Effects of insulin on skeletal muscle glucose storage, oxidation, and glycolysis in humans DAVID E. KELLEY, THIEMO VENEMAN,

JAMES P. REILLY, AND LAWRENCE

J. MANDARIN0

Departments of Medicine, Surgery, Ophthalmology, and Physiology, University of Pitt&u& School of Medicine, Presbyterian- University Hospital, Eye and Ear Institute of Pittsburgh, Pittsburgh 15213; and Veterans Administration Hospital, Pittsburgh, Pennsylvania 15240

KELLEY, DAVID E., JAMES P. REILLY, THIEMO VENEMAN, AND LAWRENCE J. MANDARINO. Effects of insulin on skeletal muscle glucose storage, oxidation, and glycolysis in humans. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E923-E929, 1990.-

The effects of physiological hyperinsulinemia (-75 mu/l) on glucosestorage, oxidation, and glycolysis in skeletal muscle were assessedwith euglycemic clamps performed in seven healthy volunteers, in conjunction with legbalance for glucose, lactate, alanine, 02, and CO,. Infusion of insulin increasedleg glucoseuptake, storage, and oxidation but did not alter net releaseof lactate and alanine. The respiratory quotient (RQ) acrossthe leg increasedfrom a basal value of 0.74 & 0.02 to 0.99 & 0.02 during hyperinsulinemia. Under conditions of insulin stimulation, 49 t 5% of leg glucoseuptake was stored, 37 t 4% was oxidized, and 14 t 2% was releasedas lactate and alanine. We concludethat during physiological hyperinsulinemia and euglycemia1) skeletal musclelipid oxidation is nearly entirely suppressed and glucosebecomesthe primary oxidative substrate of muscle,2) glucosestorage and oxidation are the major pathways of skeletal muscleglucosemetabolismand are quantitatively similar at physiological insulin levels, and 3) the majority of insulin-stimulated glycolysis is oxidized, with only a small portion releasedas lactate or alanine. hyperinsulinemia;euglycemia;glucosedisposal

GLUCOSE TRANSPORTED into human skeletal muscle can be stored as glycogen or can undergo glycolysis and be oxidized in the tricarboxylic acid cycle or released as lactate, alanine, or pyruvate. It is generally accepted that the majority of glucose disposal during euglycemic physiological hyperinsulinemia occurs in skeletal muscle (8, 33). However, the relative contributions of oxidative and nonoxidative pathways to skeletal muscle glucose disposal under these conditions in humans have not been directly assessed. Instead, the pattern of muscle glucose metabolism has been inferred from results of studies that utilize whole body indirect calorimetry and isotopically determined rates of glucose disposal to estimate systemic rates of oxidative and nonoxidative glucose metabolism (8, 33-35). This approach may yield inaccurate conclusions about muscle glucose metabolism. Rates of systemic glucose oxidation determined by whole body indirect calorimetry reflect not only true carbohydrate oxidation but also include components attributable to gluconeogenesis and lipogenesis, processes that do not 0193-1849/90

$1.50 Copyright

occur to a significant extent in muscle (12). In the postabsorptive condition, because skeletal muscle glucose utilization accounts for only 1525% of total glucose disposal (1, 4, 8, 16, 18), it is not possible to interpolate the basal pattern of oxidative and nonoxidative metabolism of skeletal muscle from systemic studies. Therefore, a method is needed to more directly assess the pathways of glucose metabolism in skeletal muscle. Thirty years ago, Andres and colleagues (1) established that the forearm arteriovenous balance technique could be used to quantify overall glucose metabolism and, in particular, oxidative metabolism across a muscle bed in humans. Measurement of forearm glucose uptake, lactate release, oxygen consumption, and carbon dioxide production (forearm indirect calorimetry) demonstrated that under postabsorptive conditions in humans, forearm glucose uptake is small and lipid is the predominant substrate for oxidation in muscle. The limb balance technique has been extensively used to measure muscle glucose uptake (1, 33), lactate release (26), free fatty acids (FFA) uptake (28), and amino acid fluxes (24). Despite these earlier studies, few investigators have employed the techniques of limb indirect calorimetry (1, 3, 6, 18, 21). This may partially be attributable to the cumbersome Van Slyke technique previously used for determining whole blood CO2 content (31), which has limited the number of blood CO2 determinations that could be performed over a short time period, decreasing the reliability in estimates of limb oxidative metabolism. It is generally thought that glycogen formation is the major pathway of glucose metabolism in skeletal muscle during hyperinsulinemia (5, 8, 19). This conclusion may be dependent on the insulin concentration. In the studies cited, arterial insulin exceeded 100 mu/l, a value above the physiological range for healthy nondiabetic humans. At a plasma insulin concentration of -50 mu/l, which is within the physiological range, systemic glucose oxidation was equivalent to glucose storage (20,30). In these studies, as in all investigations in which systemic indirect calorimetry is employed, nonoxidative glucose metabolism included lactate generation and therefore glucose “storage” was overestimated to an unknown extent. Lactate release can be measured with the limb balance technique and thus this ambiguity can be resolved. The present studies were therefore undertaken to es-

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tablish a method to more directly determine, during postabsorptive conditions and during steady-state euglycemic physiological hyperinsulinemia, the relative importance of fat and glucose as oxidative substrates in the human leg and the relative contributions of glucose storage, oxidation, and nonoxidative glycolysis to leg glucose disposal. To reliably perform limb indirect calorimetry, we employed multiple arterial and venous blood sampling with rapid assessment of blood CO2 and 02 content using a precisely calibrated blood gas instrument and oximeter. METHODS

Subjects. Informed written consent was obtained from seven healthy subjects who had no family history of diabetes mellitus. Clinical characteristics are shown in Table 1. Subjects were instructed not to exercise the day before the study and to eat a diet containing at least 200 g carbohydrate/day for 3 days preceding admission. Protocol. Subjects were admitted to the University of Pittsburgh General Clinical Research Center the evening before the study. After eating a lo-kcal/kg evening meal (50% carbohydrate, 35% protein, and 15% fat), the subjects fasted overnight. In the morning a catheter was placed in an antecubital vein (18 gauge, Jelco, Tampa, FL) for infusion of glucose and insulin. A radial artery (20 gauge, Jelco) and a femoral vein (16 gauge, Arrow International, Reading, PA), were catheterized for intermittent blood sampling. Blood flow to the leg was measured hourly during the entire study period using electrocapacitance plethysmography (model 2560, UFI, Morro Bay, CA). During a basal period of 30 min and during the final 30 min of a 4-h infusion of insulin at 30 mu. rnm2. min-’ (U-100, Humulin R, Eli Lilly, Indianapolis, IN), simultaneous arterial and venous blood samples were obtained at lo-min intervals for determinations of glucose, lactate, and alanine concentrations. During the insulin infusion, arterial glucose was measured every 5 min, and euglycemia was maintained with a variable infusion of 50% dextrose. Patients remained supine throughout the study. Leg indirect calorimetry. During the 30-min basal period and during the final 30 min of the euglycemic clamp simultaneous arterial and femoral venous blood samples were collected at 5-min intervals for measurement of O2 and C02. These repetitive blood gas samples

1. Clinical characteristics of subjects

TABLE

Basal

M M M F M F M Means BMI,

Yr

BMI, kg/m2

25 25 32 32 32 32 48

22 26 22 21 25 23 26

Age,

Sex

k SE

body

32k2.9

mass index.

24kO.8

Insulin, mU/l

Glucose, mmol/l

6

5.14 5.28 4.78 5.28 5.08 5.26 5.54

15

8 10

14 14 11 11t1.3

5.19t0.09

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were collected into heparinized syringes, immersed in ice, and analyzed within 5 min. Plasma CO2 content was calculated from CO2 tension and pH measured with a blood gas analyzer (System 1304 pH/blood gas analyzer, Allied Instrumentation Laboratory, Lexington, MA) with the use of established equations and a solubility constant of 0.0307. The blood gas analyzer was calibrated with a quantitative gasometric technique. The accuracy of the equipment is 0.1 mmol/l for CO2. The relationship of plasma CO2 content to whole blood CO2 content is affected by hemoglobin content, saturation, and pH. Therefore, plasma CO2 content was adjusted to whole blood CO2 content with the use of an empirically derived regression equation that gives values of whole blood CO2 content, which are in close agreement with determination of blood CO2 content by the Van Slyke method. The advantage of the automated equipment used in this study over a Van Slyke apparatus is that six samples can be analyzed in the approximate time that is required to perform a single Van Slyke determination. A cooximeter (IL282 Cooximeter System, Allied Instrument Laboratory) was used to measure hemoglobin content and saturation, and 02 content was calculated from the hemoglobin content and percent saturation, using a constant of 1.39 (2). The accuracy of this equipment is 0.3 mmol/ 1 for O2 content. The basal and clamp arterial and venous CO2 and O2 content were taken as the mean of six respective determinations for each subject. The coefficient of variation was calculated for each individual and the mean intrasubject coefficient of variation was 1.36 t 0.2% for arterial C02, 1.96 t 0.3% for femoral venous COZ, 1.52 t 0.49% for arterial 02, and 4.93 t 0.6% for femoral venous O2 content. Local indirect calorimetry performed with multiple blood gas samples averaged over a 300min period is analogous to whole body indirect calorimetry performed with multiple breath samples averaged over a 3O-min period. The accuracy and precision of these determinations allows reliable estimation of substrate oxidation rates. Leg balance of O2 (VO,) and CO2 (VCO,) were calculated as the product of the arteriovenous difference and the leg blood flow. The rates of leg carbohydrate and lipid oxidation were calculated using the equations of Frayn (12), employing a constant value for limb protein oxidation of 85 nmolmin-‘. 100 ml tissue-’ as kindly suggested by E. Ferrannini (personal communication), based on previous studies of limb protein oxidation (13, 29) Analysis. Plasma glucose was measured with a Beckman Glucose Analyzer 2, (Beckman Instruments, Fullerton, CA). Plasma lactate and alanine were determined microfluorometrically (17, 22). Arterial insulin was assayed by the method of Herbert et al. (14). Plasma FFA was determined by the method of Itaya et al. (15). Cakulations. Plasma glucose concentration was converted to whole blood glucose by the factor (1 - 0.29) x (hematocrit). The leg balance of lactate and alanine was used in conjunction with measurements of leg carbohydrate oxidation to obtain a measurement of total glycolysis. There is rapid interchange between skeletal muscle

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lactate and alanine pool (11) so that lactate and alanine balance were combined. During insulin infusion, proteolysis is suppressed (l3), so the contribution of proteolysis to alanine release is small. Lactate and alanine are taken up by muscle, produced in muscle, and released. The rate of release of lactate and alanine into the venous circulation is given by its rate of appearance in the endogenous pool minus the rate of its oxidation, with the latter measured by indirect calorimetry. Thus, with the assumption that the intracellular pool does not change significantly during resting steady-state conditions, the arteriovenous (a-v) balance of glycolytic products equals the total rate of glycolysis minus the rate of carbohydrate oxidation and is assumed to be an estimate of the rate of nonoxidized glycolysis. Pyruvate balance was not ineluded because of its minor contributions (18). Net glucose storage was calculated as the difference between glucose uptake and the sum of glucose oxidation and release of glycolytic products. This estimate of net storage can be positive or negative. If positive, it can be interpreted as net glycogen synthesis, whereas if negative, it can be interpreted as net glycogenolysis. Statistics. Data are expressed as means t SE. Statistical analysis was performed using paired t tests to cornpare basal and insulin-stimulated measurements. Bivariate correlation analysis was performed using parametric regression techniques (Pearson’s r), while multiple analysis was performed using a stepwise method (RS/l software, BBN Software, Cambridge, MA). Comparisons among relative contributions of specific metabolic pathways to overall leg glucose uptake were performed with repeated measures analysis of variance (SPSS software, SPSS, Chicago, IL).

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sion of insulin. The arterial insulin concentration was increased from a basal value of 11 t 1 mU/l to 75 t 7 by the infusion (P < 0.001). Basal leg blood flow was 2.49 t 0.2 ml* min-’ . 100 ml tissue-’ and tended to increase, though not significantly, by the 4th h of insulin infusion to 2.76 t 0.2 ml. min-lo 100 ml tissue-’ (P = 0.06). The basal arteriofemoral vein difference (a-FV) for glucose was 0.06 t 0.02 mmol/l and increased to 1.06 t 0.1 (P < 0.001) during euglycemic hyperinsulinemia. Therefore, insulin infusion increased leg glucose uptake from a basal value of 0.14 t 0.05 prnol min-l . 100 ml tissue-l to 2.92 t 0.40 (P = 0.0005). Glucose infusion rate (GIF) at the end of the study was 7.05 t 0.8 mg. min-’ . kg-‘, and leg glucose uptake was highly correlated with GIF, r = 0.95 (P < 0.001). As shown in Fig. 1, the time course of insulin stimulation of glucose uptake across the leg paralleled the rate of glucose infusion needed to maintain euglycemia. Glucose and fat oxidation (Table 3). Arteriovenous differences in blood 02 and CO2 content were used to calculate leg respiratory quotient (RQ). Leg COz production was higher during the final 30 min of insulin infusion than during basal conditions (6.35 t 0.59 vs. 4.18 t 0.46 pmol min-’ 100 ml tissue-‘, respectively, P < 0.001). Leg 02 consumption increased during insulin infusion compared with basal conditions (6.43 t 0.65 vs. 5.52 t 0.5 1 pm01 . min-l 100 ml tissue-‘), but this was not significant (P = 0.06). The mean basal RQ across the leg was 0.74 t 0.02 and increased in each subject during steady-state hyperinsulinemia to a mean value of 0.99 t 0.02, (P < 0.001) as shown in Fig. 2. During basal l

l

l

8

RESULTS

Glucose uptake (Table 2). The basal arterial glucose concentration (5.19 t 0.09 mmol/l) was kept constant (4.98 t 0.04 mmol/l) by infusion of glucose during infu2. Arterial insulin, glucose, lactate, alanine, and FFA concentrations, leg blood flow, and leg substrate balance under basal conditions and during clamp

6

TABLE

Basal

Euglycemic, Hyperinsulinemic Clamps

LL z & -2 ;

4

I? Plasma insulin, mU/l Leg blood flow, ml. min. Plasma glucose, mmol/l Arterial a-FV Leg balance Plasma lactate (mmol/l) Arterial a-FV Leg balance Plasma alanine, mmol/l Arterial a-FV Leg balance Plasma FFA, mmol/l

100 ml-’

Values are means t SE. Leg balance blood values by method of Dillon (9); tissue-‘. * P < 0.001 clamp vs. basal. t

llk1.3 2.49t0.2

75t7.4* 2.76k0.2

5.19&0.09 0.06kO.02 0.14t0.05

4.98kO.04 1.06&O. lO* 2.92*0.40*

0.83kO.09 -0.18kO.03 -0.46kO.09

1.27&0.091 -0.24kO.05 -0.70&O. 16

0.24t0.03 -0.06&0.01 -0.15kO.02 0.5820.04

0.28*0.02t -0.05t0.01 -0.15~0.03 0.19t0.05*

values are corrected to whole units are pmol emin-’ 100 ml P < 0.05 clamp vs. basal. l

2

n 60

120

180

240

Time FIG. 1. Mean &SE glucose infusion rate (GIF) required to maintain euglycemia in mg. min-’ kg-’ and mean &SE leg glucose uptake (LGU) in pm01 min-’ 100 ml tissue-’ during 240 min of euglycemic hyperinsulinemic clamp. l

l

l

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3. Arterial O2 and CO2 content, a-FV differences, rates of leg 02 consumption and CO2production, leg RQ, and substrate oxidation rates

TABLE

Euglycemic Hyperinsulinemic Clamp

Basal COZ, mmol/l Arterial a-FV

vco2

26.5kO.90 -1.64t0.06 4.18t0.046

25.7t0.91 -2.30t0.13* 6.35+0.59t

7.52t0.30 2.2OkO.06 5.52t0.51 0.7420.02 0.17~0.08

7.21k0.38 2.34t0.16 6.43t0.65 0.99+0.03-f 1.01+0.02t

0.19&0.02

0.01+0.02~

02, mmol/l Arterial a-FV VO'L

Leg RQ

Glucose oxidation, pmol min-’ -100 ml tissue-’ Fat oxidation, pmol . min. 100 ml tissue-’ Values are means clamp vs. basal.

t

l

SE. * P < 0.01 clamp

vs. basal.

t

P < 0.001

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infusion (0.42 t 0.09 vs. basal rate of 0.30 t 0.05 prnol. min-l *lo0 ml tissue-l, P = 0.30). Combined release of lactate and alanine was positively correlated with skeletal muscle glucose oxidation during basal (r = 0.81, P < 0.01) and insulin-stimulated conditions (r = 0.83, P < O.Ol), as shown in Fig. 3. Glucose storage (Table 4). Net glucose storage was calculated as glucose uptake minus the sum of glucose oxidation and lactate and alanine release. During basal conditions, the sum of glucose oxidation and net release of lactate and alanine exceeded leg glucose uptake; therefore, net glucose storage was negative at -0.33 t 0.11 pmol glucose. min-’ 100 ml leg tissue-‘, indicating glycogenolysis was proceeding at a greater rate than glycogen synthesis. During the final 30 min of insulin infusion the net rate of glucose storage was 1.49 t 0.32 prnol min-l . 100 ml leg tissue? l

Contributions of oxidation, storage, and glycolytic release to insulin-stimulated leg glucose uptake (Table 4 and 1.5

1.0

.7 .6

i,

240



Time 2. Leg respiratory (0) and during euglycemic FIG.

quotient (RQ) hyperinsulinemic

in each subject clamp (240).

at base line

0.4

0.2

conditions leg glucose oxidation was 0.17 t 0.07 prnol. min-’ JO0 ml tissue-’ and accounted for 18% of leg oxygen consumption. Basal lipid oxidation (0.19 t 0.07 pm01 . min-lo 100 ml tissue-‘) accounted for the majority of oxygen consumption. Infusion of insulin suppressed skeletal muscle lipid oxidation to a value of 0.01 t 0.02 pmol. min-’ 100 ml tissue-’ (P = 0.0005 vs. basal rates) and increased leg glucose oxidation to 1.01 t 0.09 pmol . min-’ JO0 ml tissue-l (P = 0.0005 vs. basal rates). Hyperinsulinemia suppressed arterial FFA by 74% from a basal level of 0.58 t 0.04 to 0.19 t 0.05 mmol/l (P < 0.001). During steady-state hyperinsulinemic conditions, carbohydrate oxidation accounted for 94% of leg oxygen, whereas lipid oxidation accounted for only 3%. Glycolysis (Table 2). Arterial lactate concentrations increased during the 1st h of insulin infusion, then reached a plateau that was -50% greater than basal (0.83 t 0.09 vs. 1.27 t 0.09 mmol/l, P < 0.01). Arterial alanine concentration rose slightly during insulin infusion (0.24 t 0.03 vs. 0.28 t 0.02 mmol/l, P < 0.05). Combined lactate and alanine net release, expressed as glucose equivalents, did not significantly change during insulin

0.6

0.8

Non-Oxidized Glycolysis 3. Relationship between leg glucose oxidation (pmol. min. 100 ml tissue-‘) and leg nonoxidized glycolysis (pmol. min-’ 100 ml tissue-‘), at base line (0) and during last 30 min of euglycemic hyperinsulinemic clamp (m). Regression equations: oxidized = 1.23 nonoxidized - 0.20; and oxidized = 0.85 nonoxidized + 0.65; for base line (time = -30 to 0 min) and clamp (time = 210 to 240 min), respectively. FIG.

l

l

l

TABLE 4. Pathways of leg glucose metabolism in healthy subjects during basal and euglycemic hyperinsulinemic conditions

Basal Glucose uptake Nonoxidative glycolysis Oxidation Storage

0.14t0.05 0.30t0.05 0.17t0.08 -0.33kO.11

Euglycemic Hyperinsulinemic Clamp

‘36 Glucose Uptake

2.92&0.40* 0.42*0.09 1.01~0.02t 1.47+0.32?

100 14t2 37t4 4925

Values are means 2 SE in ~molomin-’ -100 ml tissue-‘, except nonoxidative glycolysis, which is expressed as glucose equivalents. Rates of glycolysis, oxidation, and storage during clamp are also given in parentheses as percentages of glucose uptake. * P < 0.001 clamp vs. basal. t P < 0.01 clamp vs. basal.

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Fig. 4). During steady-state insulin stimulation, the proportion of leg glucose uptake that was stored was 49 t 5%, that oxidized was 37 t 4%, and that released from the leg as lactate and alanine was 14 t 2%. The proportion of glucose uptake due to net balance of lactate and alanine was less than either glucose oxidation or storage (P < 0.001, repeated measures ANOVA), whereas the proportions due to oxidation and storage were not significantly different from each other. Rates of the pathways of glucose metabolism are depicted in Fig. 4 for each subject during the insulin infusion. There were significant simple correlations during insulin infusion between leg glucose uptake and net lactate and alanine balanced (r = 0.76, P = 0.05), and glucose storage (r = 0.90, P = 0.005), but the correlation between glucose uptake and glucose oxidation was not statistically significant (r = 0.49, P = 0.26). Because glucose oxidation and net lactate and alanine balance were also strongly correlated (r = 0.83, P = 0.01) we compared the pathways of muscle glucose metabolism using stepwise multiple regression analysis. When the correlation between lactate and alanine balance and glucose oxidation was taken into account statistically by these means, it was found that glucose oxidation and glucose storage independently ex lained nearly all of the variability in glucose uptake (r P = 0.99, P = 0.001). The multiple regression equation was uptake = 1.11. (storage) + 1.79. (oxidation) - 0.535. The partial regression coefficients for both glucose oxidation and storage were highly significant (each < 0.001). DISCUSSION

The present studies were undertaken to determine in healthy humans the relative effects of physiological hy-

rl

C

‘.:.~.:. .5-A-2 .v.-.v 2.v.v El

FIG.

final min-’

.

d Subjects

e

Non-Oxidized

Glycolysis

Ia

Glucose Oxidation

cl

Glucose Storage

4. Pathways of leg glucose metabolism in 7 subjects during 30 min euglycemic hyperinsulinemic clamp. Units are prnol100 ml tissue-‘. l

IN

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perinsulinemia on skeletal muscle glucose storage, oxidation, and nonoxidative glycolysis and also to determine the principal oxidative substrate of skeletal muscle during euglycemic physiological hyperinsulinemia. Baltzan and colleagues (3) used local indirect calorimetry across the human forearm to assess oxidative metabolism of muscle during basal conditions. A mean RQ of 0.76 in 45 young male subjects established that lipid is the primary oxidative substrate of resting muscle during postabsorptive conditions. The present data confirm this earlier finding, as the basal RQ across the leg was 0.74 t 0.02. In this study, insulin infusion increased the RQ across the leg to 0.99 t 0.04. There was a sevenfold increase in the rate of skeletal muscle glucose oxidation, whereas lipid oxidation was suppressed. O2 consumption across the leg increased by 20%, and although this did not attain statistical significance, it would be consistent with a thermic effect of infused insulin and glucose. However, there was a greater increase in CO2 production than in 02 consumption, and this produced the increase in leg RQ. Our results thus provide direct evidence that glucose becomes the primary oxidative substrate in skeletal muscle during physiological hyperinsulinemia in humans. Previously Rabinowitz et al. (25) found that forearm glucose oxidation did not increase during a brief (26min) infusion of insulin into the brachial artery in healthy male subjects. The insulin infusion used by these workers may have been too brief to affect substrate oxidation. Another factor that could account for the differences between that study and the current one is that the localized infusion of insulin used by Rabinowitz et al. did not increase systemic insulin and consequently did not decrease arterial FFA levels. In contrast, in our studies the systemic insulin infusion produced marked suppression of arterial FFA. This could mean that suppression of plasma FFA is necessary for insulin stimulation of muscle glucose oxidation and would be consistent with a glucose-FFA competition for substrate oxidation as hypothesized by Randle (27). However, because plasma FFA uptake by muscle was not directly measured isotopically in this study and because muscle may also oxidize endogenous FFA (6), the results of this study do not provide data to test this hypothesis directly. The present studies were also undertaken to determine the relative contributions of glucose storage, oxidation, and glycolysis to muscle glucose uptake. Our results indicate that glucose oxidation and glucose storage are the two major pathways of insulin-stimulated glucose disposal by muscle during euglycemia. It should be emphasized that glucose oxidation and storage are approximately equivalent in magnitude during physiological hyperinsulinemia. The current results should be interpreted cautiously, because a plasma insulin concentration of 75 pU/ml is rarely sustained for 4 h in normal subjects. However, these results are consistent with our previous findings that skeletal muscle glucose oxidation is equivalent to muscle glucose storage during metabolism of an oral glucose load in healthy humans (18). The current findings during insulin infusion are similar to those reported for whole body glucose metabolism (30), which was expected because the majority of the whole

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body glucose disposed is accounted for by muscle (8). We conclude that impairments in skeletal muscle glucose oxidation may be quantitatively as important as impairment in glucose storage under physiological conditions in circumstances of insulin resistance. This speculation requires further investigation. The relative magnitude of glucose oxidation and glucose storage depends on the specific arterial insulin level achieved (34), which in this study was at the upper physiological range for healthy humans (23). Had a higher insulin level been used, it is likely that storage would have been predominant because at the insulin level attained the leg RQ was -1.0. Higher rates of carbohydrate oxidation would therefore have depended on increased rates of metabolism, which would not be likely during resting conditions. Conversely, it is possible that if a lower insulin level had been employed, glucose oxidation might have exceeded glucose storage. It should be emphasized that the results of the present study imply that, under the conditions of hyperinsulinemia employed, glucose storage and oxidation are the two major pathways of leg glucose metabolism and that they are roughly the same in magnitude. Release of glycolytic products represented a minor portion of glucose uptake. However, if the sum of glucose oxidation and release of glycolytic products is used as an estimate of total glycolysis, the present results can be interpreted to indicate that of the glucose taken up by skeletal muscle at an insulin concentration of -75 mU/ 1, about one-half is stored and about one-half undergoes glycolysis. Of that undergoing glycolysis, the majority is oxidized during hyperinsulinemia. The results of these studies have implications regarding the control of glycolysis by insulin in skeletal muscle. Although net balance of lactate and alanine did not change during hyperinsulinemia compared with the basal conditions, muscle glucose oxidation increased substantially. This finding of differential activation by insulin of nonoxidative and oxidative glycolysis suggests that at the bifurcation of these pathways there is an insulininduced increase of pyruvate flux into oxidative metabolism. It has previously been shown that infusion of insulin in humans increases the activity of skeletal muscle pyruvate dehydrogenase (PDH) assayed in biopsies (ZO), and PDH has been proposed to be the regulatory enzyme for entry of pyruvate into the tricarboxylic acid cycle for oxidation (27). This suggests that a key regulatory step in the effects of insulin on muscle glucose metabolism is activation of PDH. A second finding from these studies that has bearing on the regulation of glycolysis in muscle is the positive correlation between glucose oxidation and nonoxidized glycolysis, which was found during basal and insulin-stimulated conditions, as shown in Fig. 3. This is consistent with additional regulation of pyruvate oxidation by substrate availability. We conclude from these results that the rate of glucose oxidation in muscle increases in response to insulin by means of both increased PDH activity and increased pyruvate availability, although the relative importance of these two mechanisms is uncertain. Glucose storage was negative in the basal state, which

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we assume to be a reflection of ongoing net glycogenolysis. Studies conducted under basal conditions using systemic indirect calorimetry and isotopically determined glucose disposal do not permit an assessment of muscle glycogenolysis because these techniques cannot discriminate between lactate formation from glucose and glycogen breakdown. During hyperinsulinemia, not only did glucose uptake increase dramatically but a shift from net glycogenolysis to glycogen synthesis occurred, such that about one-half of glucose uptake was converted to glycogen. Glucose storage in skeletal muscle was positively correlated with glucose uptake during infusion of insulin, suggesting that glycogen synthesis is influenced by the rate of glucose uptake. Several investigators have shown that insulin increases the activity of muscle glycogen synthase (5, 20). Therefore, the correlation between glucose uptake and glucose storage could also reflect the effect of insulin to stimulate both glucose transport and glycogen synthase. However, because glucose storage is calculated in part from the rate of glucose uptake, this correlation should be interpreted with caution. There was considerable variability in the rate of insulin stimulated leg glucose uptake, as shown in Fig. 4. The variability in leg glucose uptake was attributable to differences among individuals in both glucose oxidation and glucose storage, which together accounted for 99% of variability in leg glucose uptake. Glucose storage accounted for most of the variability (81%), although this may partly result from the calculation of glucose storage from glucose uptake. This finding is analogous to previous studies in healthy humans in which variability between individuals in whole body glucose disposal (measured isotopically) during hyperinsulinemic euglycemic clamps was mostly due to variability in nonoxidative glucose metabolism (measured by whole body indirect calorimetry) (19, 34). However, in those studies, as in the present study, the rates of glucose oxidation were maximally activated by insulin, whereas the rates of glucose storage were not. It is possible that this may tend to somewhat exaggerate variability in glucose storage relative to that in glucose oxidation. It is also possible that some variability in leg glucose storage may have been due to variability in exercise on the day before admission, as Devlin and Horton (7) have shown that insulin-stimulated glucose storage was increased by exercise 12-14 h before study. However, in that study (7) exhaustive, glycogen-depleting exercise was performed. The subjects in the current study were instructed not to exercise for 24 h before admission and stated that they followed instructions. This would tend to minimize any effect of exercise. In summary, in the present study we have used the leg balance method in conjunction with multiple arterial and venous blood sampling for performing leg indirect calorimetry to directly assess the pathways of leg muscle glucose metabolism during postabsorptive conditions and hyperinsulinemia. Basally, leg glucose uptake was minimal, net glycogenolysis was occurring, and the leg was obtaining ~20% of its energy requirement from glucose oxidation. Physiological hyperinsulinemia increased

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GLUCOSE

METABOLISM

skeletal muscle glucose uptake, oxidation, and storage, and suppressed fat oxidation, but did not alter net release of glycolytic products. We conclude that at this level of hyperinsulinemia (-75 mu/l), increased glycolysis is directed nearly entirely to glucose oxidation and that glucose oxidation is of equivalent importance to glucose storage in accounting for insulin-stimulated skeletal muscle glucose disposal. The ability to more directly assess the pathways of muscle metabolism should provide the mechanisms an important tool for characterizing responsible for defects in insulin action in skeletal .m .uscle of patients with insulin-resistant conditions such as noninsulin-dependent diabetes mellitus or obesity. We gratefully acknowledge the technical assistance of R. Thorne, L. Henry, C. Korbanic, L. Weiss, and the nursing staff of the General Clinic Research Center at the University of Pittsburgh. The excellent editorial assistance of J. Smith is appreciated. This study was supported by the American Diabetes Association, the Medical Research Services of the Veterans Administration, Research to Prevent Blindness, and the Eye and Ear Institute of Pittsburgh. Address for reprint requests: D. Kelley, Univ. of Pittsburgh School of Medicine, 930 Scaife Hall, Pittsburgh, PA 15261. Received

10 July

1989; accepted

in final

form

29 January

1990.

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Effects of insulin on skeletal muscle glucose storage, oxidation, and glycolysis in humans.

The effects of physiological hyperinsulinemia (approximately 75 mU/l) on glucose storage, oxidation, and glycolysis in skeletal muscle were assessed w...
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