Search

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.

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)

Comparison of hindlimb muscles of rats flown on Spacelab-3 or tail-traction-suspended showed that 11-17 h reloading post-flight might have altered the results. Soleus atrophied, plantaris, gastrocnemius and extensor digitorum longus grew slower, and tibialis anterior grew normally. In both flight and simulated soleus and plantaris, higher tyrosine and greater glutamine/glutamate ratio indicated negative protein balance and increased glutamine production, respectively, relative to controls. Aspartate was lower in these muscles. Reloading generally decreased tyrosine, but increased aspartate and glutamine/glutamate. These data showed that at 12 h of reloading after flight is characterized by reversal to varying extents of effects of unloading.