What Factors Increase Glycogen Synthase Activity Quizlet

in vivo and in vitro studies demonstrate that musculus wrinkle enhances insulin stimulation of glucose disposal when contracting muscle is stimulated with insulin (10, 11). This enhanced glucose disposal persists for several hours afterward the abeyance of exercise (33, 36). Notwithstanding, the mechanism for this enhancement remains unknown. Although insulin and musculus contraction independently stimulate GLUT-4 translocation and glucose uptake, the mechanisms by which they bring virtually these effects are distinct. Insulin acts past bounden to its receptor, resulting in tyrosine phosphorylation of the receptor and insulin receptor substrates (primarily IRS-1), which serve as docking proteins for proteins containing Src homology (SH2) domains. Association of the SH2 domain of the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) with IRS-one activates the catalytic subunit of PI3-kinase. Numerous studies have shown that insulin-stimulated glucose uptake is dependent on the activation of PI3-kinase (half dozen, 20). A potential downstream effector of PI3-kinase is Akt, a serine/threonine kinase too known every bit PKB (iii, 23, 34). Unlike insulin, wrinkle stimulates glucose uptake contained of PI3-kinase (iii, 27). Although the signaling mechanisms are unclear, increased five′-adenosine monophosphate-activated protein kinase activity has been linked to contraction-induced glucose uptake (17).

Because of the singled-out upstream signaling mechanisms utilized by insulin and contraction, it is non surprising that the combination of the two stimuli increases glucose uptake to a degree greater than either stimulus alone. A previous written report in human subjects showed that do, when performed simultaneously with an insulin infusion, synergistically increased whole trunk glucose disposal (10). This result was contributed to enhanced blood flow to the working muscle, resulting in a greater presentation of insulin and glucose to muscle (10). Information technology is non clear, however, whether the synergistic increase in glucose disposal is due solely to a greater mass of muscle existence perfused or whether there are qualitative effects on the musculus also. One logical mechanism to increase insulin-stimulated glucose disposal might be to increase skeletal muscle insulin receptor signaling.

Information from studies in rodents (15) and humans (27) bespeak strongly that do or musculus contraction alone does not enhance insulin signaling in the absence of an increase in insulin concentration. Even so, a unmarried tour of practise can increase the subsequent response of insulin signaling to an insulin infusion. For case, 24 h subsequently a single bout of practice, insulin receptor and IRS-i tyrosine phosphorylation are increased in response to insulin in insulin-resistant subjects compared with unexercised controls. These increases were non accompanied past an increase in PI3-kinase activity or glucose disposal, however (9). Because there were effects 24 h after exercise, the results raise the possibility that exercise during an insulin infusion might farther increase insulin receptor signaling at more than distal steps, thereby mediating an increase in glucose disposal. Glycogen synthase activity was also increased (9), suggesting that down-stream effectors are also involved.

The present written report was undertaken, therefore, to decide whether, during a simultaneous insulin infusion and exercise bout, enhanced glucose disposal can be attributed to increased insulin receptor signaling or glycogen synthase action.

METHODS

Subjects. 16 nondiabetic and seven Type ii diabetic subjects participated in the study (Table ane). A consummate history was obtained from each subject, and each subject underwent a physical exam, including a 75-chiliad oral glucose tolerance test to determine the presence or absence of diabetes using established American Diabetes Association criteria. All nondiabetic subjects denied a family history of Blazon ii diabetes and had normal glucose tolerance. Other than having diabetes, diabetic subjects were in proficient wellness. Three of the seven diabetic subjects were taking glyburide, which was withdrawn 3 days before clinical studies. The remaining four diabetic subjects were treated with diet alone. No subject field was taking any other medication known to affect glucose metabolism. A normal resting electrocardiogram reading was a prerequisite for participation. Subjects were instructed to consume a diet containing ≥200 g of carbohydrate per 24-hour interval for the 3 days preceding clinical studies and to not exercise on the 24-hour interval earlier the studies. Trunk fat percentages were determined using bioimpedance (31). The study protocol was approved past the Institutional Review Board of the Academy of Texas Wellness Scientific discipline Center at San Antonio, and all subjects gave written informed consent.

Table 1. *Subject area characteristics*
	Nondiabetic (n = sixteen)	Diabetic (due north = 7)
Age, yr	35 ± ii	45 ± 4^*
BMI, kg/m^two	28.3 ± 0.four	xxx.8 ± 1.2^*
HbA_1c	iv.9 ± 0.1	7.viii ± 0.9†
Fasting plasma glucose, mg/dl	94 ± 2	149 ± 16‡
Fasting plasma insulin, uU/ml	9 ± 3	12 ± 3
Cholesterol, mmo/50	186 ± 11	192 ± iv
Triglycerides, mmol/fifty	106 ± 12	319 ± 140^*
Ethnicity	7C/7H/1AA/1A	2C/5H

Elevation aerobic capacity. None of the subjects had participated in a regular practice program for ≥1 yr before entering the study. Peak aerobic chapters [i.e., peak O_ii uptake (V̇o _{2 acme})] was adamant using an incremental bicycle ergometer protocol. Criteria for test completion were a respiratory exchange ratio >1.one and no further increase in O₂ uptake (V̇o ₂) and/or middle charge per unit. The center charge per unit corresponding to 70% V̇o _{2 height} was used for the insulin clamp + exercise protocol.

Insulin clamp without simultaneous exercise. At least 1 wk after the V̇o _{two peak} test, the subjects reported to the General Clinical Enquiry Center (GCRC) at 8 AM after consuming nothing but h2o since the prior evening (Fig. iA). An antecubital vein was cannulated for infusion of [iii-³H]glucose, twenty% dextrose, and insulin (Humulin, Eli Lilly, Indianapolis, IN). A mitt vein was cannulated in a retrograde fashion, and the paw was placed in a heated box (60°C) for sampling of arterialized blood. To ensure isotopic equilibrium, a primed [(25 μCi × fasting plasma glucose)/90], continuous (0.25 μCi/min) infusion of [3-³H]glucose was started two h (nondiabetic subjects) or 3 h (diabetic subjects) before the start of insulin infusion. At lx min before the start of insulin infusion, a percutaneous biopsy of the vastus lateralis muscle was obtained with a Bergstrom cannula nether local anesthesia (2). Muscle biopsy specimens were immediately blotted free of blood, frozen, and stored in liquid nitrogen until used. Arterialized blood was sampled for measuring plasma glucose, insulin, and [3-³H]glucose specific activity. Blood samples were obtained at baseline and every 10 min during the terminal thirty min of the isotopic equilibration flow. Continuous indirect calorimetry was performed with a ventilated hood system (DeltaTrac, Sensor Medics, Anaheim, CA) during the terminal 30 min of the tracer equilibration (basal) and insulin clamp periods for measurement of saccharide and lipid oxidation rates. Leg blood flow was measured using straingauge plethysmography basally and later 55 and 120 min of insulin infusion. After completion of the tracer equilibration menstruation, a primed, continuous infusion of insulin was started at 40 mU · grand^-2 · min^-one for 120 min. Plasma glucose was measured every 5 min throughout the report with a glucose oxidase analyzer (Beckman Instruments, Fullerton, CA) and maintained at euglycemia (ninety-100 mg/dl) using a variable infusion of 20% dextrose. After 30 min of insulin infusion, a 2nd biopsy was obtained from the reverse vastus lateralis muscle.

**Fig. 1.**Study blueprint for euglycemic, hyperinsulinemic clamps. Subjects underwent 2 euglycemic, hyperinsulinemic clamps (40 mU · one thousand^-2 · min^-i) without (A) and with (B) concomitant exercise. During each clench, tritiated glucose was infused throughout a basal period of 120 min for control subjects or 180 min for diabetic subjects and during 120-min insulin infusion. Glucose levels were maintained at euglycemia (xc-100 mg/dl) past a variable glucose infusion. When exercise was performed, subjects performed moderately intense exercise equivalent to 70% of their private elevation O₂ uptake on a stationary recumbent bike during the initial 30 min of insulin infusion. Muscle biopsies (Bx) of vastus lateralis muscle were performed basally (-lx min) and during insulin infusion (+30 min). Indirect calorimetry was measured for 30 min at the end of the basal period and insulin infusion.

Insulin clench with simultaneous exercise. On another day ≥2 wk after the first euglycemic, hyperinsulinemic clamp, subjects returned to the GCRC at viii AM after consuming nada but water since the prior evening. On this occasion, the previous protocol was followed with the following modifications. At the initiation of the insulin infusion, subjects began exercising on a recumbent cycle ergometer for 30 min at a centre charge per unit respective to lxx% V̇o _{ii summit} (Fig. oneB). Heart rate was monitored by electrocardiography, and exercise intensity was adjusted every bit necessary to maintain the target heart rate. Afterward xxx min of exercise, a 2nd biopsy of the vastus lateralis muscle was performed as shortly as possible. The clamp then connected as described in Insulin clamp without simultaneous practise.

Materials. Polyclonal anti COOH-terminal IRS-1 and polyclonal antiphospho-Akt (Ser⁴⁷³) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). A polyclonal anti-Akt antibody was purchased from Cell Signaling (Beverly, MA). Platelet-derived growth factor-stimulated NIH 3T3 L1 cell lysate (Upstate Biotechnology) served every bit a positive control for phospho-Akt (Ser⁴⁷³) immunoblotting. Rat liver homogenate served equally a standard control for the PI3-kinase assay. Goat anti-rabbit and rabbit anti-sheep antibodies coupled to horseradish peroxidase (Amersham, Piscataway, NJ) were used as secondary antibodies. Protein A and phosphatidylinositol were purchased from Sigma Chemical (St. Louis, MO), and [γ-³²P]ATP was obtained from NEN Life Science Products (Boston, MA).

Muscle processing. Muscle samples were weighed while still frozen and homogenized in ice-cold lysis buffer (1:10, wt/vol) containing 50 mM HEPES (pH 7.vi), 150 mM NaCl, xx mM sodium pyrophosphate, 20 mM β-glycerophosphate, x mM NaF, 2 mM Na_threeVO₄, 2 mM EDTA (pH 8.0), 1% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM MgCl₂, one mM CaCl_two, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. A Polytron homogenizer (Brinkman Instruments, Westbury, NY) ready on maximum speed for 30 s was used for homogenization. Homogenates were incubated on ice for 20 min and so centrifuged at 15,000 g for 20 min at four°C. Muscle debris was removed, and protein concentrations of crude extracts were estimated by the method of Lowry et al. (28). Supernatant was stored at -eighty°C until used.

SDS-PAGE and immunoblotting. For phospho-Akt (Ser⁴⁷³), equal amounts of protein were resolved on 7.5% (Akt) SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Afterwards they were blocked, the membranes were incubated with antibodies, and poly peptide bands were visualized using an enhanced chemiluminescence detection arrangement co-ordinate to the manufacturer'south protocol (Amersham). Images were digitized by scanning, and band intensity was quantified using Image Tool Software (University of Texas Wellness Science Center at San Antonio). For determining Akt expression, the phospho-Akt (Ser⁴⁷³) immunoblot was stripped using a buffer containing 0.7% β-mercaptoethanol, 7 mM SDS, and 6 mM Tris · HCl (pH 6.7) for 20 min, washed with Tris-buffered saline three times for x min each, blocked with Tris-buffered saline + Tween 20 containing 5% milk, and reprobed with anti-Akt antibody overnight. The detection procedures were the same as those described above.

PI3-kinase assay. Muscle protein (250 μg) was immunoprecipitated with anti-IRS-1 antibody, and PI3-kinase activity was measured past determining incorporation of [³²P]ATP into [³²P]phosphatidylinositol phosphate, as previously described (viii).

Glycogen synthase activeness. Glycogen synthase (GS) activities were assayed using 0.1 mM (GS_0.1) and 10 mM (GS₁₀) glucose 6-phosphate, as previously described (32). Glycogen synthase fractional velocity (GS_FV) was calculated equally the ratio of GS_0.1 to GS₁₀. Changes in GS_FV are indicative of insulin's effects.

Laboratory analyses. Plasma insulin concentration was determined by radioimmunoassay (Diagnostic Products, Los Angeles, CA). Plasma tritiated glucose specific activity was determined on barium hydroxide-zinc sulfate-precipitated plasma samples.

Calculations. Glucose disposal rates were calculated using steady-state equations or, where appropriate for non-steadystate conditions, Steele'south equation (fourteen). Glucose and fatty oxidation rates were calculated from V̇o ₂ and CO_two output data past the equations of Frayn (13). Insulin clearance was calculated as previously described by DeFronzo et al. (x).

Statistical analysis. Values are means ± SE. A t-test was used to test for differences betwixt study groups with regard to subject clinical characteristics. Statistical differences amongst groups were adamant past 2-way repeated-measures assay of variance and Fisher's post hoc tests using StatView four.0 software. Correlation analysis was performed by the Pearson product-moment method. For all analyses, P < 0.05 was considered to exist statistically significant.

RESULTS

Subjects. The characteristics of the subjects are given in Table 1. As expected, diabetic subjects had elevated HbA1_c and fasting plasma glucose levels compared with control subjects. There was no divergence in fasting insulin levels between the groups. Diabetic subjects were somewhat older (P < 0.05) and had higher triglyceride levels than control subjects. Although both groups were moderately obese, diabetic subjects had a slightly college body mass index. Maximal aerobic capacity, work, and heart rate, besides equally values achieved when exercise was performed during the insulin infusion, are given in Table 2. Diabetic subjects had reduced maximum aerobic capacity (V̇o _{2 pinnacle}) compared with control subjects (P < 0.001). In conjunction with the decreased aerobic capacity of the diabetic subjects, less work was performed during a V̇o _{2 superlative} exam (P < 0.01). Maximum center rate accomplished during the V̇o _{2 superlative} examination, as well as the heart rate at which subjects exercised during the insulin infusion, was not significantly unlike betwixt the groups.

Tabular array two. *Do characteristics of subjects*
	Nondiabetic			Diabetic
	Peak	Exercise during clamp	Elevation	Exercise during clamp
V̇o ₂, ml O₂·kg FFM^-ane·min^-i	35.4±1.vii†	24.nine±i.3	23.5±1.9†‡	16.four±ane.iii‡
Heart rate, beats/min	166±four†	136±4	155±5†	131±3
Work, West	169±12†	116±ten	98.5±eight^*†	70±10^*

Euglycemic clamps. Subjects underwent two euglycemic, hyperinsulinemic clamps (40 mU · m^-2 · min^-1) on separate occasions, once without and in one case with concomitant exercise (Fig. 1). When exercise was performed together with insulin infusion, subjects exercised at 70% V̇o _{ii peak} for 30 min beginning at the offset of the insulin infusion (Table 2). Considering the exercise intensity, past pattern, was relative to an individual'due south V̇o _{2 superlative}, the diabetic subjects on average exercised at a lower V̇o ₂ (P < 0.001) and performed less work (P < 0.01) than the command subjects during the clamp (Table two).

Plasma glucose levels were maintained at euglycemia by a variable glucose infusion throughout the clamps for control subjects (Fig. 2). Because of the increased fasting glucose levels of the diabetic subjects, their glucose concentrations were significantly greater during the initial part of the clamps than those of the control subjects (Fig. 2). During insulin lone, plasma glucose concentrations were significantly greater in the diabetic than in the control subjects during the first forty min of insulin infusion (P < 0.05), whereas insulin + exercise was associated with a more rapid autumn in glucose concentrations in the diabetic subjects and so that, by xxx min, glucose concentrations were not significantly different from those of control subjects.

Plasma insulin concentrations were determined during the initial 30 min and last xxx min of the clamps. In the control subjects, plasma insulin concentrations were elevated during insulin + practise compared with insulin alone (Fig. 3A). Plasma insulin concentrations peaked at 76 ± iv μU/ml and cruel to 61 ± iii μU/ml during the first 30 min of insulin alone, whereas concentrations peaked at 90 ± 5 μU/ml and cruel to 72 ± 2 μU/ml during the same period of insulin + exercise. Because the insulin infusion rate was the same on both occasions, nosotros calculated the insulin clearance rates. Whole body insulin clearance was decreased when practise was performed forth with insulin infusion (Fig. threeB). Later on completion of the exercise, plasma insulin concentrations fell and insulin clearance increased to match values reached during insulin alone. In dissimilarity, do did not affect insulin concentrations or insulin clearance in the diabetic subjects (Fig. 3, C and D).

**Fig. 3.**Plasma insulin concentrations (A and C) and insulin clearance (B and D) for control and diabetic subjects. Subjects underwent 2 euglycemic, hyperinsulinemic clamps (forty mU · chiliad^-2 · min^-ane) once without and one time with concomitant exercise. See methods for calculation of insulin clearance. ▪, Command, insulin alone; □, command, insulin + do; ▴, diabetic, insulin alone; ▵, diabetic, insulin + practice. ^* P < 0.05; †P < 0.01 vs. insulin alone.

Glucose disposal. In control subjects, insulin alone gradually increased the rate of glucose disposal calculated using tritiated glucose to 4.6 ± 0.4 mg · kg fatty-free mass (FFM)^-one · min^-1 at 30 min so further to an average of vi.vi ± 0.7 mg · kg FFM^-one · min^-1 over the concluding 30 min of insulin infusion (90-120 min). When exercise was performed during the first 30 min of insulin infusion in control subjects, the rate of glucose disposal increased steeply to nine.5 ± 0.8 mg · kg FFM^-i · min^-ane after 30 min and and so decreased gradually after cessation of exercise to an average of 8.i ± 0.vii mg · kg FFM^-1 · min^-one over the last 30 min of insulin infusion. At each time indicate, the rate of glucose disposal was greater when practise was performed during the offset xxx min of insulin infusion (Fig. 4A). In insulin-infused nonexercised diabetic subjects, the rate of glucose disposal increased gradually to 4.3 ± 1.0 mg · kg FFM^-one · min^-one at 30 min and and so remained constant throughout the insulin infusion, with an average of four.ii ± 0.6 mg · kg FFM^·-ane · min^-1 over the last 30 min of insulin infusion (90-120 min). When practice was performed during the first 30 min of insulin infusion in diabetic subjects, the rate of glucose disposal increased steeply to 7.9 ± 0.7 mg · kg FFM^-i · min^-ane afterwards 30 min and then quickly decreased to 6.0 ± 0.8 mg · kg FFM^-1 · min^-ane and remained constant throughout the elapsing of the insulin infusion (Fig. 4B). Throughout the studies, the charge per unit of glucose disposal was significantly decreased in the diabetic subjects compared with the control subjects during insulin solitary and insulin + practice.

Fig. 4. — **Fig. four.**Effect of exercise on glucose disposal rates for control (A) and diabetic subjects (B). Subjects underwent 2 euglycemic, hyperinsulinemic clamps (xl mU · thou^-two · min^-1), one time without (-Ex) and one time with (+Ex) concomitant exercise, during which tritiated glucose was infused for determination of glucose disposal. Encounter methods for calculation. ▪, Control, insulin alone; □, control, insulin + exercise; ▴, diabetic, insulin solitary; ▵, diabetic, insulin + do. ^* P < 0.001; §P < 0.01; ‡P < 0.05 vs. insulin lone. C: event of exercise on insulin-stimulated whole torso glucose metabolism. Whole torso glucose disposal (height of bar) was determined using tritiated glucose, and indirect calorimetry was used to determine whole trunk glucose oxidation (open bars). Nonoxidative glucose storage (solid confined) represents difference between total glucose disposal and glucose oxidation. Basal measurements were subtracted from stimulated measurements for each bailiwick to decide consequence of insulin solitary and insulin + exercise. FFM, fat-free mass. Values are means ± SE for seven command and 7 diabetic subjects. ^a P < 0.05; ^c P < 0.01 vs. insulin solitary; ^b P < 0.05 vs. control.

The rate of whole trunk glucose oxidation was determined basally (-thirty to 0 min) and during the last thirty min of insulin infusion (90-120 min) using V̇o ₂ and CO₂ output measured by systemic indirect calorimetry. Glucose storage was calculated as the difference between the rate of glucose disposal and oxidation. Results (Table 3, Fig. fourC) signal that, every bit described above, the rate of insulin-stimulated glucose disposal was increased in both groups when exercise was performed during the showtime 30 min of insulin infusion. Nonetheless, the rate of systemic glucose oxidation was non increased (60-xc min later exercise had ceased) in either grouping. Basal and insulin-stimulated rates of glucose oxidation remained significantly decreased in diabetic subjects compared with control subjects, whether or not practise was performed. Therefore, in command and diabetic subjects, exercise significantly enhanced the rate of insulin-stimulated glucose storage (glycogen synthesis). This was particularly true for the diabetic subjects, in whom insulin alone failed to increase glucose storage above basal values, only exercise increased the rate of insulin-stimulated glucose storage to a value not significantly different from that of control subjects studied during insulin infusion solitary. The effect of practise was strongly correlated with enhancement of glucose disposal and glucose storage (r = 0.93, P < 0.001).

Table three. *Glucose metabolism during euglycemic, hyperinsulinemic clamps*
	Nondiabetic							Diabetic
	—Exercise			+Exercise			—Practice		+Exercise
	Basal	Insulin	Basal	Insulin	Basal	Insulin	Basal	Insulin
Oxidation	2.3±0.2	4.0±0.5^a	ii.0±0.four	3.7±0.7^a	1.4±0.5	ii.5±0.five^b,e	1.0±0.4^east	2.0±0.6^a,e
Storage	0.5±0.ii	2.9±0.9^a	0.8±0.3	iv.nine±i.one^a,c	1.five±0.four	1.7±0.v	i.ix±0.4^{due east}	four.2±0.8^a,d
Disposal	ii.eight±0.1	half dozen.9±one.3^b	2.8±0.ii	eight.6±i.5^b,d	two.9±0.2	4.2±0.vi^a,e	two.9±0.2	half dozen.2±1.0^a,d

Insulin signaling. To evaluate the outcome of practice on insulin'southward ability to stimulate the PI3-kinase signaling pathway, muscle biopsies were performed in the basal period and during insulin stimulation with and without simultaneous exercise. IRS-ane-associated PI3-kinase activity and Akt serine phosphorylation and poly peptide expression were measured in lysates from these muscle biopsies. Muscle samples from insulin and insulin + do clamps for each subject were analyzed on the same immunoblot to reduce variability.

The event of exercise on insulin stimulation of IRS-i-associated PI3-kinase activity is shown in Fig. v (normalized to 100% for insulin lone for each group). Consequent with their full general level of insulin resistance, insulin alone modestly but significantly increased IRS-1-associated PI3-kinase activeness in both groups. Exercise significantly increased insulin-stimulated IRS-1-associated PI3-kinase activity compared with insulin alone in the command subjects (P < 0.05); however, this was non the case in the diabetic subjects.

Fig. 5. — **Fig. five.**Effect of practise on insulin stimulation of insulin receptor substrate 1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase. Data are expressed relative to boilerplate value for insulin solitary for each group. Open bars, basal values; solid bars, insulin-stimulated values. ^* P < 0.05 vs. basal; †P < 0.05 vs. insulin (Ins) alone.

Insulin infusion alone increased Akt Ser⁴⁷³ phosphorylation x-fold in control and diabetic subjects (Fig. 6). To decide whether Akt protein expression was different between the two groups, muscle samples obtained during the basal menstruation of the insulin-alone clamp were analyzed on the same immunoblot to reduce variability. Akt protein expression was decreased 23 ± 10% in the diabetic subjects compared with the command subjects (P = 0.05). In command subjects, practise performed during the insulin infusion significantly increased Akt Ser⁴⁷³ phosphorylation (P < 0.05), but this effect of exercise did not occur in the diabetic subjects.

Glycogen synthase activeness. Glycogen synthase activity was assayed using GS_0.ane and GS_x to decide active and total forms of the enzyme. Under basal conditions, neither GS_0.1 nor GS_FV differed with the insulin-lonely and insulin + exercise for command or diabetic subjects (Table iv, Fig. vii). Insulin alone stimulated GS_0.1 and GS_FV to a similar extent in the obese control and diabetic subjects consistent with their caste of insulin resistance. Do performed during the first thirty min of insulin infusion resulted in a significant increase in GS_0.1 and GS_FV compared with the value achieved during insulin alone (P < 0.05) for the obese command and diabetic patients.

Table iv. *Glycogen synthase action for nondiabetic and diabetic subjects*
	-Do			+Exercise
	Basal	Insulin	Basal	Insulin
Nondiabetic (n = eight)
GS_0.1	1.3 ± 0.3	2.4 ± 0.5^*	1.5 ± 0.four	3.9 ± 0.five†‡
GS₁₀	xiii.2 ± 1.ix	14.iii ± 1.9	12.1 ± ane.2	11.seven ± 0.9
Diabetic (n = vi)
GS_0.1	2.two ± 0.v	3.6 ± 0.7^*	two.3 ± 0.6	4.one ± 0.9†
GS₁₀	xv.two ± 2	15.six ± 1.iii	thirteen.5 ± 2.2	13.0 ± 1.8

**Fig. 7.**Consequence of insulin and insulin + exercise on glycogen synthase activity for control (A) and diabetic (B) subjects. Basal (open confined) and insulin-stimulated (solid bars) glycogen synthase activity in muscle samples without and with concomitant exercise was assayed in the presence of 0.1 and x mM glucose 6-phosphate (GS_0.i and GS₁₀). Glycogen synthase fractional velocity was and so calculated equally the ratio of GS_0.1 to GS_ten for eight command and 6 diabetic subjects. ^* P < 0.01; §P < 0.05 vs. basal. ‡P < 0.01; †P < 0.05 vs. no exercise.

DISCUSSION

Do performed simultaneously with an insulin infusion synergistically increases glucose disposal compared with insulin infusion solitary (10). Astute exercise can heighten subsequent (24 h later) insulin stimulation of proximal insulin receptor signaling and glycogen synthase activity (9). We sought to determine whether the synergistic effect of do on insulin-stimulated glucose disposal can be attributed to increased insulin signaling or glycogen synthase activity. Nosotros chose to study insulin-resistant subjects to gain insight into the mechanism of the ability of practise to overcome insulin resistance.

Results of the nowadays written report ostend that practise performed simultaneously with insulin administration increases glucose disposal to a degree greater than that predicted on the basis of their separate private contributions (x). For case, glucose disposal at the end of insulin + practice was 9.5 ± 0.eight mg · kg FFM^-i · min^-1, an increment of 4.9 ± 0.6 mg · kg FFM^-1 · min^-1 over that accomplished with insulin lone. Previous studies in our laboratory using similar exercise intensity (27) evidence that if the effect of exercise and insulin were condiment, one would look an increase of only 1.5 ± 0.4 mg · kg FFM^-1 · min^-ane. Therefore, this study pattern results in exercise synergistically increasing insulin-stimulated glucose disposal in nondiabetic subjects. Similarly, in the diabetic subjects, glucose disposal at the end of insulin + exercise was 7.9 ± 0.7 mg · kg FFM^-1 · min^-1, an increase of iii.7 ± 0.8 mg · kg FFM^-1 · min^-1 over that achieved by insulin alone. Minuk et al. (xxx) demonstrated that do-induced glucose disposal in diabetic subjects is similar to that measured in nondiabetic subjects. Therefore, if we assume that exercise alone stimulates glucose disposal to the same extent every bit in obese nondiabetic subjects (27), the predicted additive effect of insulin and exercise would be v.8 ± i.1 mg · kg FFM^-1 · min^-1, suggesting that exercise synergistically increases insulin-stimulated glucose disposal in diabetic subjects likewise.

With regard to insulin receptor signaling, we chose to assess distal components of the PI3-kinase signaling pathway, that is, IRS-1-associated PI3-kinase activity and Akt Ser⁴⁷³ phosphorylation. Our laboratory previously showed that the consequence of exercise can be dissociated from changes in insulin receptor and IRS-1 tyrosine phosphorylation (nine), so the present study was designed to assess the involvement of the distal portion of the PI3-kinase pathway. We and other investigators showed that voluntary (27) exercise lone and electrically stimulated musculus contraction (15) do not increase IRS-1-associated PI3-kinase in the absence of insulin or in the presence of low insulin concentrations. However, whether practise performed simultaneously with insulin infusion, leading to synergistic increases in glucose disposal, is associated with increased IRS-1-associated PI3-kinase activity is not known. In the nowadays study, do performed in conjunction with insulin infusion increased IRS-one-associated PI3-kinase activity over that obtained with insulin alone in non-diabetic subjects. Considering do alone does not increment IRS-1-associated PI3-kinase activity (15, 27), the increase in response to insulin + exercise is synergistic past definition. Therefore, in nondiabetic subjects, a synergistic increase in IRS-ane-associated PI3-kinase activeness is consistent with the synergistic increase in glucose disposal. In dissimilarity, fifty-fifty though simultaneous exercise enhanced insulin-stimulated glucose disposal in the diabetic group, there was no increase of IRS-i-associated PI3-kinase activity. Therefore, in dissimilarity to the nondiabetic subjects, it is unlikely that increased IRS-i-associated PI3-kinase activity is involved in the synergistic effect of exercise on insulinstimulated glucose uptake in patients with Type 2 diabetes.

Contrary to IRS-1-associated PI3-kinase activeness, insulin alone significantly increased Akt Ser⁴⁷³ phosphorylation in both groups. These results are comparable to those of Kim et al. (22). Similar to PI3-kinase activity, exercise performed in conjunction with insulin infusion significantly increased Akt serine phosphorylation only in the nondiabetic group. Considering practise alone does non stimulate Akt serine phosphorylation (three, 35), similar to the results for PI3-kinase, exercise synergistically increased insulin-stimulated Akt Ser⁴⁷³ phosphorylation in the nondiabetic subjects (27) but had no effect in the diabetic subjects. Taken together, the results of the present study signal that practice synergistically enhances signaling through PI3-kinase and Akt in nondiabetic, but non Type two diabetic, subjects.

Contempo studies suggest that decreased insulin stimulation of Akt isoforms 2 and 3 is involved in the development of insulin resistance and diabetes (4, seven). In the present study, nosotros were unable to place individual Akt isoforms, and therefore we are unable to decide whether there is a difference betwixt the ii study groups with regard to the effects of insulin or exercise on individual isoforms of Akt.

There are several possible explanations for the difference between the obese control and Type 2 diabetic subjects. 1) Because of their lower V̇o _{2 summit}, work was performed at a lower absolute rate by the diabetic subjects, and possibly this rate was insufficient to induce the insulin signaling increase observed in the control subjects. 2) Insulin signaling abnormalities are more profound in patients with Blazon 2 diabetes than in obese control subjects (nine). These defects may be too profound to be overcome past a single bout of exercise. iii) The diabetic subjects were, on boilerplate, older than the nondiabetic control subjects; thus age may take contributed to the discrepancies betwixt the two groups. However, when nosotros investigated this matter, we found no correlation betwixt age and the effects of exercise on insulin action and signaling. Nevertheless, glucose disposal was increased in both groups of subjects when practice was performed together with an insulin infusion. This indicates that if effects on insulin signaling are involved, they only partially explain the phenomenon.

Downstream mediators of glucose metabolism, such as glycogen synthase (24, 27, 29) and GLUT-4 (xvi), are also influenced past exercise. Cusi et al. (9) demonstrated that 24 h after an astute bout of exercise insulin-stimulated glycogen synthase activity was enhanced, even though insulin-stimulated IRS-1-associated PI3-kinase activeness was unaffected. In the present study, insulin lonely modestly increased glycogen synthase activity to the aforementioned extent in nondiabetic and diabetic subjects. Withal, insulin-stimulated glycogen synthase activeness was significantly increased when practise was performed during the first 30 min of insulin infusion compared with insulin stimulation alone in both groups. Because exercise performed in conjunction with insulin stimulation enhanced glycogen synthase activeness to the same extent in nondiabetic and diabetic subjects, exercise is likely to mediate its furnishings on glycogen synthase independent of its effects on insulin stimulation of IRS-ane-associated PI3-kinase and Akt under these weather. Compared with the effect of insulin or exercise alone (27) to independently increase glycogen synthase activity, do performed during the offset 30 min of insulin infusion approximately additively increased glycogen synthase activity in nondiabetic subjects. These results are consistent with reports that exercise and insulin stimulate glycogen synthase by distinct mechanisms in skeletal muscle (1, 37).

In the present study, enhanced glucose disposal when practise was performed in conjunction with insulin infusion was associated with increased rates of glucose storage (glycogen synthesis). This is especially true for the diabetic subjects, in whom insulin alone merely minimally stimulated glucose storage. Glucose oxidation rates for diabetic subjects were significantly reduced compared with those for nondiabetic subjects basally and during insulin stimulation with or without exercise. This finding is consistent with previous studies and may exist contributed to decreased pyruvate dehydrogenase action basally and during insulin stimulation (21). Still, exercise did not increase subsequent insulin stimulation of glucose oxidation in either group. Therefore, the exercise-induced increase in insulin-stimulated glucose uptake was selectively shunted toward glycogen synthesis in nondiabetic and diabetic subjects. It is probable that increased routing of intracellular glucose to glycogen synthesis was due to the increase in glycogen synthase activity. However, it is less articulate that the increase in glycogen synthase activity led to increased glucose disposal. Rather, because insulin and muscle contraction induce translocation of Overabundance-four transporters to the plasma membrane of muscle cells (16, nineteen), information technology is likely that the increment in glucose disposal was due to increased Overabundance-4 translocation, even though this was not measured in the present study. It has been proposed that practise and insulin recruit Glut-4 from distinct pools of transporters (12). If this is the example, it might be predicted that practice performed in conjunction with insulin infusion would event in an condiment increase in glucose uptake. Because the issue of exercise on insulin-stimulated glucose disposal was synergistic in the present study, mechanisms in improver to Overabundance-four translocation are likely to be involved. It is also possible that an exercise-induced increase in hexokinase action contributed to the increase in glucose uptake (xviii, 26).

In that location were articulate indications of changes in blood flow during practise. For instance, insulin clearance was decreased in command subjects, suggesting that an increase in blood menses to working muscle may accept resulted in decreased splanchnic claret period. This did not occur in the diabetic subjects, possibly because of the lower absolute work rate. The cyberspace result was slightly higher insulin concentration with practice + insulin in the obese nondiabetic control subjects. This may accept contributed to the increment in insulin signaling in this group. Nevertheless, results from higher insulin infusion rates indicate that the magnitude of increase in insulin concentrations was bereft to take much of an effect (unpublished observation). Nevertheless, blood flow to working muscle was doubtlessly increased (10).

In summary, exercise performed in conjunction with insulin administration synergistically increases glucose disposal compared with the effect of insulin alone in nondiabetic and diabetic subjects. The practice-induced increase in glucose uptake is then selectively routed toward glycogen synthesis. This event is probable mediated past increased glycogen synthase activity. At least in the nondiabetic group, increased PI3-kinase signaling may contribute to the exercise-induced synergistic enhancement of glucose disposal. Therefore, although increased blood flow to working musculus undoubtedly increases commitment of insulin and glucose, qualitative changes within the muscle contribute to the increment in glucose uptake and routing of this boosted glucose to glycogen stores.

DISCLOSURES

The enquiry was supported in part by National Institutes of Health Grants R01 DK-47936 (to L. J. Mandarino) and RR-01346 (to GCRC, Audie 50. Murphy Veterans Affairs Hospital) and a grant from the American Diabetes Association (to 50. J. Mandarino).

FOOTNOTES

The authors gratefully acknowledge the exceptional technical assistance of C. Muñoz, S. Taylor, and M. Military camp and the first-class nursing assistance of P. Wolf, North. Diaz, J. Rex, and J. Kincade.

References

1 Aschenbach WG, Suzuki Y, Breeden K, Prats C, Hirshman MF, Dufresne SD, Sakamoto K, Giuseppe Vilardo P, Steele M, Kim JH, Jing SL, Goodyear LJ, and DePaoli-Roach AA. The muscle-specific protein phosphatase PP1G/R(GL)(M(Thou)) is essential for activation of glycogen synthase past exercise. J Biol Chem 276: 39959-39967, 2001.
Crossref | PubMed | ISI | Google Scholar
2 Bergstrom J. Muscle electrolytes in homo determined past neutron activation analysis on needle biopsy specimens: a study in normal patients, kidney patients, and patients with chronic diarrhea. Scand J Clin Lab Invest Suppl 68: one-110, 1962.
Google Scholar
3 Brozinick JT and Birnbaum MJ. Insulin, merely non wrinkle, activates Akt/PKB in isolated rat skeletal muscle. J Biol Chem 273: 14679-14682, 1998.
Crossref | PubMed | ISI | Google Scholar
4 Brozinick JTJ, Roberts BR, and Dohm GL. Defective signaling through Akt-2 and Akt-3 but not Akt-1 in insulin-resistant human skeletal muscle. Potential role in insulin resistance. Diabetes 52: 935-941, 2003.
Crossref | PubMed | ISI | Google Scholar
6 Cheatham B, Vlahos CJ, Cheatham 50, Wang Fifty, Blenis J, and Kahn CR. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, Deoxyribonucleic acid synthesis and glucose transporter translocation. Mol Cell Biol xiv: 4902-4911, 1994.
Crossref | PubMed | ISI | Google Scholar
vii Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, Crenshaw EB III, Kaestner KH, Bartolomei MS, Shulman GI, and Birnbaum MJ. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the poly peptide kinase Akt2 (PKBb). Science 292: 1728-1731, 2003.
ISI | Google Scholar
8 Christ CY, Hunt D, Hancock J, Garcia-Macedo R, Mandarino LJ, and Ivy JL. Exercise grooming improves muscle insulin resistance but non insulin receptor signaling in obese Zucker rats. J Appl Physiol 92: 736-744, 2002.
Link | ISI | Google Scholar
ix Cusi One thousand, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T, DeFronzo RA, Kahn CR, and Mandarino LJ. Insulin resistance differentially affects the PI iii-kinase- and MAP kinase-mediated signaling in human being muscle. J Clin Invest 105: 311-320, 2000.
Crossref | PubMed | ISI | Google Scholar
10 DeFronzo R, Ferrannini Eastward, Sato Y, Felig P, and Wahren J. Synergistic interaction between do and insulin on peripheral glucose uptake. J Clin Invest 68: 1468-1474, 1981.
Crossref | PubMed | ISI | Google Scholar
11 Dela F, Mikines KJ, Larsen JJ, and Galbo H. Glucose clearance in aged trained skeletal muscle during maximal insulin with superimposed exercise. J Appl Physiol 87: 2056-2067, 1999.
Google Scholar
12 Douen AG, Ramlal T, Rastogi S, Bilan PJ, Cartee GD, Vranic One thousand, Holloszy JO, and Klip A. Do induces recruitment of the "insulin-responsive glucose transporter." Evidence for distinct intracellular insulin- and exercise-recruitable transporter pools in skeletal muscle. J Biol Chem 265: 13427-13430, 1990.
Crossref | PubMed | ISI | Google Scholar
13 Frayn K. Adding of substrate oxidation rates in vivo from gaseous substitution. J Appl Physiol 55: 628-634, 1983.
Link | ISI | Google Scholar
fourteen Gastaldelli A, Coggan AR, and Wolfe RR. Assessment of methods for improving tracer estimation of non-steady-state rate of advent. J Appl Physiol 87: 1813-1822, 1999.
Link | ISI | Google Scholar
15 Goodyear L, Giorgino F, Balon T, Condorelli K, and Smith R. Effects of contractile activeness on tyrosine phosphoproteins and PI3-kinase action in rat skeletal muscle. Am J Physiol Endocrinol Metab 268: E987-E995, 1995.
Link | ISI | Google Scholar
16 Goodyear L, Hirshman M, and Horton E. Exercise-induced translocation of skeletal muscle glucose transporters. Am J Physiol Endocrinol Metab 261: E795-E799, 1991.
Link | ISI | Google Scholar
17 Goodyear LJ. AMP-activated poly peptide kinase: a critical signaling intermediary for practice-stimulated glucose send? Exerc Sport Sci Rev 28: 113-116, 1999.
Google Scholar
eighteen Halseth AE, Bracy DP, and Wasserman DH. Overexpression of hexokinase Two increases insulin- and exercise-stimulated muscle glucose uptake in vivo. Am J Physiol Endocrinol Metab 276: E70-E77, 1999.
Link | ISI | Google Scholar
nineteen Hirshman MF, Goodyear LJ, Wardzala LJ, Horton ED, and Horton ES. Identification of an intracellular puddle of glucose transporters from basal and insulin-stimulated rat skeletal musculus. J Biol Chem 265: 987-991, 1990.
Crossref | PubMed | ISI | Google Scholar
20 Kanai F, Ito K, Todaka 1000, Hayashi H, Kanohara S, Ishii 1000, Okada T, Hazeki O, Ui M, and Ebina Y. Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-one and the activity of PI iii-kinase. Biochem Biophys Res Commun 195: 762-768, 1993.
Crossref | PubMed | ISI | Google Scholar
21 Kelley DE, Mokan M, and Mandarino L. Intracellular defects in glucose metabolism in obese patients with non-insulin-dependent diabetes mellitus. Diabetes 41: 698-706, 1992.
Crossref | PubMed | ISI | Google Scholar
22 Kim YB, Nikoulina SE, Ciaraldi TP, Henry RR, and Kahn BB. Normal insulin-dependent activation of Akt/poly peptide kinase B, with diminished activation of phosphoinositide 3-kinase, in musculus in type two diabetes. J Clin Invest 104: 733-741, 1999.
Crossref | PubMed | ISI | Google Scholar
23 Kitamura T, Ogawa West, Sakaue H, Hino Y, Kuroda South, Takata M, Matsumoto M, Maeda T, Konishi H, Kikkawa U, and Kasuga M. Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose ship. Mol Prison cell Biol 18: 3708-3717, 1998.
Crossref | PubMed | ISI | Google Scholar
24 Kochan RG, Lamb DR, Lutz SA, Perrill CV, Reimann EM, and Schlender KK. Glycogen synthase activation in human skeletal musculus: effects of diet and exercise. Am J Physiol Endocrinol Metab Gastrointest Physiol 236: E660-E666, 1979.
Link | ISI | Google Scholar
26 Koval JA, DeFronzo RA, O'Doherty RM, Printz R, Ardehali H, Granner DK, and Mandarino LJ. Regulation of hexokinase 2 activeness and expression in homo muscle by moderate exercise. Am J Physiol Endocrinol Metab 274: E304-E308, 1998.
Link | ISI | Google Scholar
27 Koval JA, Maezono K, Patti ME, Pendergrass M, DeFronzo RA, and Mandarino LJ. Effects of practice and insulin on insulin signaling proteins in homo skeletal muscle. Med Sci Sports Exerc 31: 998-1004, 1999.
Crossref | PubMed | ISI | Google Scholar
28 Lowry OH, Rosebrough NJ, Fan AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951.
Crossref | PubMed | ISI | Google Scholar
29 Markuns JF, Wojtazsewski JFP, and Goodyear LJ. Insulin and exercise subtract glycogen synthase kinase-3 activity by different mechanisms in rat skeletal muscle. J Biol Chem 274: 24896-24900, 1999.
Crossref | PubMed | ISI | Google Scholar
30 Minuk HL, Vranic M, Marliss EB, Hanna AK, Albisser AM, and Zinman B. Glucoregulatory and metabolic response to practice in obese non-insulin-dependent diabetes. Am J Physiol Endocrinol Metab 240: E458-E464, 1981.
Link | ISI | Google Scholar
31 Nyboer J, Murray P, and Sedersky J. Blood menses indices in amputee and control limbs past mutual electrical impedance plethysmography. Am Eye J 87: 704-710, 1974.
Crossref | ISI | Google Scholar
32 Pratipanawatr T, Cusi K, Ngo P, Pratipanawatr W, Mandarino LJ, and DeFronzo RA. Normalization of plasma glucose concentration by insulin therapy improves insulin-stimulated glycogen synthesis in type 2 diabetes. Diabetes 51: 462-468, 2002.
Crossref | PubMed | ISI | Google Scholar
33 Richter EA, Garetto LP, Goodman NN, and Ruderman NB. Enhanced muscle glucose metabolism later on exercise: modulation by local factors. Am J Physiol Endocrinol Metab 246: E476-E482, 1984.
Link | ISI | Google Scholar
34 Wang Q, Somwar R, Bilan PJ, Liu Z, Jin J, Woodgett JR, and Klip A. Poly peptide kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol Jail cell Biol 19: 4008-4018, 1999.
Crossref | PubMed | ISI | Google Scholar
35 Widegren U, Jiang XJ, Krook A, Chibalin AV, Bjornholm Grand, Tally M, Roth RA, Henriksson J, Wallberg-Henriksson H, and Zierath JR. Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal musculus. FASEB J 12: 1379-1389, 1998.
Crossref | PubMed | ISI | Google Scholar
36 Wojtaszewski J, Hansen B, Gade J, Kiens B, Markuns J, Goodyear L, and Richter E. Insulin signaling and insulin sensitivity after exercise in human skeletal muscle. Diabetes 49: 325-331, 2000.
Crossref | PubMed | ISI | Google Scholar
37 Wojtazsewski JFP, Nielson P, Kiens B, and Richter EA. Regulation of glycogen synthase kinase-three in man skeletal muscle. Effects of food intake and bicycle exercise. Diabetes 50: 265-269, 2001.
Crossref | PubMed | ISI | Google Scholar