10 days of unloading, the mean IL/B ratio was significantly lower in unloaded (3.92), unloaded plus exercise (4.18), and unloaded plus GH (5.25) groups than in the ambulatory control (6.39) group. On the opposite, the mean IL/B ratio in the group treated with both exercise and GH (7.3) was similar to control. These findings indicate that the interaction between exercise and GH treatments attenuates the changes in MTJ structure that result from chronic unloading and thus can be used as a countermeasure to these adaptations.

The effect of purified compared with nonpurified diet on bone changes induced by hindlimb suspension of female rats.

The purpose of this study was to compare the bone changes induced by unloading in rats fed different diets, because space flight studies use a semipurified diet, whereas space flight simulation studies typically use nonpurified diets. Female Sprague-Dawley rats were fed a purified American Institute of Nutrition (AIN) 93G diet or a standard nonpurified diet and kept ambulatory or subjected to unloading by hindlimb suspension (HLS) for 38 days. Bone mineral content (BMC), mechanical strength, and factors related to the diet that affect bone (i.e., urinary calcium excretion, estradiol, and corticosterone) were measured. Average food intakes (grams per day) differed for diets, but caloric intake (kilocalories per day) and the final body masses of treatment groups were similar. The HLS-induced decrease in femoral BMC was not statistically different for rats fed a nonpurified diet (–8.6%) compared with a purified AIN-93G diet (–11.4%). The HLS-induced decrease in femoral mechanical strength was not statistically different for rats fed a nonpurified diet (–24%) compared with a purified AIN-93G diet (–31%). However, bone lengths were decreased (P < 0.05) in rats fed a nonpurified diet compared with a purified diet. Plasma estradiol levels were lower (P < 0.05) in the HLS/AIN-93G group but similar in the HLS and ambulatory rats fed a nonpurified diet. Plasma estradiol was related to femoral BMC (r = 0.85, P < 0.01). Urinary calcium excretion was higher (P < 0.05) in rats fed a nonpurified diet than those fed a purified AIN-93G diet, which is consistent with the higher level of calcium in the nonpurified diet. Urinary corticosterone levels were higher (P < 0.05) in rats fed a nonpurified diet than rats fed the AIN-93G diet. Although the osteopenia induced by unloading was similar in both diet groups, there were differences in longitudinal bone growth, calcium excretion, plasma estradiol levels, and urinary corticosterone levels. Results indicate that the type of standard diet used is an important factor to consider when measuring bone end points.