increased muscle glucose uptake after exercise no - diabetes

Increased Muscle Glucose UptakeAfter ExerciseNo Need for Insulin During ExerciseERIK A. RICHTER, THORKIL PLOUG, AND HENRIK GALBO

SUMMARYIt has recently been shown that insulin sensitivity ofskeletal muscle glucose uptake and glycogen synthesisis increased after a single exercise session. The pres-ent study was designed to determine whether insulin isnecessary during exercise for development of thesechanges found after exercise. Diabetic rats and con-trols ran on a treadmill and their isolated hindquarterswere subsequently perfused at insulin concentrationsof 0, 100, and 20,000 jxlJ/ml. Exercise increased insulinsensitivity of glucose uptake and glycogen synthesisequally in diabetic and control rats, but insulin respon-siveness of glucose uptake was noted only in controls.Analysis of intracellular glucose-6-phosphate, glucose,glycogen synthesis, and glucose transport suggestedthat the exercise effect on responsiveness might bedue to enhancement of glucose disposal. After electri-cal stimulation of diabetic hindquarters in the presenceof insulin antiserum, insulin sensitivity of 3-O-methyl-glucose transport was increased to the same extent asin muscle from healthy rats stimulated in the presenceof insulin at 50 |xU/ml. Furthermore, in muscle depletedof glycogen by contractions, transport of 3-O-methyl-glucose was increased in the presence of insulin anti-serum and in the absence of increased regional perfus-ate flow. It is concluded that after exercise, increasedsensitivity of muscle glucose metabolism to insulin canbe found in the absence of insulin during exercise, butstill involves increased membrane transport of glucose.At maximal insulin concentrations, the enhancing effectof exercise on glucose uptake may involve enhance-ment of glucose disposal, an effect that is probablyless in muscle from diabetic rats. Finally, after exer-cise, increased glucose transport in glycogen-depletedmuscle does not require increased muscle blood flowand, therefore, involves increased membrane permea-bility for glucose. The presence of insulin is not nec-

From the Department of Medical Physiology B, The Panum Institute, Universityof Copenhagen, DK-2200 Copenhagen N, Denmark.Address reprint requests to Erik A. Richter, M.D., D.M.Sc. Institute for theTheory of Gymnastics, August Krogh Institute, Universitetsparken 13, DK-2100Copenhagen 0, Denmark.Received for publication 17 July 1984 and in revised form 8 March 1985.

essary for this effect of exercise. DIABETES 1985;34:1041-48.

It has previously been shown that insulin sensitivity ofmuscle glucose transport and uptake, and glycogen syn-thesis are enhanced after a single exercise session.13 Ithas also been shown that glucose transport and uptake,

and glycogen synthesis after exercise are increased whenmuscles are perfused with a perfusate to which no insulin isadded, as long as muscle glycogen concentration is belowresting levels.2~4 The role of insulin during exercise for thedevelopment of the abovementioned phenomena is un-known. In light of the concept that a small, so-called per-missive concentration of insulin has to be present for musclecontractions to elicit increased glucose uptake,56 the pres-ence of insulin during exercise might be necessary for de-velopment of increased glucose uptake and increased insulinsensitivity found after exercise.

From a clinical point of view, it is of interest knowingwhether insulinopenic diabetic patients will increase the in-sulin sensitivity of their muscles by exercising as normal sub-jects do. We therefore studied glucose metabolism in per-fused muscle isolated from either exercised or rested ratsthat were in a state of streptozocin (STZ)-induced diabeticketoacidosis or were healthy controls. Postcontraction uptakeof 3-O-methylglucose (a measure of glucose transport) wasalso studied in perfused hindquarters from diabetic rats thathad been washed out and, during electrically induced mus-cle contractions, had been perfused with insulin antiserumadded to the perfusate, since even in severe ketoacidosissome insulin may still be present in plasma.

Increased glucose uptake after exercise could be due toincreased capillary perfusate flow, providing greater avail-ability of glucose and/or insulin to the muscle cells, ratherthan to enhancement of muscle membrane permeability toglucose by direct effects of exercise on the membrane, evenwhen measured in vitro. To examine this question, distributionof capillary flow was estimated using the radioactive micro-

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sphere technique in perfused muscle at rest and after con-tractions.

In accordance with our recent finding that insulin is notneeded for muscle to increase glucose uptake during con-tractions,7 we report herein studies that provide evidence thatpresence of insulin is also not needed during exercise forincreased muscle glucose metabolism (including increasedinsulin sensitivity) in muscle after exercise. Furthermore, theincrease in glucose transport measured in vitro after musclecontractions was found to be due to increased membranepermeability for glucose rather than increased perfusate flow.

MATERIALS AND METHODSAnimals. Male Wistar rats weighing 240-260 g were injectedvia a tail vein with either streptozocin (STZ, 100 mg/kg bodywt) in 0.8 ml citrate buffer (pH 4.5) or only the buffer duringanesthesia with Hypnorm (Janssen Pharma, Copenhagen,Denmark, containing fentanyl and fluanisone). During thenext 3 days, STZ-treated rats were subcutaneously (s.c.) in-jected with 7, 6, and 2 U, respectively, of a long-acting por-cine insulin preparation (Ultralente, pH 5.5, modified for usein rats, kindly donated by Novo Research Institute, Copen-hagen, Denmark). On the following 2 days, insulin was with-held. The insulin treatment prevented development of ke-toacidosis during the first 4 days after STZ injection. The ratswere used in experiments 6 days after injection of STZ and2.5 days after the last injection of insulin.In vivo exercise exeriments. To accustom the rats to thetreadmill, they were run 10 min daily at a speed of 19 m/minfor 5 days preceding the experiments. On the morning of theexperiments, the fed rats were randomly divided into restingand exercising groups. The latter ran for 45 min on a motor-driven treadmill at 19 m/min at an inclination of 0%. Imme-diately after running or rest, the rats were anesthetized byan intraperitoneal (i.p.) injection of pentobarbital (5 mg/kgbody wt) and were surgically prepared for hindquarter per-fusion, as described previously.8 The perfusion medium con-sisted of Krebs-Henseleit solution that also contained reju-venated, aged human erythrocytes9 at a hematocrit of 40%;5% bovine serum albumin (Sigma, St. Louis, Missouri) di-alyzed for 24 h (pore size 10-15 kdaltons) against 33 vol ofKrebs-Henseleit solution; 6 mM glucose; 0.15 mM pyruvate;and 4-5 mM lactate originating from the erythrocytes. ThepH of the perfusate was adjusted to 7.8 with NaOH to obtaina pH of 7.4 in the gassed arterial perfusate (PC02 —30 mmtorr). The initial volume of perfusate was 130 ml. Before place-ment in the perfusion apparatus, the rats were heparinizedby injection of 500 IU heparin into the inferior vena cava.Blood for determination of glucose, ketone bodies, and in-sulin was then obtained from the perfusion catheter in theinferior vena cava, and urine was expressed from the blad-der. The rat was then killed by an intracardial injection ofpentobarbital and placed in the perfusion apparatus. OnlySTZ-injected rats with blood glucose values >13.9 mmol/L(250 mg/dl) and glucosuria and ketonuria (Labstix, Ames,Elkhart, Indiana) were considered diabetic.

The first 25 ml of perfusate passed through the preparationwas discarded, whereupon the medium was recirculated ata flow of 7 ml/min. After 15 min of preperfusion, musclebiopsies of one leg were obtained. The superficial part of thegastrocnemius muscle containing mainly fast-twitch whitefibers10 was freeze-clamped in situ with aluminum clamps

cooled in liquid nitrogen. Thegastrocnemius-soleus-plantarismuscle group was then reflected, and the soleus muscleconsisting mainly of slow-twitch red fibers1011 was clamped.Finally, a portion of the deep part of the medial head of thegastrocnemius consisting mainly of fast-twitch red fibers10

was cut out and clamped. The common iliac vessels sup-plying the biopsied leg were subsequently tied off, and astring was tied tightly around the proximal part of the leg.Perfusate flow was then reduced to 5.5 ml/min. At this flow,fractional extraction of O2 was approximately 15% and thepressure in the perfusion system equal to the prebiopsy pres-sure (total O2 content in arterial and venous perfusate wasmeasured with a Lex-O2-Con Analyzer, Lexington Instru-ments, Lexington, Massachusetts). Porcine insulin (lot num-ber S-835158, kindly donated by Novo Research Institute),yielding concentrations of 0, 100, and 20,000* |xU/ml cell-free perfusate, and 15-30 |xCi 3-3H-glucose (New EnglandNuclear, Boston, Massachusetts) were then added. The ex-perimental period began 5 min later, since the transit timefrom the reservoir to the hindquarter was ~4 min at the flowused. At 0 min and 45 min, samples of perfusate were taken.Aliquots were deproteinized in ice-cold HCIO4 for glucoseand lactate determinations, and the rest was placed in chilledtubes for insulin determinations. At 45 min, biopsies of theremaining leg were obtained.In vitro "exercise" experiments. To study muscle glucosetransport after contractions in an insulin-free environment,hindquarters of diabetic rats were perfused at a flow of 12.5ml/min for 20 min with a glucose-free medium containing 125IJLI guinea pig anti-insulin serum (kindly donated by Dr. J. J.Hoist, Copenhagen, Denmark). In this series, the hematocritwas 30%; the initial volume of perfusate was 150 ml and thefirst 25 ml of perfusate passed through the hindquarter wasdiscarded. The amount of antibody was calculated to pro-vide five times the binding capacity needed to neutralize100 |JLU insulin/ml cell-free perfusate. Furthermore, the bind-ing ability of the anitbodies was verified in experiments withhindquarters from treadmill-exercised rats: insulin at 100 |xU/ml increased glucose uptake from 3.0 ± 0.4 |xmol/g/h(mean ± SEM, N = 7) to 8.3 ±1.1 (N = 4, P < 0.05), andthis effect was totally blocked by the insulin antiserum(2.5 ± 0.4, N = 4). In addition, twice the amount of antiserum(250 |xl) did not further decrease glucose uptake (3.1 ± 1.1,N = 4). Thus, the amount of antibody (125 JJLI antiserum)added to the perfusate was able to fully inhibit the biologicaction of insulin at a concentration of at least 100 fill/ml cell-free perfusate. Fed, nondiabetic control rats had insulin at50 |xU/ml cell-free perfusate added in place of the antiserum.After the preperfusion period, muscles of one hindlimb wereelectrically stimulated to contract via the sciatic nerve,whereas the other leg was left unstimulated. Stimulation was200-ms trains of 100 Hz, each impulse in the train being 0.1ms. The trains were delivered at a rate of 1/s at a supra-maximal voltage (6-10 V) for 2 x 5 min, with a 1-min restbetween the stimulation periods. During stimulation, tensiondeveloped by the gastrocnemius-plantaris-soleus musclegroup was recorded on a Harvard Instruments Isometric Mus-cle Transducer (Harvard Instruments, Millis, Massachusetts)

*This concentration of insulin has previously been shown to elicit maximaleffects on glucose metabolism in the perfused hindquarter.12

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E A RICHTER. T PLOUG. AND H GALBO

connected to an Astro-Med recorder (Atlan Tol Industries,West Warwick, Rhode Island). During the initial contractions,muscle length was adjusted to achieve maximal contractileactive tension. Two minutes after stimulation, the perfusate wasswitched to fresh medium (150 ml) without added antiserum.The first 25 ml of the new perfusate that passed through thehindquarter was discarded, whereupon 10 |xCi of 3H-inulin(New England Nuclear) for determination of extracellularspace, and porcine insulin yielding concentrations of 0, 200,and 20,000 fxU/ml cell-free perfusate were added, and theperfusate was recirculated. After 20 min, 7.5 |xCi of 14C-3-O-methylglucose (New England Nuclear) along with 3 mM ofunlabeled 3-O-methylglucose were added. When the 3-O-methylglucose reached the hindquarter, the perfusion wasswitched to a flow-through perfusion, and samples of theperfusate were collected in HCIO4 for determination of radio-activity. After another 5 min, flow to the hindquarter wasstopped, and biopsies of the three different fiber types fromboth legs were cut out, trimmed of connective tissue andvisible blood vessels, washed in saline, blotted, and clamp-frozen. The 5-min perfusion time for measurement of 3-O-methylglucose uptake was chosen as a compromise be-tween obtaining a reasonable accuracy at low uptake ratesand not obtaining a too-high intracellular concentration of 3-O-methylglucose at high uptake rates. In our hands, uptakeof 3-O-methylglucose is indistinguishable from linearity atleast as long as the concentration in intracellular musclewater is <30% of the perfusate concentration (3 mM). In someexperiments, the antiserum-containing perfusate was not re-placed by a new medium, and no insulin was added aftercontractions. Aside from that, the perfusion was conductedas described above.

For measurement of distribution of perfusate flow to mus-cles that had either been rested or electrically stimulated,90,000 (1|xCi) 57Co-tagged, 15-[xm large microspheres (NewEngland Nuclear) dissolved in 0.1 ml saline containing 1%Tween were, after 30 s of sonication, quickly injected as abolus into the perfusion tubing just before the flow meter.This point of injection was chosen because the turbulent flowpassing the floater in the flow meter would provide thoroughmixing of the microspheres in the perfusate. The injectionwas performed 20 min after electrical stimulation of one hind-limb, i.e., when measurements of uptake of 3-O-methylglu-cose started in other experiments. While the venous outflowwas collected, perfusion was continued for 3 min after themicrospheres reached the hindquarter to clear the big ves-sels of microspheres. Muscles from both hindlegs were thenremoved, blotted, and put into preweighed, 7-counting tubesthat were then weighed again and counted in a 7-counter(Palle Medicoteknik, Copenhagen, Denmark). Samples of themixed venous outflow were also counted.Analyses, calculations, and statistics. Glucose, glucose-6-phosphate, lactate, and (3-hydroxybutyrate were measuredwith standard enzymatic methods.12 For determination of glu-cose and glucose-6-phosphate in intracellular muscle waterin in vivo exercise experiments, the volume of the extracellularspace was determined in separate perfusions using 3H-inulin.The concentration of glucose in intracellular muscle waterwas calculated as:

C, =Cm - Cp x E

0.79 - E

where C, = jximol glucose/ml cell water, Cm = (xmol glucose/g wet wt, Cp = ixmol glucose/ml of perfusate plasma water,E = extracellular space (ml/g wet wt), and 0.79 = the watercontent of perfused muscle (ml/g).13 The concentration ofglucose-6-phosphate in muscle water was calculated as:Cm/(0.79 - E), where Cm = ixmol glucose-6-phosphate/gwet wt. Insulin in cell-free perfusate was measured with acommercially available radioimmunoassay kit (Novo) usingrat insulin as the standard. The detection limit was 2.5 jxU/ml.

Incorporation of labeled glucose into glycogen was de-termined by digestion of muscle in 30% KOH followed byprecipitation of glycogen (including 10 mg carrier glycogen/muscle) with 96% alcohol. After two washes with 96% al-cohol, the precipitate was hydrolyzed in 2 N boiling HCI andcounted in a liquid scintillation counter (Searle Mark III, SearleAnalytical, Des Plaines, Iowa). For estimation of net glycogensynthesis, the specific activity of glucose was determined inthe 0-min sample. Uptake of 14C-3-O-methylglucose in intra-cellular muscle water in individual muscles was determinedin perchloric acid extracts and was corrected for dry matter(21% wt/wt13) and for counts in the extracellular space de-termined with 3H-inulin. Samples were counted for radioac-tivity in a dual-channel Searle Mark III liquid scintillationcounter with automatic correction of spillover of 14C-countsin the 3H-channel. Glycogen in muscle was determined asdescribed previously.14

Rates of glucose uptake and lactate release were deter-mined from changes in their concentrations in the perfusateand the volume of perfusate, and are expressed per gramof muscle perfused. The amount of perfused muscle wasdetermined by injection of Evans blue into the arterial line atthe end of the perfusion and subsequent weighing of thestained tissue. Perfused muscle mass in diabetic and controlrats was found to be a similar percentage of body weight.Rates of lactate release were corrected for lactate productionby the erythrocytes, whereas glucose uptake by the red cellswas not measurable. The data were analyzed by Student'sf-test, paired or unpaired as applicable.

RESULTSMetabolic status of diabetic and control rats. In diabeticrats, blood glucose concentrations before perfusion were16.8 ± 0.4 mmol/L, mean ± SEM (N = 67), compared with7.2 ± 0.2 mmol/L (N = 49), in controls. There was no dif-ference between rested and treadmill-exercised rats. Plasmainsulin concentration was also similar in rested and exercisedrats, averaging 3.7 ± 0.7 |x(J/ml (N = 22) in diabetic ratsand 19.3 ± 2.2 |xU/ml (N = 13) in controls. In diabetic rats,(3-hydroxybutyrate in blood was 6.7 ± 1.0 mmol/L (N = 15)in rested rats and 10.4 ± 1.6 mmol/L (N = 11) in exercisedrats (P < 0.1). In control rats, p-hydroxybutyrate in blood washigher (P < 0.001) in exercised rats (0.72 ± 0.04 mmol/L,N = 10) than in rested rats (0.47 ± 0.03 mmol/L, N = 10).Glycogen depletion induced by treadmill running. Musclesamples obtained 5 min before glucose uptake measure-ments were begun indicated that, in hindquarters from ex-ercised, diabetic rats, the fast-twitch red and slow-twitch redfibers were somewhat glycogen depleted, whereas the fast-twitch white fibers were not significantly depleted (Table 1).In hindquarters from control rats, the slow-twitch red and fast-twitch white fibers were glycogen depleted, whereas the fast-


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INCREASED MUSCLE GLUCOSE UPTAKE AFTER EXERCISE

TABLE 1Muscle glycogen concentrations in rat hindquarters perfusedafter exercise or rest in vivo (jimol/g wet wt)

TABLE 2Glucose uptake in perfused rat hindquarters ((xmol/g perfusedmuscle/h)

Fiber type

Slow-twitch redFast-twitch redFast-twitch white

Control

Rested Exercised

34 ± 1 23 ±42 ± 1 40 ±47 ± 1 42 ±

2*1r

Diabetic

Rested

27 d34 d40 d

t 1t- 1t- 1t

Exercised

14 ±24 ±35 ±

1*t2*|2*f

Diabetic and control rats were either run for 45 min on a treadmill ata speed of 19 m/min or remained resting. The rats were then anes-tetized with pentobarbital and prepared surgically for hindquarterperfusion. Muscle samples were taken after 15-min perfusion withoutinsulin in the perfusate. Values are means ± SEM of 18-19 controlrats or 15-16 diabetic rats.*P < 0.05 compared with values at rest.fP < 0.05 compared with corresponding values in control rats.

twitch red fibers were not significantly depleted (Table 1).Glycogen concentrations in diabetic rats were lower than incontrols (Table 1) before as well as after exercise.Glucose uptake. As shown in Table 2, in the absence ofinsulin glucose uptake was similar in hindquarters from di-abetic and control rats whether they were exercised or not.Insulin at 100 (xU/ml increased glucose uptake significantlymore in hindquarters from exercised rats than in hindquartersfrom rested rats. These results were similar in diabetic andin control rats. At maximal insulin concentration, (20,000 |xU/ml), glucose uptake in hindquarters from control rats wasincreased by prior exercise, whereas this was not the casein hindquarters from diabetic rats.Glucose disposal. Exercise enhanced the ability of insulinat 100 |xU/ml to stimulate glycogen synthesis in hindquartersfrom both normal and diabetic rats in the slow-twitch red and

Insulin(nU/ml)

0100

20,000

Control

Rested

3.3 :5.4 :

13.4 :

t 0.5t 0.7t 1.0

Exercised

3.3 d8.7 d

16.8 d

t 0.6t 1.0*t 1.1*

Diabetic

Rested

1.9 ± 0.75.0 ± 0.7

11.4 ± 1.4

Exercised

2.6 d7.6 d

12.2 d

t 0.6i: 0.7*i 1.9f

After 25 min of preperfusion, glucose uptake was measured over a45-min period. Insulin, when present, was added after 20. min ofpreperfusion. Values are means ± SEM of 7-10 observations.*P < 0.05 compared with values at rest.fP < 0.05 compared with corresponding values in control rats.

fast-twitch red fibers but not in fast-twitch white fibers (Table3). The maximal response to insulin was increased by ex-ercise only in the slow-twitch red fibers, but of both normaland diabetic muscle (Table 3). Exercise did not increaseincorporation of glucose into glycogen in the absence ofinsulin.

In the present study, in which perfusate lactate due to thehigh concentration of erythrocytes (40%) was 4-5 mM, therewas a small net uptake of lactate that did not differ signifi-cantly among the groups (data not shown). At an insulinconcentration of 20,000 |xU/ml cell-free perfusate, glucose-6-phosphate was generally lower in fast-twitch white than inslow-twitch red fibers (Table 4). In controls, the concentrationof glucose-6-phosphate in slow-twitch red fibers was lowerin exercised than in rested muscle, whereas in diabetic ratsthe decrease after exercise did not reach statistical signifi-cance (Table 4). Intracellular muscle glucose was not de-tectable in fast-twitch white fibers in any of the groups (Table

TABLE 3Incorporation of 3-3H-glucose into glycogen in muscles of perfused rat hindquarters ((xmol/g wet wt/h)

Inci il in11 loUlll 1(ji/ml)

0

100

20,000

Fiber type

Slow-twitchred

Fast-twitchred

Fast-twitchwhite

Slow-twitchred

Fast-twitchred

Fast-twitchwhite

Slow-twitchred

Fast-twitchred

Fast-twitchwhite

Rested

0.20 ± 0.02

0.09 ± 0.01

0.06 ± 0.01

1.6 ±0.6

0.89 ± 0.30

0.21 ± 0.06

11.6 ±1.4

11.9 ± 1.5

1.9 ±0.4

Control

Exercised

0.49 ± 0.13f

0.10 ± 0.01

0.09 ± 0.02

4.7 ± 1.2*

3.7 ± 0.9*

0.28 ± 0.05

17.3 ± 2.5t

12.1 ± 1.4

2.4 ± 0.3

Rested

0.14 ± 0.02

0.19 ± 0.08

0.07 ± 0.02

1.2 ±0.3

1.3 ±0.4

0.19 ± 0.05

10.1 ± 0.9

8.4 ± 1.0

1.5 ±0.5

Diabetic

Exercised

0.22 ± 0.05

0.26 ± 0.06}

0.07 ± 0.01

3.6 ± 1.0*

5.5 ± 1.3*

0.46 ± 0.19

18.8 ± 1.3*

8.1 ± 1.3t

1.3 ±0 .4*

Diabetic and control rats were either run for 45 min on a treadmill at a speed of 19 m/min or remained resting. Immediately afterward,the rats were anesthetized with pentobarbital and prepared surgically for hindquarter perfusion. After 25 min of perfusion and musclebiopsies of one leg, the hindquarters were perfused for another 45 min in the presence of 3-3H-glucose. Values are means ± SEM of 7-10 observations.*P < 0.05 compared with value in rested hindquarters.fP < 0.1 compared with value in rested hindquarters.t-P < 0.05 compared with corresponding value in controls.


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ER. T PLOUG. AND H, GALBO

TABLE 4Concentrations of glucose-6-phosphate and glucose in intracellular muscle water of perfused rat hindquarters ((imol/ml)

Fiber type

Glucose-6-phosphateSlow-twitch

redFast-twitch

whiteGlucose

Slow-twitchred

Fast-twitchwhite

Rested

1.06 ± 0.08

0.43 ± 0.06

1.27 ± 0.28*

-0.16 ± 0.03

Control

Exercised

0.74 ± 0.06f

0.41 ± 0.04

0.08 ± 0.19f

-0.09 ± 0.06

Rested

0.85 ± 0.11

0.45 ± 0.07

1.25 ± 0.31*

-0.17 ± 0.12

Diabetic

Exercised

0.71 ± 0.09

0.38 ± 0.06

0.29 ± 0.20f

-0.18 ± 0.05

Muscle samples were obtained after 25 min of preperfusion followed by 45 min of perfusion at an insulin concentration of 20,000 nil/ml.Concentrations in intracellular muscle water were calculated as described in the text. Fast-twitch red fibers were not analyzed, since thesefibers had to be excised before freezing, which results in increased concentrations of hexose monophosphates. Values are means ± SEMof 7-10 observations.*Glucose values are significantly (P < 0.05) larger than zero.fP < 0.05 compared with values at rest.

4). In slow-twitch red fibers, free intracellular glucose ac-cumulated in muscles from rested diabetic and control rats(Table 4), but was not significantly different from zero in hind-quarters from either diabetic or control rats after exercise(Table 4).In vitro stimulation. Electrical stimulation markedly de-creased the glycogen concentration in the fast-twitch red andfast-twitch white fibers, but only slightly in the slow-twitch redfibers (Table 5). Tension development during electrical stim-ulation, expressed per gram perfused muscle, was similar inhindquarters from diabetic and control rats (data not shown).In nonstimulated muscle, uptake of 3-O-methylglucose wasnot detectable after the 5-min perfusion time in the absenceof insulin (Table 6). As shown in Table 6, prior stimulationincreased 3-O-methylglucose uptake identically in hind-quarters from normal and diabetic rats. The effect of electricalstimulation was seen in the absence of insulin in both fast-twitch red and fast-twitch white fibers, in which glycogenconcentrations were markedly reduced, but not in slow-twitchred fibers, which were only slightly depleted (Tables 5 and6). The increment in 3-O-methylglucose uptake induced byinsulin at 200 fjiU/ml was increased by prior contractions inslow-twitch red fibers. This was also the case in the fast-twitch red fibers, since at this insulin concentration uptakeof 3-O-methylglucose was maximal in stimulated, but not inunstimulated, muscles (Table 6). Since the effect of maximalinsulin (insulin responsiveness) was not increased by con-tractions (Table 6), the increased response to a submaximalinsulin concentration indicates a shift to the left of the dose-response curve and consequently an increase in insulin sen-sitivity after contractions. At maximal insulin concentrations,uptake in fast-twitch red and white fibers was higher in stim-ulated than in unstimulated muscle (Table 6), but in fast-twitchwhite fibers, contractions increased uptake of 3-O-methyl-glucose to a value that was not increased significantly byinsulin (Table 6). In this fiber type, the effect of insulin onuptake of 3-O-methylglucose in resting muscle was small(Table 6).

In hindquarters from diabetic rats, the insulin antiserum-containing perfusate was, in most perfusions, replaced aftercontractions by a new perfusate without antiserum. However,

to ensure that uptake of 3-O-methylglucose in the absenceof added insulin after electrical stimulation was not due tobinding of insulin that might possibly be present in the newperfusate albumin, the perfusate was not switched to onewithout antiserum after electrical stimulation in some hind-quarters from diabetic rats. During 5 min of perfusion, uptakeof 3-O-methylglucose in the stimulated fast-twitch red andfast-twitch white muscles was 0.25 ± 0.07 and 0.25 ± 0.03limol/ml intracellular water, N = 6, respectively, in the pres-ence of insulin antiserum. In the other muscles, uptake of 3-O-methylglucose was not detectable. These results are iden-tical to results obtained when, after electrical stimulation inhindquarters from diabetic rats, the perfusate was switchedto one without insulin antiserum (Table 6) and, in hindquartersfrom control rats, it was switched to one without insulin(Table 6).

Perfusate flow 20 min after electrical stimulation was similarin the stimulated muscles compared with unstimulated mus-cles except for the fast-twitch red fibers, in which it wasmodestly increased by prior electrical stimulation (Table 7).

TABLE 5Effect of electrical stimulation on muscle glycogen concentrationsin perfused rat hindquarters (^mol/g wet wt)

Fiber type

Slow-twitchred

Fast-twitchred

Fast-twitchwhite

Rested

27 :

45 :

44 :

t 2

b 3

t 2

Control

Stimulated

20 ± 1*

17 ± 2*

15 ± 1*

Diabetic

Rested

21 ± 2f

33 ± 2f

36 ± 3f

Stimulated

16 ± 2*

15 ± 2*

13 ± 2*

After 20 min of preperfusion, one leg of the hindquarter was stimu-lated for 2 x 5 min via the sciatic nerve and the other leg was leftunstimulated. The perfusate contained no glucose, but insulin anti-serum in diabetic hindquarters and insulin at 50 |xU/ml in nondiabetichindquarters. After electrical stimulation, the hindquarters were per-fused for 25 min with a perfusate without antiserum, insulin, or glu-cose, whereafter muscle samples were obtained. Values aremeans ± SEM of 8-9 observations.*P < 0.05 compared with value at rest.fP < 0.05 compared with corresponding value in control rats.


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TABLE 6Effect of electrical stimulation on uptake of 3-0-methylglucose in muscles of perfused hindquarters (|xmol/ml intracellular water)

1 noi 111nII IbUIII 1

(jtU/ml)

0

200

20,000

Fiber type

Slow-twitchred

Fast-twitchred

Fast-twitchwhite

Slow-twitch. redFast-twitch

redFast-twitch

whiteSlow-twitch

redFast-twitch

redFast-twitch

white

Rested

ND

ND

ND

0.31 ± 0.11t

0.12 ± 0.04f

ND

0.76 ± 0.06f

0.50 ± 0.06f

0.10 ± 0.03f

Control

Stimulated

ND

0.31 ± 0.05*

0.16 ± 0.03*

0.63 ± 0.09*f

0.57 ± 0.06*f

0.18 ± 0.03*

0.90 ± 0.11

0.74 ± 0.09*

0.24 ± 0.05*

Diabetic

Rested

ND

ND

ND

0.20 ± 0.03t

0.16 ± 0.04t

ND

0.68 ± 0.08t

0.34 ± 0.04f*

0.10 ± 0.02t

Stimulated

ND

0.32 ± 0.06*

0.24 ± 0.03*

0.41 ± 0.06* f

0.55 ± 0.08*f

0.31 ± 0.03*t-

0.81 ± 0.11f

0.69 ± 0.06*

0.27 ± 0.06*

After electrical stimulation as described in Table 5, the hindquarters were perfused for 25 min with a glucose-free perfusate containinginsulin at 0, 200, or 20,000 ^ll/ml cell-free perfusate and 10 |xCi of 3H-inulin. 14C-3-O-methylglucose (7.5 (juCi) along with 3 mM of cold 3-O-methylglucose was added so that, at the. end of the perfusion, the muscles had been perfused with 14C-3-O-methylglucose-containingperfusate for 5 min. Results are means ± SEM of 8-9 observations.ND: not detectable.*P < 0.05 compared with value in rested muscles.fP < 0.05 compared with value at insulin concentrations one level lower.IP < 0.05 compared with corresponding value in controls.

DISCUSSIONThe findings in the present study indicate that insulin sen-sitivity of glucose uptake and glycogen synthesis in muscleis increased by treadmill running similarly in ketotic diabeticrats and in controls (Tables 2 and 3). This suggests that anormal plasma insulin concentration during exercise is not aprerequisite for the development of a postexercise increasein insulin sensitivity in muscle. This finding is of clinical in-terest, since it suggests that insulinopenic diabetic patients,by exercising, can expect to achieve the same increase ininsulin sensitivity of their muscles as subjects with normal

TABLE 7Distribution of perfusate flow in muscles of perfused rathindquarters (cpm/mg wet muscle)

Fiber type Rested Stimulated

Slow-twitchred

Fast-twitchred

Fast-twitchwhite

56 ± 6

62 ± 7

30 ± 5

56 ± 11

101 ± 15*

25 ± 3

Hindquarters of normal rats were preperfused for 25 min and thenone hindlimb was electrically stimulated via the sciatic nerve for2 x 5 min. The other hindlimb was left uns.timulated. Twenty minutesafter stimulation, 1 jxCi (—90,000) of 57Co-tagged microspheres of adiameter of 15 ^m were injected into the perfusate tubing. Samplesof the three different fiber types from both hindlegs were taken 4 minlater. Two and one-half percent of the injected radioactivity was re-covered in the venous outflow. Values are means ± SEM of 7 ob-servations.*P < 0.05 compared with value in rested muscle.

plasma insulin concentrations. This may in part account forthe tendency to hypoglycemia after exercise in insulin-de-pendent diabetic subjects.15

However, since the diabetic rats were not totally insulindeficient, the finding does not prove whether increased sen-sitivity to insulin can develop in the total absence of insulin;e.g., it could be that insulin has a permissive effect on thedevelopment of increased insulin sensitivity. Although re-cently disproved,716 such a permissive effect of insulin hasbeen claimed to exist for the increase in muscle glucoseuptake during exercise.56 To study whether increased insulinsensitivity in muscle after contractions requires the presenceof insulin during contractions, hindquarters from diabetic ratswere washed out and subsequently perfused with a mediumcontaining insulin antiserum before and during electricallyinduced contractions. In spite of this treatment, insulin sen-sitivity of 3-O-methylglucose transport (a measure of glucosetransport) was increased to an extent similar to that found inmuscles from nondiabetic rats that, before and during con-tractions, were perfused with insulin at 50 fjiU/ml cell-freeperfusate (Table 6). This suggests that increased insulin sen-sitivity of skeletal muscle glucose uptake after contractionscan develop without the presence of insulin during contrac-tions, since at submaximal insulin concentrations glucosetransport, at least in nondiabetic muscle, is the rate-limitingstep in glucose utilization after exercise.12

In hindquarters from diabetic rats, prior treadmill runningdid not increase glucose uptake at maximal insulin concen-trations, whereas glucose uptake was increased in hind-quarters from nondiabetic rats (Table 2). The latter finding isin accordance with a previous study.1 Increased responsive-


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E. A. RICHTER. T. PLOUG, AND H. GALBO

ness of glucose uptake could be due to increased membranetransport and/or increased intracellular disposal of glucoseat maximal insulin after exercise. Glucose transport was notmeasured after in vivo exercise, but it was measured afterelectrical stimulation in vitro. In vitro, the maximal effect ofinsulin (insulin responsiveness) on glucose transport was un-changed by electrically induced contractions in hindquartersfrom both diabetic and control rats (Table 6). This is in ac-cordance with a previous study in healthy rats electricallystimulated in vivo.2 Although unlikely,23 it can not be excludedthat in vivo exercise affects muscle glucose transport differ-ently than electrically induced contractions. Nevertheless, thefinding of no greater maximal effect of insulin on glucosetransport in vitro after electrically induced contractions sug-gests that the increase in responsiveness of glucose uptakeafter in vivo exercise in healthy rats (Table 2) probably in-volves factors other than increased responsiveness of glu-cose transport, e.g., increased disposal of glucose. In ac-cordance with this view, free intracellular glucose was foundin slow-twitch red fibers of rested control hindquarters (Table4). This indicates that, at maximal insulin concentrations, glu-cose transport in this fiber type is not the rate-limiting stepin glucose utilization. In slow-twitch red fibers of exercisedhindquarters, however, free intracellular glucose was not de-tectable and the concentration of glucose-6-phosphate waslower than in rested hindquarters (Table 4). Furthermore, inthis fiber type, glycogen synthesis from glucose at maximalinsulin concentrations was increased after exercise (Table3). In fast-twitch white fibers, however, no such exercise-induced changes were found (Tables 3 and 4). In fast-twitchred fibers, glucose-6-phosphate and intracellular glucosewere not measured due to technical reasons (Table 4). Inhindquarters from diabetic rats, exercise also enhanced glu-cose disposal in slow-twitch fibers as judged by the decreasein free intracellular glucose and increase in glycogen syn-thesis (Tables 3 and 4) at maximal insulin concentrations. Glu-coses-phosphate, however, did not decrease significantly(Table 4). Since transport of glucose at maximal insulin con-centrations after electrically induced contractions was similarin hindquarters from diabetic and control rats (Table 6), thereason for the failure of in vivo exercise to increase insulinresponsiveness of glucose uptake in hindquarters from di-abetic rats (Table 2) is not clear. It is possible that hindquarterglycolysis and glucose oxidation, which were not measuredin this study, might have been affected differently by priorexercise in diabetic rats compared with controls, e.g., dueto the humoral milieu during exercise being different in dia-betic rats compared with controls.

It has previously been found that glucose transport anduptake is increased in perfused skeletal muscle after exercisein the absence of added insulin as long as muscle glycogenconcentrations are below resting levels.24 This finding wasessentially confirmed (Tables 5 and 6) in the present study,although in slow-twitch fibers a small but significant glycogendepletion (Table 5) was not accompanied by measurablyincreased 3-O-methylglucose transport (Table 6). However,to prevent high intracellular 3-O-methylglucose concentra-tions in those muscles that had the fastest rate of 3-O-methylglucose transport, exposure of the hindquarters to3-O-methylglucose was short. This again indicates that theprecision of the measurements of 3-O-methylglucose trans-

port in those muscles with the lowest uptake rate is not sat-isfactory. In fact, in other experiments with longer exposureto 3-O-methylglucose, the contraction-induced glycogen de-pletion found in slow-twitch fibers in the present study (Table5) was accompanied by an increase in 3-O-methylglucosetransport (data not shown) in the absence of insulin.

It has been previously speculated that increased transportand uptake of glucose in deglycogenated muscle in vitromight in fact be due to increased binding of insulin remainingin the interstitial fluid in the glycogen-depleted muscles.3 Itis, however, not possible to ascertain the precise concentra-tion of insulin in the interstitial fluid, and hence at the receptorsite. However, the increase in 3-O-methylglucose transportafter contractions was identical whether the perfusate con-tained either insulin at 50 |xU/ml (controls) or insulin anti-serum (diabetic) during contractions (Table 6). Furthermore,3-O-methylglucose uptake was uninfluenced in glycogen-depleted muscle by the presence of insulin antibody afterexercise at zero insulin (Table 6 and RESULTS). These findingsretake it extremely unlikely that insulin is needed during orafter contractions as a prerequisite for the increase in glucosetransport seen in muscle deglycogenated by exercise.

It can be speculated that increased glucose transport inmuscle after contractions can in part be due to increasedperfusate flow increasing availability of insulin and/or glu-cose to the muscle cells, and that this, in turn, increasesglucose uptake. However, markedly increased glucosetransport in the presence or absence of insulin was seenafter contractions (Table 6) in the face of unaltered (fast-twitch white and slow-twitch red fibers) or only moderatelyincreased (fast-twitch red fibers) perfusate flow (Table 7).This indicates that increased glucose transport after exercisecan be explained by increased membrane permeability anddoes not require increased availability of insulin and/or glu-cose.

It is concluded that the development of increased insulinsensitivity of skeletal muscle glucose transport and uptake,and glycogen synthesis after exercise does not require thepresence of insulin during contractions. Furthermore, in mus-cles depleted of glycogen by contractions, glucose transportis increased without the presence of insulin. This is also inagreement with recent findings from this laboratory and an-other that insulin is not required to increase glucose transportand uptake during contractions in glycogen-depleted mus-cle.716 In contrast with findings at submaximal insulin con-centrations, glucose disposal rather than glucose transportmay be the rate-limiting step in glucose utilization in restingmuscle at maximal insulin concentrations and, after exercise,glucose utilization may be increased at maximal insulin con-centrations by enhancement of glucose disposal. Finally,after exercise, muscle glucose transport in the absence ofinsulin may be enhanced in the absence of increased bloodflow, suggesting a direct effect of exercise on membranepermeability for glucose.

ACKNOWLEDGMENTSHanne Overgaard and Lisbeth Kail performed skilled tech-nical assistance.

This study was supported by grants from the Danish Med-ical Research Council, the Novo Fund, Landsforeningen forSukkersyge, and the Danish Medical Association.


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REFERENCES1 Richter, E. A., Garetto, L. P., Goodman, M. N., and Ruderman, N. B.:

Muscle glucose metabolism following exercise in the rat. Increased sensitivityto insulin. J. Clin. Invest. 1982; 69:785-93.

2 Richter, E. A., Garetto, L. P., Goodman, M. N., and Ruderman, N. B.:Enhanced muscle glucose metabolism following exercise: modulation by localfactors. Am. J. Physiol. 1984; 246:E476-82.

3 Garetto, L. P., Richter, E. A., Goodman, M. N., and Ruderman, N. B.:Enhanced muscle glucose metabolism after exercise in the rat: the two phases.Am. J. Physiol. 1984; 246:E471-75.

"Fell, R. D., Terblanche, S. E., Ivy, J. L, Young, J. C, and Holloszy,J. 0.: Effect of muscle glycogen content on glucose uptake following exercise.J. Appl. Physiol. 1982; 52:434-37.

5 Berger, M., Hagg, S., and Ruderman, N. B.: Glucose metabolism inperfused skeletal muscle. Interaction of insulin and exercise on glucose up-take. Biochem. J. 1975; 146:231-38.

6Vranic, M., and Berger, M.: Exercise and diabetes mellitus. Diabetes1979; 28:147-63.

7 Ploug, T., Galbo, H., and Richter, E. A.: Increased muscle glucoseuptake during contractions: no need for insulin. Am. J. Physiol. 1984;247:E726-31.

8 Ruderman, N. B., Houghton, C. R. S., and Hems, R.: Evaluation of theisolated perfused rat hindquarter for the study of muscle metabolism. Biochem.J. 1971; 124:639-51.

9 Ruderman, N. B., Kemmer, F. W., Goodman, M. N., and Berger, M.:Oxygen consumption in perfused skeletal muscle. Effect of perfusion withaged, fresh and age-rejuvenated erythrocytes on oxygen consumption, tissuemetabolites and inhibition of glucose utilization by acetoacetate. Biochem. J.1980; 190:57-64.

10 Hickson, R. C, Heusner, W. W., Van Huss, W. D., Taylor, J. F., andCarrow, R. E.: Effects of an anabolic steroid and sprint training on selectedhistochemical and morphological observations in rat skeletal muscle types.Eur. J. Appl. Physiol. 1976; 35:251-59.

11 Ariano, M. A., Armstrong, R. B., and Edgerton, V. R.: Hindlimb musclefiber populations of five mammals. J. Histochem. Cytochem. 1973; 21:51-55.

12 Lowry, 0. H., and Passonneau, J. V.: A Flexible System of EnzymaticAnalysis. New York, Academic Press, 1972.

13 Richter, E. A., Ruderman, N. B., Gavras, H., Belur, E. R., and Galbo,H.: Muscle glycogenolysis during exercise: dual control by epinephrine andcontractions. Am. J. Physiol. 1982; 242:E25-32.

14 Karlsson, J., Diamant, B., and Saltin, B.: Muscle metabolites duringsubmaximal and maximal exercise in man. Scand. J. Clin. Lab. Invest. 1971;26:385-94.

15 Richter, E. A., Ruderman, N. B., and Schneider, S. H.: Diabetes andexercise. Am. J. Med. 1981; 70:201-209.

16 Wallberg-Henriksson, H., and Holloszy, J. 0.: Contractile activity in-creases glucose uptake by muscle in severely diabetic rats. J. Appl. Physiol.1984; 57:1045-49.


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