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Hindlimb suspension (HS) of rats is a model of simulated weightlessness and induces dynamic alterations in insulin action. In the present study, the effect of acute (1-day) HS on whole-body glucose tolerance and insulin action on skeletal muscle glucose transport was assessed in juvenile, female Sprague-Dawley rats. Compared to weight-bearing control rats, 1-day HS animals displayed significantly decreased glucose tolerance and diminished whole-body insulin sensitivity. Glucose transport activity in the 1-day unweighted soleus muscle was significantly decreased (P <.05) compared to weight-bearing control muscles both in the absence and presence of insulin (2 mU/mL). Insulin-mediated glucose transport activity in the extensor digitorum longus (EDL) muscles also tended (P =.09) to be lower. There was no change in the protein expression of insulin receptor beta-subunit (IR-beta), insulin receptor substrate-1 (IRS-1), IRS-2, the p85 subunit of phosphatidylinositol-3 kinase (PI3-kinase), Akt, and glucose transporter protein 4 (GLUT-4). The activities of these proteins were also unchanged, as insulin-stimulated IR-beta tyrosine phosphorylation, IRS-1 tyrosine phosphorylation, IRS-1-associated p85, and Akt serine phosphorylation were similar to controls. However, basal Akt phosphorylation was significantly depressed (P <.05) in the 1-day HS soleus. In addition, the protein expression and basal phosphorylation of the stress-activated p38 mitogen-activated protein kinase (p38 MAPK) were significantly elevated (P <.05) in the 1-day unweighted soleus. These results indicate that the development of insulin resistance in the 1-day unweighted soleus is not due to impaired functionality of elements involved in the IR/IRS-1/PI3-kinase/Akt signaling pathway. However, activation of p38 MAPK may play a role in this response.

Hindlimb suspension (HS), a model of simulated weightlessness, enhances insulin action on glucose transport in unweighted rat soleus muscle. In the present study, we tested the hypothesis that these changes in glucose transport in 3- and 7-day HS soleus of juvenile, female Sprague-Dawley rats were due to increased functionality of insulin signaling factors, including insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3-kinase), and Akt. Insulin-stimulated (2 mU/ml) glucose transport was significantly (P < 0.05) enhanced in 3- and 7-day HS soleus by 59 and 113%, respectively, compared with weight-bearing controls. Insulin-stimulated tyrosine phosphorylation of IR and Ser(473) phosphorylation of Akt was not altered by unweighting. Despite decreased (34 and 64%) IRS-1 protein in 3- and 7-day HS soleus, absolute insulin-stimulated tyrosine phosphorylation of IRS-1 was not diminished, indicating relative increases in IRS-1 phosphorylation of 62 and 184%, respectively. In the 7-day HS soleus, this was accompanied by increased (47%) insulin-stimulated IRS-1 associated with the p85 subunit of PI3-kinase. Interestingly, the enhanced insulin-stimulated glucose transport in the unweighted soleus was not completely inhibited (89-92%) by wortmannin, a PI3-kinase inhibitor. Finally, protein expression and activation of p38 MAPK, a stress-activated serine/threonine kinase associated with insulin resistance, was decreased by 32 and 18% in 7-day HS soleus. These results indicate that the increased insulin action on glucose transport in the 7-day unweighted soleus is associated with increased insulin signaling through IRS-1 and PI3-kinase and decreased p38 MAPK protein expression. However, PI3-kinase-independent mechanisms must also play a small role in this adaptive response to HS.

Analogs of human insulin have been used to discriminate between responses of metabolic and mitogenic (growth-related) pathways. This study compared the stimulatory effects of human insulin (HI) and 2 analogs (X2, B-Asp(9), B-Glu(27) and H2, A-His(8),B-His(4),B-Glu(10), B-His(27)) on glucose uptake and protein synthesis in rat soleus muscle in situ. Glucose uptake, estimated by intramuscular (IM) injection of 2-deoxy[1,2-3H]glucose with or without insulin, was maximally increased at 10(-6) mol/L for HI and X2 and 10(-7) mol/L for H2. HI had a larger effect (318%) than either X2 (156%) or H2 (124%). The half-maximal effect (ED(50)) values for HI, X2, and H2 were 3.3 x10(-8) mol/L, 1.7 x 10(-7) mol/L, and 1.6 x 10(-9) mol/L, respectively. Protein synthesis, estimated by protein incorporation of [(3)H]phenylalanine injected into muscles with or without insulin, was maximally increased at 10(-5) mol/L for HI and 10(-6) for X2 and H2. HI had a larger effect in stimulating protein synthesis (34%) than either X2 (25%) or H2 (19.8%). The ED(50) values for HI, X2, and H2 were 3.0 x 10(-7) mol/L, 3.2 x 10(-7) mol/L, and 1.0 x 10(-9) mol/L, respectively. The biological potency of each analog (ED(50)insulin/ED(50)analog) showed X2 to be less potent than HI for both glucose uptake (0.2) and protein synthesis (0.9), whereas H2 is more potent than HI with ratios of 20 and 300, respectively. These data suggest that this approach for studying insulin responsiveness in a single muscle in situ may be a useful tool for investigating insulin signaling in muscle in vivo., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

We examined the mechanism of accelerated proteolysis in denervated rat soleus muscles. The soleus was denervated by severing either the tibial nerve (proximal, short stump) or sciatic nerve (distal, long stump) at 24, 48, 72, or 96 h before excision. Twenty-four hours after denervation, the extent of atrophy was similar for proximal and distal denervation, although lysosomal latency declined in both groups. After 48 and 72 h, denervation resulted in a decline in protein content, an increase in in vitro protein degradation, and a decline in lysosomal latency, all of which were greater in proximally denervated than in contralateral distally denervated muscles. These differences between acute responses of proximally and distally denervated muscles suggest the retention of some factor in the longer nerve stump that attenuates atrophy. After 96 h, total protein loss, protein degradation, and lysosomal latency were similar for proximal and distal denervation, suggesting the loss of axoplasmic flow from the long nerve stump.

Unweighting atrophy of immature soleus muscle occurs rapidly over the first several days, followed by slower atrophy coinciding with increased sensitivity to insulin of in vitro protein metabolism. This study determined whether this increased sensitivity might account for the diminution of atrophy after 3 days of tall-cast hindlimb suspension. The physiological significance of the increased response to insulin in unweighted muscle was evaluated by analyzing in vivo protein metabolism for day 3 (48 to 72 hours) and day 4 (72 to 96 hours) of unweighting in diabetic animals either injected with insulin or not treated. Soleus from nontreated diabetic animals showed a similar loss of protein during day 3 (-16.2%) and day 4 (-14.5%) of unweighting, whereas muscle from insulin-treated animals showed rapid atrophy (-14.5%) during day 3 only, declining to just -3.1% the next day. Since fractional protein synthesis was similar for both day 3 (8.6%/d) and day 4 (7.0%/d) of unweighting in insulin-treated animals, the reduction in protein loss must be accounted for by a slowing of protein degradation due to circulating insulin. Intramuscular (IM) injection of insulin (600 nmol/L) stimulated in situ protein synthesis similarly in 4-day unweighted (+56%) and weight-bearing (+90%) soleus, even though unweighted muscle showed a greater in situ response of 2-deoxy-[3H]glucose uptake to IM injection of either insulin (133 nmol/L) or insulin-like growth factor-I (IGF-I) (200 nmol/L) than control muscle. These findings suggest that unweighted muscle is selectively more responsive in vivo to insulin, and that the slower atrophy after 3 days of unweighting was due to an increased effect of insulin on inhibiting protein degradation.

The potential role of neurotrophic factors in the decline of glucose transporter (GLUT-4) protein levels and citrate synthase (CS) activity was studied by comparing distally with proximally denervated juvenile rat soleus muscle. Severing of the tibial nerve produced distal (long stump) or proximal (short stump) denervation. GLUT-4 levels and CS activities were measured at 24-h intervals for up to 96 h after denervation. No differences were observed in GLUT-4 or CS activity between soleus muscles left with short or long nerve stumps at any time point. However, within just 24 h, denervation decreased (P < 0.05). GLUT-4 and CS (67.4 +/- 3.3 and 63.4 +/- 1.7% of innervated control values, respectively). Both parameters continued to decline up to 96 h (44.4 +/- 3.1 and 48.7 +/- 4.0%, respectively). There was a significant correlation between the GLUT-4 protein level and CS activity over this 96-h period of denervation (r = 0.653, P < 0.001). A similar response in the 24-h denervated soleus of adult rats was observed. In contrast, 24-h denervation of red gastrocnemius (type IIa fibers) left with a long nerve stump resulted in a prevention of the decline of GLUT-4 and CS seen in red gastrocnemius left with a short nerve stump in both juvenile and adult animals. These results suggest that unlike type IIa muscles, the decline in GLUT-4 level and CS activity in type I soleus muscle after denervation results from a lack of coordinated electrical activity but likely does not involve a neurotrophic agent. These results also support the hypothesis that there is coregulation of decreased expression of GLUT-4 protein and CS activity in this model of reduced neuromuscular activity.

The purpose of this study was to determine whether there are local regions in resting skeletal muscle in which the oxygen delivery is insufficient to support oxidative metabolism. This hypothesis was tested by stopping the blood supply to the exteriorized cat sartorius muscle for 5 min while monitoring NADH fluorescence in localized tissue areas 15-25 microns in diameter. A rise in fluorescence was taken to indicate a shift to anaerobic metabolism. Tissue sites in the arteriolar and venular regions of the capillary network were selected for study. After flow stoppage, fluorescence did not change for an average of 48 +/- 22 (SD) s and then rose over a period of 61 +/- 27 s to an average value 55 +/- 19% above control for arteriolar and venular sites combined. Fluorescence began to rise within 5 s of flow stasis in only 1 of 61 sites and within 10 s in 2 sites. There was no difference in the time course or magnitude of fluorescence changes at arteriolar and venular sites. The data indicate that in resting skeletal muscle, oxygen supply appears sufficient to support oxidative metabolism in over 95% of the tissue.

The most studied skeletal muscles which depend on gravity, "antigravity" muscles, are located in the posterior portion of the legs. Antigravity muscles are characterized generally by a different fiber type composition than those which are considered nonpostural. The gravity-dependent function of the antigravity muscles makes them particularly sensitive to weightlessness (unweighting) resulting in a substantial loss of muscle protein, with a relatively greater loss of myofibrillar (structural) proteins. Accordingly alpha-actin mRNA decreases in muscle of rats exposed to microgravity. In the legs, the soleus seems particularly responsive to the lack of weight-bearing associated with space flight. The loss of muscle protein leads to a decreased cross-sectional area of muscle fibers, particularly of the slow-twitch, oxidative (SO) ones compared to fast-twitch glycolytic (FG) or oxidative-glycolytic (FOG) fibers. In some muscles, a shift in fiber composition from SO to FOG has been reported in the adaptation to spaceflight. Changes in muscle composition with spaceflight have been associated with decreased maximal isometric tension (Po) and increased maximal shortening velocity. In terms of fuel metabolism, results varied depending on the pathway considered. Glucose uptake, in the presence of insulin, and activities of glycolytic enzymes are increased by space flight. In contrast, oxidation of fatty acids may be diminished. Oxidation of pyruvate, activity of the citric acid cycle, and ketone metabolism in muscle seem to be unaffected by microgravity.

The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult.

Unweighting the hindlimbs by tail suspension of juvenile rats leads to atrophy of the soleus muscle, especially during the third day of unweighting. Although a previous study using adrenalectomized animals suggested a minimal role of glucocorticoids in this atrophy, the inability of adrenalectomized animals to release other adrenal hormones could have been important. Therefore, the influence of oral administration of RU38486 [11-beta-(4-dimethylaminophenyl) 17-beta-hydroxy 17-alpha(prop-1-ynyl) estra 4,9-dien 3-one], a selective glucocorticoid antagonist, on protein metabolism in the unweighted soleus was studied. The effectiveness of RU38486 treatment was demonstrated in hindlimb-suspended rats as the drug abolished the increase in soleus glutamine synthetase activity shown previously to be caused by elevated circulating glucocorticoids. The slower weight gain of suspended rats was unaffected by the drug. After 3 days of unweighting, the difference in protein content from weighted soleus muscle was not diminished significantly by RU38486 (-25%, vehicle only; -18%, RU38486-treated). However, in both weight-bearing and suspended animals, RU38486 seemed to promote protein accretion between days 2 and 3 of the experiment; ie, unweighted muscle seemed to lose less protein. All suspended animals showed slower (-58% to -64%) fractional in vivo rates of synthesis. RU38486 did not affect these percent differences in fractional protein synthesis after either 2 or 3 days of unweighting. The apparent improvement in protein balance likely resulted from a decline in protein degradation in both the weight-bearing (-26%) and unweighted (-35%) soleus.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of exercise training with or without subsequent unweighting on wet weight, protein content, and in vivo fractional protein synthesis were studied in soleus and plantaris muscles of juvenile female Sprague-Dawley rats under the following four conditions: normal weight bearing (N), voluntary-activity wheel running (WR) for up to 4 weeks, mechanical unweighting for 7 days via hindlimb suspension (HS), or wheel running followed by 7 days of hindlimb suspension (WR-HS). Fractional protein synthesis was determined by the 3H-phenylalanine flooding method. Increases (P < .05) in wet weight and protein content were detected in the soleus after just 1 week of running, with no increase in fractional protein synthesis. Two weeks of running were required for an increase in protein synthesis in this muscle. Significant increases in these parameters were first observed in the plantaris after 2 weeks of running. Maximal increases occurred by 3 weeks in both muscles. Reductions (P < .05) in soleus and plantaris parameters were observed in both HS and WR-HS groups compared with N and WR groups, respectively. However, protein content and fractional synthesis were maintained at significantly higher levels in WR-HS muscles compared with HS muscles. These results indicate that (1) wheel training represents a noninvasive method for inducing rapid hypertrophy of the skeletal muscles studied, in part by increasing fractional protein synthesis; (2) unweighting decreases protein content and synthesis to the same extent whether the muscles are trained; and (3) previously hypertrophied muscles display higher protein contents and fractional protein synthesis following unweighting compared with unweighted muscles of untrained animals.

The present study provides experimental data which indicate that the neutrophil is ideal for studying programmed cell death or apoptosis in vitro. Neutrophils can be obtained from human peripheral blood in large numbers with minimal experimental manipulation and are easily separated from other leukocytes, providing nearly pure cell suspensions. The neutrophil life span in vitro is sufficiently short to allow observations to be made within eight hours after experimental manipulation. Neutrophils can also be easily maintained in serum-free, chemically defined media which can be systematically altered, thereby defining specific variables that influence the apoptotic process. Since the neutrophils do not need an exogenous trigger to undergo programmed cell death, it is also an excellent model to study senescence. It was determined from this study that neutrophils undergo apoptosis most efficiently at 37 degrees C, a temperature requirement for physiologic cell death. Neutrophils undergo apoptosis at a slightly faster rate and maintain membrane integrity better when incubated in a tissue culture medium (e.g., RPMI 1640) compared with a balanced salt solution (e.g., HBBB). Cycloheximide, an inhibitor of protein synthesis, was shown to accelerate apoptosis in a dose-dependent manner. The presence of Zn++ significantly decreased the rate of apoptosis, whereas the presence of Ca++ and Mg++ had no apparent effect. These studies indicate that the process of senescence, culminating in cell death, is subject to modulation by a variety of agents and experimental conditions. In addition, the ultrastructural features of neutrophils undergoing programmed cell death in vitro were compared in detail to those occurring in vivo and were found to be comparable.

The adaptive responses of myocardial protein metabolism to chronic increases in work load were evaluated in juvenile female Sprague-Dawley rats. Rats were studied under four conditions: normal weight bearing (N), voluntary wheel running (WR) for < or = 4 wk, head-down-tilt suspension for 7 days (HS), or wheel running (2 or 3 wk) followed by 7 days of suspension (WR-HS). WR activity plateaued after 2 wk at 16 km/day and was maintained through week 4. WR did not affect normal whole body growth. Protein metabolism was studied by measuring heart protein content and in vivo fractional rate of protein synthesis with the [3H]phenylalanine "flooding dose" method. Two weeks of WR increased (P < 0.05) absolute heart protein content (22%) and protein synthesis (21%) relative to age-matched N group values. These differences in protein content and synthesis were maintained for > or = 4 wk. Rats failed to gain significant body weight during suspension. Heart protein content increased (P < 0.05) by 12% to 26% as did protein synthesis (14% to 22%) in HS compared with N group. In WR-HS group, cardiac protein content and protein synthesis were maintained at significantly elevated levels. These findings indicate that 1) high-volume WR by young rats provides a convenient noninvasive method for producing rapid and substantial cardiac hypertrophy, which results, at least in part, from enhanced cardiac protein synthesis; and 2) head-down suspension of sedentary juvenile rats leads to increased cardiac protein synthesis, which helps to increase cardiac protein content despite a lack of whole body growth.

Recent evidence by Kandarian et al. (J. Appl. Physiol. 71: 910-914, 1991) indicates that prolonged (28 days) unweighting of the rat soleus by hindlimb suspension results in a substantial increase in interstitial fluid volume (IFV), as defined by the inulin space. The lack of any significant difference in absolute IFV values between unweighted and control groups suggested that this elevated IFV was a consequence of muscle atrophy. Using young female rats, we directly tested this hypothesis by comparing the early responses of soleus muscle weight and IFV with unweighting by tail-cast suspension or actual exposure to microgravity during spaceflight. Significant differences from control were first observed after 3 days of suspension unweighting for soleus wet weight (-14%; P < 0.01) and IFV (+35%; P < 0.01) and increased further after 6 days (-32% and +53%, respectively; both P < 0.001). After 5.4 days of spaceflight, soleus wet weight was 38% less and IFV was 52% greater than control (both P < 0.001). A highly significant negative correlation between soleus wet weight and IFV for all groups was observed (r = -0.70, P < 0.001). These data indicate that elevated soleus IFV develops at an early time point during unweighting and that there is a direct relationship between the magnitude of this increase in IFV and the extent of muscle atrophy. This relationship also exists in soleus muscles unweighted by exposure to a microgravity environment.

Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of 3H-phenylalanine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show (1) acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and (2) sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.

Our knowledge of the effects of unweighting on skeletal muscle of juvenile rapidly growing rats has been obtained entirely by using hindlimb-suspension models. No spaceflight data on juvenile animals are available to validate these models of simulated weightlessness. Therefore, eight 26-day-old female Sprague-Dawley albino rats were exposed to 5.4 days of weightlessness aboard the space shuttle Discovery (mission STS-48, September 1991). An asynchronous ground control experiment mimicked the flight cage condition, ambient shuttle temperatures, and mission duration for a second group of rats. A third group of animals underwent hindlimb suspension for 5.4 days at ambient temperatures. Although all groups consumed food at a similar rate, flight animals gained a greater percentage of body mass per day (P < 0.05). Mass and protein data showed weight-bearing hindlimb muscles were most affected, with atrophy of the soleus and reduced growth of the plantaris and gastrocnemius in both the flight and suspended animals. In contrast, the non-weight-bearing extensor digitorum longus and tibialis anterior muscles grew normally. Earlier suspension studies showed that the soleus develops an increased sensitivity to insulin during unweighting atrophy, particularly for the uptake of 2-[1,2-3H]deoxyglucose. Therefore, this characteristic was studied in isolated muscles within 2 h after cessation of spaceflight or suspension. Insulin increased uptake 2.5- and 2.7-fold in soleus of flight and suspended animals, respectively, whereas it increased only 1.6-fold in control animals. In contrast, the effect of insulin was similar among the three groups for the extensor digitorum longus, which provides a control for potential systemic differences in the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Unweighting, but not denervation, of muscle reportedly "spares" insulin receptors, increasing insulin sensitivity. Unweighting also increases beta-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and beta-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a beta-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular [IM] injection) to a greater degree (P less than .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased beta-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (Bmax) for [125I]iodo-(-)-pindolol in particulate preparations of unweighted (420.10(-18) mol/mg muscle) than of control or denervated muscles (285.10(-18) mol/mg muscle). The three dissociation constant (Kd) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the "sparing" and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished cAMP accumulation in the presence of theophylline in unweighted muscle (-66% +/- 2%) more than in controls (-42% +/- 6%, P less than .001). These results show that insulin affects cAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18-30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.

A food bar diet is used for rats in space flight. Since ground based studies have only been performed with the typical rat chow in dry pellet form, we tested whether the food bar diet allows normal growth and normal response of muscle protein content to unloading. These parameters were measured in normal and tail-cast hind limb suspended rats fed standard pellets or food bars. Body mass following 5 d of hind limb unloading was similar in bar-fed (97.9 +/- 4.8 g) and pellet-fed (92.6 +/- 3.4 g) animals (p greater than 0.05). In addition, gains in body mass were comparable between bar-fed (5.3 g/d) and pellet-fed (5.1 g/d) animals. Food bar consumption over 6 d increased from 10.5 to 12.0 g/d animal. During 5 d of hind limb suspension, food bar consumption increased from 13.2 +/- 1.4 to 19.1 +/- 1.4 g/d per animal. In agreement with previous studies, hind limb unloading reduced soleus muscle mass and protein content per 100 g body mass in both diet groups (p less than 0.05). Protein content per 100 g body mass was unchanged for the plantaris, extensor digitorum longus and tibialis anterior muscles during suspension in both diet groups. Rodent consumption of a food bar diet results in normal gains in body mass and muscle protein when compared to a standard pellet diet, and does not alter the atrophic response of skeletal muscle to unloading.

The effects of insulin on carbohydrate metabolism in atrophied rat soleus muscle are increased after unweighting by tail-cast suspension. This work has been extended by testing the effect of unweighting on the response of carbohydrate metabolism to isoproterenol, a beta-adrenergic agonist. Isoproterenol promoted glycogen degradation more in the unweighted than in the weight-bearing soleus but showed no differences in the extensor digitorum longus, which is unresponsive to hindlimb unweighting. In soleus muscles depleted of glycogen, to avoid varied inhibitory effects of glycogen on glycogen synthesis, isoproterenol inhibited this process more in the unweighted muscle. Isoproterenol did not have a greater inhibitory effect on net uptake of 2-deoxy-D[1,2-3H]glucose by the unweighted muscle. Measurements of intracellular 2-deoxy-[3H]glucose 6-phosphate and 3-O-methyl-D-[1-3H]glucose, which cannot be phosphorylated, showed that isoproterenol inhibited glucose phosphorylation but not transport. This effect could be explained by an increase of glucose 6-phosphate, an inhibitor of hexokinase. At 100 microU insulin/ml but not at a lower amount (10 microU/ml), isoproterenol inhibited hexose phosphorylation more in the control than in the unweighted muscle. This result may be explained by greater insulin antagonism in the unweighted muscle owing to increased insulin sensitivity. However, insulin antagonism of isoproterenol stimulation of glycogenolysis or inhibition of glycogenesis was not altered by unweighting. Therefore, for some aspects of carbohydrate metabolism, the unweighted muscle has an increased response to beta-adrenergic activation, just as this muscle shows increased responses to insulin.

Previous reports indicated that glucose uptake in denervated muscle is resistant to insulin, while in non-weight-bearing (unweighted) muscle this effect of insulin is enhanced. To extend the comparison of these differences, insulin effects on amino acid uptake and protein metabolism were studied in soleus muscles subjected to denervation or unweighting. Denervated muscle showed insulin resistance of both 2-deoxy[1,2-3H]glucose and alpha-[methyl-3H]aminoisobutyric acid uptake whereas unweighted muscle showed an increased or normal response, respectively. Atrophy was greater in denervated than in unweighted muscle, apparently due to faster protein degradation. The stimulation of protein synthesis and the inhibition of protein degradation by insulin was generally less in denervated than in unweighted muscle. Since metabolic measurements in denervated-unweighted muscles did not differ from those in denervated-weight-bearing muscles, effects of denervation must be independent of leg posture.

Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteolysis were more rapid and lysosomal latency was lower in denervated than in unweighted muscle. In vitro, lysosomotropic agents (eg, chloroquine, methylamine) did not lessen the increase in proteolysis caused by unweighting, but abolished the difference in proteolysis between denervated and unweighted muscle. Leucine methylester, an indicator of lysosome fragility, lowered latency more in denervated than in unweighted muscle. 3-Methyladenine, which inhibits phagosome formation, increased latency similarly in all muscles tested. Mersalyl, a thiol protease inhibitor, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which antagonizes sarcoplasmic reticulum release of Ca2+, reduced accelerated proteolysis caused by unweighting without diminishing the faster proteolysis due to denervation. Calcium ionophore (A23187) increased proteolysis more so in unweighted than control muscles whether or not Ca2+ was present. Different mechanisms of accelerated proteolysis were studied further by treating muscles in vivo for 24 hours with chloroquine or mersalyl. Chloroquine diminished atrophy of the denervated but not the unweighted muscle, whereas mersalyl prevented atrophy of the unweighted but not of the denervated muscle, both by inhibiting in vivo proteolysis. These results suggest that (1) atrophy of denervated, but not of unweighted, soleus muscle involves increased lysosomal proteolysis, possibly caused by greater permeability of the lysosome, and (2) cytosolic proteolysis is important in unweighting atrophy, involving some role of Ca2(+)-dependent proteolysis and/or thiol proteases.

Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both "flooding-dose" and "non-carrier" techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 micromoles/insect) together with the [3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24-53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76-86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 micrograms/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.

Both left and right atria from fasted rats produced significant amounts of 14CO2 during incubation with U-14C-labeled leucine, isoleucine, valine, alanine, glutamate, glutamine, aspartate, asparagine, proline, threonine, or lysine. This pattern of amino acid metabolism resembles that of skeletal muscle. Production of 14CO2 from [1-14C]leucine was 2.5-fold greater in atria from fasted than from fed rats and was due to greater alpha-ketoisocaproic dehydrogenase activity in the tissue from fasted animals. At normal plasma concentrations, leucine reduced the oxidation of glucose and lactate in atria from fasted but not from fed rats by inhibiting pyruvate oxidation and without altering the rate of glycolysis. Leucine also reduced glucose oxidation when added in the presence of ketone bodies or other amino acids and stimulated the release of lactate into the medium. Although the leucine skeleton can be completely oxidized to CO2 and thus can serve as an alternative fuel in fasting in place of glucose, oxidation of leucine (like glucose or lactate oxidation) accounts only for a very small fraction of the total oxygen consumption of the resting atria.

Atrophy and growth failure of muscle in a tail-cast suspension model were evaluated in hindlimbs of female Sprague-Dawley rats. Based on measurements of food consumption, animal growth rate, urinary excretion of urea and ammonia, and muscle size, 6 days seemed to be the optimum duration of suspension for studying muscle unloading. After 6 days, the soleus, plantaris, and gastrocnemius muscles from suspended animals were 27, 10, and 11% smaller (P less than 0.05), respectively, than those from tail-casted weight-bearing animals. The extensor digitorum longus and tibialis anterior muscles were unaffected by suspension (less than or equal to 6 days) while the triceps brachii hypertrophied (8%, P less than 0.05). Wet weight-to-dry weight ratios were smaller in the plantaris (-0.19, P less than 0.05) and gastrocnemius (-0.19, P less than 0.05) muscles from suspended rats. In the plantaris, this difference coincided with a higher protein concentration (+12 mg/g, P less than 0.001). In vitro measurements of protein metabolism in the soleus muscles of suspended rats showed both slower protein synthesis (P less than 0.05) and faster protein degradation (P less than 0.05), whereas these processes were unaltered in the extensor digitorum longus muscles.

In isolated rat diaphragms, only those substrates that increased the tissue NADH/NAD+ ratio lowered the rate of proteolysis. However, direct inhibition of proteinase activity by leupeptin promoted oxidation of the NAD couple of the muscles. These results suggest that changes in muscle reduction-oxidation state may be important in the regulation of proteolysis.

Soft tissue injury to one hindlimb of rats was used to test the metabolic response of atrial and ventricular muscle to trauma. Effects of insulin on muscle metabolism were also studied. In myocytes and atria from normal animals, insulin increased protein synthesis and decreased protein degradation. For myocytes of rats at one and two days after trauma, this effect of insulin on proteolysis could not be detected. Over the next two days, the inhibitory effect returned to normal. Insulin also did not increase protein synthesis on day 1, but did thereafter. In atria, in contrast to heart cells, the inhibitory effect of insulin on proteolysis was enhanced at two and three days after trauma, and its stimulation of protein synthesis was unaltered. Insulin increased carbohydrate metabolism in both myocytes and atria of normal rats and traumatized rats after 2 days, and trauma did not alter this response. In myocytes, but not atria, trauma led to a faster oxidation of leucine and a significant rise in the production of alanine. Production of glutamine and glutamate was not affected in either tissue. These results show that the metabolic responses to trauma of atrial and ventricular muscle differ considerably.

One leg of tail-casted suspended rats was immobilized in a plantar-flexed position to test whether chronic shortening of posterior leg muscles affected the metabolic response to unloading. The immobilized plantaris and gastrocnemius muscles of these animals showed approximately 20% loss of muscle mass in contrast to simply a slower growth rate with unloading. Loss of mass of the soleus muscle during suspension was not accentuated by chronic shortening. Although protein degradation in the isolated soleus muscle of the plantar-flexed limb was slightly faster than in the contralateral free limb, this difference was offset by faster synthesis of the myofibrillar protein fraction of the chronically shortened muscle. Total adenine nucleotides were 17% lower (P less than 0.005) in the chronically shortened soleus muscle following incubation. Glutamate, glutamine, and alanine metabolism showed little response to chronic shortening. These results suggest that, in the soleus muscle, chronic shortening did not alter significantly the metabolic responses to unloading and reduced activity.

Isolated soleus muscle deprived of oxygen produces more lactate and alanine than oxygen-supplied muscle. Oxygenated muscle synthesized glutamine, while anoxic muscle used this amino acid. Oxygen deprivation decreased adenine nucleotides leading to the efflux of nucleosides. Protein synthesis and degradation responded differently to anoxia. Synthesis almost completely ceased, while proteolysis increased. Therefore, protein degradation in soleus muscle is enhanced when energy supplies and oxygen tension are low.

Soft tissue injury to one hindlimb produced trauma in rats without affecting their food intake or weight gain. Histologic examination showed damage to the soleus and gastrocnemius muscles but not to the extensor digitorum longus muscle. The protein content of the injured soleus muscle was lower than that of the contralateral soleus at one day after injury, and was reflected in vitro by a faster rate of protein degradation. The injured soleus also showed greater rates of protein synthesis, glucose uptake, glycolysis, oxidation of glucose, pyruvate, and leucine, and de novo synthesis of alanine. During three days after the injury, urinary nitrogen excretion increased progressively and was paralleled by a faster rate of protein degradation in uninjured muscles incubated with glucose, insulin, and amino acids. In these muscles, the inhibition of protein degradation by insulin diminished, while its stimulation of protein synthesis was unaffected. This insensitivity of proteolysis to insulin in trauma can explain the increased rate of this process. The oxidation of glucose and pyruvate were lower in the diaphragms of traumatized than of normal rats incubated with leucine, while glycolysis and uptake of 2-deoxyglucose did not differ. The degradation of leucine and isoleucine was greater in the diaphragms of traumatized animals and was associated with a faster de novo synthesis of alanine. For the uninjured soleus muscles of the traumatized rats, the slower rates of oxidation of glucose, glycolysis, and uptake of 2-deoxyglucose in the presence of insulin showed an insensitivity of glucose metabolism to this hormone. In contrast, no differences were seen in these various metabolic processes between the extensor digitorum longus muscles of traumatized and normal rats. These data suggest that the response of skeletal muscles to trauma may depend on their physiologic and biochemical characteristics.

Protein synthesis and degradation were measured simultaneously in epididymal fat pads of rats by use of the incorporation of [14C]phenylalanine into protein and the sum of net protein breakdown and protein synthesis, respectively. Neither glucose nor insulin altered protein synthesis, but together they promoted this process; pyruvate could be substituted for glucose. Separately, glucose or insulin diminished proteolysis, and these effects were additive. In the presence of glucose and insulin, leucine, alanine, glutamine, glutamate, and aspartate lowered protein degradation to varying degrees but did not alter protein synthesis. Glutamate, but not leucine or alanine, was inhibitory without glucose and insulin present. When aminooxyacetic acid was provided to decrease the rate of transamination of amino acids, the inhibitory effects of leucine, alanine, and aspartate, but not of glutamate, appeared to be diminished. alpha-Ketoglutarate, but neither alpha-ketoisocaproate nor pyruvate, could diminish proteolysis. Inhibition of proteolysis was associated with a higher tissue content of glutamate and a greater production of glutamate and glutamine. These results suggest that glutamate itself may inhibit proteolysis in adipose tissue and mediate, at least in part, the effects of other amino acids.

Effects of stretching on muscle amino acids were tested in unloaded soleus by casting the foot in dorsiflexion on one limb of tail-casted, hindquarter-suspended rats. For comparison with unloading, amino acids also were measured in shortened extensor digitorum longus (EDL) in the same casted limb and in denervated leg muscles. Concentrations of tyrosine and glutamate were lower, while aspartate, ammonia, and the ratio of glutamine to glutamate were greater in the stretched than in the freely moving, unloaded soleus, but stretched did not differ from weight-bearing, control muscle. Therefore, stretching the soleus muscle prevented changes in certain amino acids due to unloading. Aspartate, ammonia, glutamine, and the ratio of glutamine to glutamate were lower in the shortened EDL than in the freely moving muscle of the contralateral limb, or in the control muscle. When denervated, these leg muscles also showed lower aspartate, ammonia, and ratio of glutamine to glutamate relative to innervated muscles. Since muscle shortening or denervation produced amino acid changes that mimicked the effects of unloading on the soleus, these responses must reflect the effect of muscle disuse. These data suggested that lower ammonia might cause the lower ratio of glutamine to glutamate with disuse. Because the fresh muscle energy charge, one factor which controls AMP deaminase, generally was not affected by disuse, altered deamination of glutamate via glutamate dehydrogenase may explain the variations in muscle ammonia.(ABSTRACT TRUNCATED AT 250 WORDS)

The time course of glycogen changes in soleus muscle recovering from 3 days of nonweight bearing by hindlimb suspension was investigated. Within 15 min and up to 2 h, muscle glycogen decreased. Coincidentally, muscle glucose 6-phosphate and the fractional activity of glycogen phosphorylase, measured at the fresh muscle concentrations of AMP, increased. Increased fractional activity of glycogen synthase during this time was likely the result of greater glucose 6-phosphate and decreased glycogen. From 2 to 4 h, when the synthase activity remained elevated and the phosphorylase activity declined, glycogen levels increased (glycogen supercompensation). A further increase of glycogen up to 24 h did not correlate with the enzyme activities. Between 24 and 72 h, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that reloading transiently uncoupled glycogen control of this enzyme. These data suggest that the activities of glycogen synthase and phosphorylase, when measured at physiological effector levels, likely provide the closest approximation to the actual enzyme activities in vivo. Measurements made in this way effectively explained the majority of the changes in the soleus glycogen content during recovery from nonweight bearing.

The effect of acutely reduced weight bearing (unloading) on the in vitro uptake of 2-[1,2-3H]deoxy-D-glucose was studied in the soleus muscle by tail casting and suspending rats. After just 4 h, the uptake of 2-deoxy-D-glucose fell (-19%, P less than 0.01) and declined further after an additional 20 h of unloading. This diminution at 24 h was associated with slower oxidation of [14C]glucose and incorporation of [14C]glucose into glycogen. Unlike after 1 day, at 3 days of unloading basal uptake of 2-deoxy-D-glucose did not differ from control. Reloading of the soleus after 1 or 3 days of unloading increased uptake of 2-deoxy-D-glucose above control and returned it to normal within 6 h and 4 days, respectively. These effects of unloading and recovery were caused by local changes in the soleus, because the extensor digitorum longus from the same hindlimbs did not display any alterations in uptake of 2-deoxy-D-glucose or metabolism of glucose. This study demonstrates that alterations in contractile activity, brought about by unloading or recovery from unloading, can influence the regulation of glucose transport in the soleus.

Degradation of branched-chain amino acids was studied in muscles of unloaded hind limbs from rats subjected to six days of tail-cast suspension. The total production of 14CO2 from uniformly labeled 14C-leucine, isoleucine, or valine, and the fluxes through leucine aminotransferase and alpha-ketoisocaproate dehydrogenase, which were measured using L-1-14C-leucine, were generally greater in the soleus and extensor digitorum longus muscles of unloaded than of weight-bearing hind limbs. Adrenalectomy abolished any difference in flux through the aminotransferase, whereas the administration of cortisol to adrenalectomized animals restored the greater flux in the unloaded soleus muscle. Adrenalectomy partially diminished the greater flux through alpha-ketoisocaproate dehydrogenase in the unloaded soleus, whereas cortisol (2 mg/100 g body weight) treatment increased this difference. In the extensor digitorum longus, adrenalectomy abolished the differences in both enzyme fluxes due to hind limb suspension. In this muscle, cortisol treatment increased these fluxes to a similar extent in both weight-bearing and suspended, adrenalectomized animals so that the normal difference was not restored. These results suggest that leucine catabolism in hind limb muscles of suspended rats was influenced primarily by increased circulating glucocorticoid hormones, which are elevated twofold to fourfold in these animals.

Amino acid metabolism was investigated in atrophied soleus muscle from rats subjected to six days of tail-cast, hindlimb suspension. The fresh-frozen unloaded muscle showed higher concentrations of tyrosine and glutamate but lower amounts of aspartate, glutamine, ammonia, and a lower ratio of glutamine to glutamate than normal muscle. The atrophied muscle also showed faster in vitro production of alanine and tyrosine, and slower utilization of glutamate and aspartate. Despite a greater activity of glutamine synthetase, synthesis of glutamine was slower in the soleus muscle of suspended rats than in control muscle. Provision of ammonium chloride and/or glutamate showed that this slower synthesis of glutamine in the atrophied soleus probably was due to limiting amounts of free ammonia and not of glutamate. Flux through AMP deaminase was probably slower as demonstrated by the maintenance of a greater pool of total adenine nucleotides and by the slower release of nucleosides by the incubated soleus muscle of suspended v control rats. The extensor digitorum longus muscles of suspended animals showed greater glutamine production, glutamine synthetase activity, and aspartate utilization than control muscles. Data from muscles of intact, adrenalectomized and adrenalectomized, cortisol-treated rats suggested that the greater glutamine synthetase activity was mediated possibly by higher circulating glucocorticoid hormones and a greater response of the soleus muscle to these hormones. Glutamine synthesis in skeletal muscle may be regulated primarily by the availability of ammonia, which is associated with the degradation of adenine nucleotides, and secondarily by the amount of glutamine synthetase and glutamate in the tissue.