Insulin sensitivity and responsiveness of epitrochlearis and soleus muscles from fed and starved rats. Recognition of differential changes in insulin sensitivities of protein synthesis and glucose incorporation into glycogen.

The insulin sensitivity of protein synthesis and glucose incorporation into glycogen by the soleus and epitrochlearis muscles from fed rats and 24 h-starved rats was determined in vitro during the first and second hours of incubation after isolation of the muscles. Rates of protein synthesis by both muscles from fed rats in the first hour of incubation were 2-fold higher than in the second hour and were not increased by insulin. Rates of protein synthesis during the first hour in the presence of 6000 microunits of insulin/ml were increased in soleus, but not in epitrochlearis, muscles from starved rats. Rates of protein synthesis in both muscles from fed and starved rats were increased significantly by insulin during the second hour. High concentrations of insulin caused a marked stimulation of the rates of glucose incorporation by both muscles from fed and starved rats in both the first and second hours of incubation. The insulin sensitivity of glucose incorporation during the second hour, defined as the concentration of insulin causing half-maximal stimulation, was increased 10-fold for both muscle types from starved rats (soleus, 65 microunits/ml; epitrochlearis, 45 microunits/ml) relative to muscles from fed rats (soleus, 600 microunits/ml; epitrochlearis, 500 microunits/m). The insulin sensitivity of protein synthesis in the second hour was greater for soleus muscles from starved rats (65 microunits/ml) than from fed rats (500 microunits/ml). In contrast, the insulin sensitivity of protein synthesis in epitrochlearis muscles from starved rats was significantly decreased (225 microunits/ml) compared with fed rats (25 microunits/ml Maximal rates achieved by high concentrations of insulin were not different from those in the same muscle from fed rats. It is suggested that protein synthesis, in distinction to glucose utilization, may be resistant to insulin stimulation during periods of acute starvation in muscles with fibre compositions similar to the epitrochlearis, but not in muscles with fibre compositions similar to the soleus. Partial reversal of the resistance observed in vitro for epitrochlearis muscles from starved rats may be due to the loss of factors which suppress the effect of insulin in vivo.     

Catalytic activity of bovine glutamate dehydrogenase requires a hexamer structure.

Transfer of young rats from a maintenance diet to a breeding diet plus 10% sucrose in the drinking water for 4 weeks caused the development of insulin resistance. Inclusion of the enzyme adenosine deaminase or the adenosine-receptor antagonist 8-phenyltheophylline caused a marked increase in the sensitivity of the soleus-muscle strips isolated from the diet-induced insulin-resistant rats: the concentration of insulin giving 50% of maximum response of glycolysis shifted from 500 to less than 20 microunits/ml.     

Effects of analogues of adenosine and methyl xanthines on insulin sensitivity in soleus muscle of the rat

The concentration of insulin that produces half-maximal stimulation of glycolysis by stripped soleus muscle preparations is markedly increased by the adenosine analogues, 2-chloroadenosine and N6-phenylisopropyladenosine, but is markedly decreased by the methyl xanthine analogue, 8-phenyltheophylline. 2-Chloroadenosine increases the concentration of insulin required to stimulate glycolysis half maximally, from about 100 to 2000 mu units/ml. 8-Phenyltheophylline decreases this concentration of insulin from about 100 to 10 mu units/ml, an effect which is similar to that produced either by addition of adenosine deaminase to the medium or to exercise-training of the donor animals for 4 weeks.     

Effects of the beta-adrenoceptor agonist isoprenaline on insulin-sensitivity in soleus muscle of the rat.

The interactions between a beta-adrenoceptor agonist (isoprenaline) and insulin on rates of hexose transport, glucose phosphorylation, glycogen synthesis and glycogenolysis were investigated in the incubated stripped soleus-muscle preparation of the rat. In the presence of 1 microM-isoprenaline, insulin was less effective in stimulating glucose phosphorylation and glycogen synthesis. The stimulation of glycogenolysis by isoprenaline was only slightly decreased even at high (10000 microunits/ml) concentrations of insulin. Insulin-stimulated phosphorylation of 2-deoxyglucose was decreased by isoprenaline. It is suggested that this decrease in the rate of glucose phosphorylation is caused by the observed elevated concentration of glucose 6-phosphate, which inhibits hexokinase activity. This conclusion is supported by the fact that isoprenaline had no effect on the stimulation of 3-O-methylglucose transport by insulin.     

Effects of adenosine deaminase on the sensitivity of glucose transport, glycolysis and glycogen synthesis to insulin in muscles of the rat

1. Soleus, extensor digitorum longus (EDL) or hemi-diaphragm muscles of the rat were incubated in the presence of insulin and rates of the processes of glycolysis and glycogen synthesis were measured. 2. The concentrations of insulin required to cause half-maximal stimulation of glycolysis in both soleus and EDL preparations were significantly decreased by the presence of adenosine deaminase in the medium. 3. Adenosine deaminase increased the sensitivity of the process of hexose transport to insulin (in an identical manner to the change in sensitivity of glycolysis) in the EDL preparation. 4. None of the adenosine mediated effects on insulin-stimulated rates of glycolysis were observed in the hemi-diaphragm preparation or on the rates of glycogen synthesis in any of the three muscle preparations. 5. Therefore, changes in the adenosine system in skeletal muscle influence insulin sensitivity regardless of fibre type composition of the muscle.     

Effect of endurance and sprint exercise on the sensitivity of glucose metabolism to insulin in the epitrochlearis muscle of sedentary and trained rats

The effects of two types of acute exercise (1 h treadmill running at 20 m.min-1, or 6 x 10-s periods at 43 m.min-1, 0 degree inclination), as well as two training regimes (endurance and sprint) on the sensitivity of epitrochlearis muscle [fast twitch (FT) fibres] to insulin were measured in vitro in rats. The hormone concentration in the incubation medium producing the half maximal stimulation of lactate (la) production and glycogen synthesis was determined and used as an index of the muscle insulin sensitivity. A single period of moderate endurance as well as the sprint-type exercise increased the sensitivity of la production to insulin although the rate of la production enhanced markedly only after sprint exercise at 10 and 100 microU.ml-1 of insulin. These effects persisted for up to 2 h after the termination of exercise. Both types of exercise significantly decreased the muscle glycogen content, causing a moderate enhancement in the insulin-stimulated rates of glycogen synthesis in vitro for up to 2 h after exercise. However, a significant increase in the sensitivity of this process to insulin was found only in the muscle removed 0.25 h after the sprint effort. Training of the sprint and endurance types increased insulin-stimulated rates of glycolysis 24 h after the last period of exercise. The sensitivity of this process to insulin was also increased at this instant. Both types of training increased the basal and maximal rates of glycogen synthesis, as well as the sensitivity of this process to insulin at the 24th h following the last training session.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin sensitivity of rates of glycolysis and glycogen synthesis in soleus,stripped soleus,epitrochlearis, and hemi-diaphragm muscles isolated from sedentary rats

The effect of insulin concentrations on the rates of glycolysis and glycogen synthesis in four different in vitro rat muscle preparations (intact soleus, stripped soleus, epitrochlearis, and hemi-diaphragm) were investigated: the concentrations of insulin that produced half-maximal stimulation of the rates of these two processes in the four muscle preparations were similar - about 100 muunits/ml. This is at least 10-fold greater than the concentration that produced half-maximal inhibition of lipolysis in isolated adipocytes. Since 100 muunits/ml insulin is outside the normal physiological range in the rat, it is suggested that, in vivo, insulin influences glucose utilization in muscle mainly indirectly, via changes in the plasma fatty acid levels and the 'glucose/fatty acid cycle'. Consequently the view that insulin stimulates glucose utilization in muscle mainly by a direct effect on membrane transport must be treated with caution.     

Sensitivity of the soleus muscle to insulin in resting and exercising rats with experimental hypo- and hyper-thyroidism.

1. The effects of hypothyroidism (caused by surgical thyroidectomy followed by treatment for 1 month with propylthiouracil) and of hyperthyroidism [induced by subcutaneous administration of L-tri-iodothyronine (T3)] on glucose tolerance and skeletal-muscle sensitivity to insulin were examined in rats. Glucose tolerance was estimated during 2 h after subcutaneous glucose injection (1 g/kg body wt.). The sensitivity of the soleus muscle to insulin was studied in vitro in sedentary and acutely exercised animals. 2. Glucose tolerance was impaired in both hypothyroid and hyperthyroid rats in comparison with euthyroid controls. 3. In the soleus muscle, responsiveness of the rate of lactate formation to insulin was abolished in hypothyroid rats, whereas the sensitivity of the rate of glycogen synthesis to insulin was unchanged. In hyperthyroid animals, opposite changes were found, i.e. responsiveness of the rate of glycogen synthesis was inhibited and the sensitivity of the rate of lactate production did not differ from that in control sedentary rats. 4. A single bout of exercise for 30 min potentiated the stimulatory effect of insulin on lactate formation in hyperthyroid rats and on glycogen synthesis in hypothyroid animals. 5. The data suggest that thyroid hormones exert an interactive effect with insulin in skeletal muscle. This is likely to be at the post-receptor level, inhibiting the effect of insulin on glycogen synthesis and stimulating oxidative glucose utilization.     

Hormonal regulation of the rate of the glycogen/glucose-1-phosphate cycle in skeletal muscle

A method is presented which allows simultaneous estimation of rates of glycogen synthesis and glycogenolysis in an isolated incubated skeletal muscle, thus allowing measurement of the glycogen/glucose-1-phosphate substrate cycling rate. In the absence of hormonal additions, the measured rates of glycogen synthesis and breakdown were similar [respectively, 0.54 +/- 0.12 (8) and 0.74 +/- 0.10 (8) mumol glucosyl equiv. h-1 (g tissue)-1]. Incremental addition of insulin stimulated glycogen synthesis up to threefold and reduced glycogenolysis by about sevenfold; the half-maximally effective concentration of insulin on both processes was about 100 microU/ml (0.7 nM). Incremental addition of adrenaline (in the presence of 1 mU insulin/ml) caused a dramatic increase in the glycogenolytic rate (about 15-fold), but a much less marked inhibition of glycogen synthetic rate. In addition to hormonal manipulation of the muscle preparation in vitro, the effects of cold exposure, the hyperthyroid state, a single exercise bout and exercise-training of animals in vivo on the rates of glycogen synthesis and breakdown in the isolated incubated muscle preparation have been investigated. Significant changes in measured glycogen synthesis, breakdown and glycogen/glucose-1-phosphate cycling have been observed, both under basal conditions and in response to hormonal additions in vitro. The results are discussed with respect to the possible physiological importance of this substrate cycle.     

Sensitivity of glucose uptake and lipolysis of white adipocytes of the rat to insulin and effects of some metabolites.

1. Insulin increased glucose uptake and inhibited lipolysis in white adipocytes of the rat over the same concentration range of the hormone: the half-maximal effects were observed at approx. 10 microunits of insulin/ml. Thus, contrary to previous reports, no difference in sensitivity of the two processes to insulin could be found, which suggests that both these effects of insulin are important in increasing the rate of glucose utilization after a meal. 2. Adenosine deaminase, which lowers the concentration of adenosine in the incubation medium, decreased the sensitivity of both processes (lipolysis and glucose uptake) to insulin: this suggests that adenosine increases the sensitivity of both processes. Similarly, lactate and 3-hydroxybutyrate increased the sensitivity of both processes (to the same extent) to insulin. It is suggested that this increased sensitivity will improve the response (of adipose tissue) to insulin on refeeding after a prolonged period of starvation (when the hydroxybutyrate concentration is high), and after a short burst of exercise, when the blood lactate concentration is high and when large amounts of glucose are produced from lactate via gluconeogenesis in the liver.     

Effects of continuous low-carbohydrate diet after long-term exercise on GLUT-4 protein content in rat skeletal muscle.

Stimulation of AMPK and decreased glycogen levels in skeletal muscle have a deep involvement in enhanced insulin action and GLUT-4 protein content after exercise training. The present study examined the chronic effects of a continuous low-carbohydrate diet after long-term exercise on GLUT-4 protein content, glycogen content, AMPK, and insulin signaling in skeletal muscle. Rats were divided randomly into four groups: normal chow diet sedentary (N-Sed), low carbohydrate diet sedentary (L-Sed), normal chow diet exercise (N-Ex), and low carbohydrate diet exercise (L-Ex) groups. Rats in the exercise groups (N-Ex and L-Ex) were exercised by swimming for 6 hours/day in two 3-hour bouts separated by 45 minutes of rest. The 10-day exercise training resulted in a significant increase in the GLUT-4 protein content (p<0.01). Additionally, the GLUT-4 protein content in L-Ex rats was increased by 29% above that in N-Ex rats (p<0.01). Finally, the glycogen content in skeletal muscle of L-Ex rats was decreased compared with that of N-Ex rats. Taken together, we suggest that the maintenance of glycogen depletion after exercise by continuous low carbohydrate diet results in the increment of the GLUT-4 protein content in skeletal muscle.     

Adaptive response of hypertrophied skeletal muscle to endurance training

The response of hypertrophied soleus and plantaris muscle of rats to endurance training was studied. Hypertrophy was produced by bilateral extirpation of the gastrocnemius muscle. A 13-wk training program of treadmill running initiated 30 days after removal of the gastrocnemius muscle accentuated (P less than 0.01) the hypertrophy. Succinate dehydrogenase activities of the enlarged muscles of sedentary rats were similar to those of normal animals, as were the increases associated with training. Phosphorylase and hexokinase activities were unaltered as a result of the experimental perturbations. Rates of glycogen depletion during exercise were lower (P less than 0.01) in the liver and soleus and plantaris muscles of endurance-trained animals. No difference existed in the rate of glycogen depletion of normal and hypertrophied muscle within the sedentary or trained groups. These data demonstrate that extensively hypertrophied muscle responds to training and exercise in a manner similar to that of normal muscle.     

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).     

The effects of swimming and running regimens on skeletal muscle glycogen in the rat.

Endurance capacity and the effects of different post-exercise states on skeletal muscle glycogen have been studied in rats trained by swimming or running and in sedentary controls. Regular endurance exercise resulted in increased skeletal muscle glycogen stores. A greater depletion was observed in trained animals than in non-trained animals after a training bout or exhaustive exercise. While muscle glycogen levels did not reflect a differential training stimulus (running vs swimming), swimming as a measure of exhaustive exercise was deemed invalid because of the ability of trained swimmers to avoid stenuous exercise by an alteration of swimming pattern.     

Maximal activities of enzymes involved in adenosine metabolism in muscle and adipose tissue of rats under conditions of variations in insulin sensitivity

The maximal activities of 5'-nucleotidase, adenosine deaminase and adenosine kinase were measured in quadriceps or soleus muscle from animals in which the sensitivity to insulin was changed. Most conditions caused no effect on the activities but exercise-training increased the activity of adenosine deaminase and cold exposure increased the activity of 5'-nucleotidase in soleus muscle: in addition, ageing decreased markedly the activities of all three enzymes in both muscles. When the activities are based on mg protein they are much higher in both white and brown adipose tissue than in muscle, suggesting that changes in adenosine concentration may be important in changing insulin sensitivity in adipose tissue whereas changes in adenosine receptor number may be more important in muscle.     

Effect of exercise training on whole-body insulin sensitivity and responsiveness

Exercise training causes a decline in basal and glucose-stimulated plasma insulin levels and improves glucose tolerance. Furthermore evidence has been presented for effects on both insulin receptors and postreceptor events. However, it is unclear how these changes affect the in vivo dose-response relationship between insulin levels and whole-body glucose utilization. The aim was to examine the effect of exercise training on this relationship and distinguish between changes in insulin sensitivity and responsiveness. Euglycemic clamps were performed in trained (ET, running 1 h/day for 7 wk), sedentary (CON), and sedentary food-restricted ( SFR ) rats. ET rats showed no increase in maximal net glucose utilization in response to insulin (ET 29.5 +/- 0.6 vs. CON 28.2 +/- 1.5 mg X kg-1 X min-1, NS), whereas insulin sensitivity was increased as indicated by the insulin concentration causing half-maximal stimulation (ED50) (49 +/- 20 for ET and 133 +/- 30 mU/l for CON). Thus 7 wk of moderate exercise training resulted in a significant shift of whole-body insulin sensitivity to place ED50 well within the physiological range of insulin concentrations. This would undoubtedly result in improved glucose disposal in the postprandial state and emphasizes the potential benefit of exercise in obesity and type II diabetes.     

Effect of exercise on insulin action in human skeletal muscle

The effect of 1 h of dynamic one-legged exercise on insulin action in human muscle was studied in 6 healthy young men. Four hours after one-legged knee extensions, a three-step sequential euglycemic hyperinsulinemic clamp combined with arterial and bilateral femoral vein catheterization was performed. Increased insulin action on glucose uptake was found in the exercised compared with the rested thigh at mean plasma insulin concentrations of 23, 40, and 410 microU/ml. Furthermore, prior contractions directed glucose uptake toward glycogen synthesis and increased insulin effects on thigh O2 consumption and at some insulin concentrations on potassium exchange. In contrast, no change in insulin effects on limb exchange of free fatty acids, glycerol, alanine or tyrosine were found after exercise. Glycogen concentration in rested vastus lateralis muscle did not increase measurably during the clamp even though indirect estimates indicated net glycogen synthesis. In contrast, in exercised muscle estimated and biopsy-verified increases in muscle glycogen concentration agreed. Local contraction-induced increases in insulin sensitivity and responsiveness play an important role in postexercise recovery of human skeletal muscle.     

Endurance exercise training reduces lactate production

In situ muscle stimulation in trained and untrained rats was used to reevaluate whether adaptations induced by endurance exercise training result in decreased lactate production by contracting muscles. The gastrocnemius-plantaris-soleus muscle group was stimulated to perform isotonic contractions. After 3 min of stimulation with 100-ms trains at 50 Hz at 60/min, the increases in lactate concentration in the plantaris, soleus, and fast-twitch red muscle (deep portion of lateral head of gastrocnemius) were only approximately 50% as great in trained as in sedentary rats. In the predominantly fast-twitch white superficial portion of the medial head of the gastrocnemius the increase in lactate concentration was 28% less in the trained than in the sedentary group. The decreases in muscle glycogen concentration seen after 3 min of stimulation at 60 trains/min were smaller in the trained than in the untrained group. The reduction in lactate accumulation that occurred in the different muscles in response to training was roughly proportional to the degree of glycogen sparing. These results show that endurance training induces adaptations that result in a slower production of lactate by muscle during contractile activity.     

Effects of aging on the responsiveness and sensitivity of glucose metabolism to insulin in the incubated soleus muscle isolated from Sprague-Dawley and Wistar rats.

1. The effects of aging on the sensitivity and responsiveness of glucose transport, lactate formation and glycogen synthesis to insulin were studied in the incubated stripped soleus muscle isolated from aging Sprague-Dawley and Wistar rats. 2. As Sprague-Dawley rats aged from 5 to 13 weeks, there were marked increases in the concentrations of insulin that were required for half-maximal stimulation (i.e. EC50 value, which is a measure of sensitivity) of glucose transport, lactate formation and glycogen synthesis. 3. In marked contrast, there were no alterations in sensitivities of any of these processes to insulin in soleus muscle prepared from Wistar rats aged between 6 and 12 weeks. 4. However, in soleus muscles from 85-week-old Wistar rats the rates of glycogen synthesis in response to basal, sub-maximal and maximal concentrations of insulin were markedly decreased. The insulin EC50 value of glycogen synthesis was increased 4-fold, but was unchanged for lactate formation. 5. The insulin-stimulated rates of glucose transport in soleus muscles from 5- or 85-week-old Wistar rats were not significantly different.     

Voluntary exercise training enhances glucose transport but not insulin signaling capacity in muscle of hypertensive TG(mREN2)27 rats.

Male heterozygous TG(mREN2)27 rats (TGR) overexpress a murine renin transgene, display marked hypertension, and have insulin resistance of skeletal muscle glucose transport and insulin signaling. We have shown previously that voluntary exercise training by TGR improves insulin-mediated skeletal muscle glucose transport (Kinnick TR, Youngblood EB, O'Keefe MP, Saengsirisuwan V, Teachey MK, and Henriksen EJ. J Appl Physiol 93: 805-812, 2002). The present study evaluated whether this training-induced enhancement of muscle glucose transport is associated with upregulation of critical insulin signaling elements, including insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3. TGR remained sedentary or ran spontaneously in activity wheels for 6 wk, averaging 7.1 +/- 0.8 km/day by the end of week 3 and 4.3 +/- 0.5 km/day over the final week of training. Exercise training reduced total abdominal fat by 20% (P < 0.05) in TGR runners (2.64 +/- 0.01% of body weight) compared with sedentary TGR controls (3.28 +/- 0.01%). Insulin-stimulated (2 mU/ml) glucose transport activity in soleus muscle was 36% greater in TGR runners compared with sedentary TGR controls. However, the protein expression and functionality of tyrosine phosphorylation of insulin receptor and IRS-1, IRS-1 associated with the p85 regulatory subunit of phosphatidylinositol 3-kinase, and Ser473 phosphorylation of Akt were not altered by exercise training. Only insulin-stimulated glycogen synthase kinase-3beta Ser9 phosphorylation was increased (22%) by exercise training. These results indicate that voluntary exercise training in TGR can enhance insulin-mediated glucose transport in skeletal muscle, as well as reduce total abdominal fat mass. However, this adaptive response in muscle occurs independently of modifications in the proximal elements of the insulin signaling cascade.