Bradykinin does not mediate activation of glucose transport by muscle contraction. J. Appl. Physiol. 61(3): 881-884, 1986.-The purpose of this study was to eval-.
Bradykinin does not mediate activation of glucose transport by muscle contraction STEFAN H. CONSTABLE, ROLAND J. FAVIER, JENNIFER UHL, AND J. 0. HOLLOSZY Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
CONSTABLE,STEFANH.,ROLAND J. FAVIER,JENNIFER UHL, AND J. 0. HOLLOSZY. Bradykinin does not mediate activation of glucose transport by muscle contraction. J. Appl. Physiol. 61(3): 881-884, 1986.-The purpose of this study was to evaluate the report that bradykinin is the “muscle activity hypoglycemia factor” responsible for the activation of glucose transport that occurs in response to muscle contractile activity. Stimulation of rat epitrochlearis muscles to contract resulted in approximately a fourfold increase in the rate of intracellular accumulation of the nonmetabolizable glucose analog 3-0methylglucose. Incubation of the muscles with high concentrations of aprotinin (Trasylol), a polypeptide inhibitor of kallikrein which blocks formation of kinins, did not inhibit the activation of sugar transport by contractile activity. Furthermore incubation of muscles with bradykinin did not have a stimulatory effect on the uptake of 3-methylglucose either at a physiological concentration or at high concentrations. These results provide no support for the claims that aprotinin prevents the activation of sugar transport in muscle by contractile activity or that bradykinin is the muscle activity hypoglycemia factor. exercise; indomethacin; trochlearis muscle
kinins;
3-0-methylglucose;
rat epi-
I EXERCISE HAS A POWERFUL insulin-like action on glucose transport in skeletal muscle (1, 8-13, 19, 21, 23). Goldstein (9) attributed the activation of glucose transport by muscle contractile activity to a hormone produced by contracting skeletal muscles. This “Goldstein factor” has proven elusive, and whether or not such a factor exists is still not clearly established. Dietze and Wicklmayr (2) have postulated that the Goldstein factor is bradykinin, formed as the result of activation of kallikrein during exercise. This hypothesis was based on their findings, in studies on human subjects, that 1) 3 min of isometric exercise resulted in a S-fold increase in glucose uptake by the forearm, 2) infusion of the kallikrein inhibitor aprotinin (Trasylol) inhibited the increase in glucose uptake during forearm exercise, and 3) infusion of bradykinin into the brachial artery in an additional study resulted in an increase in glucose uptake by the forearm (2, 3). In a subsequent study they found that indomethacin blocked the effect of bradykinin on glucose uptake by the forearm and hypothesized that bradykinin mediates its effect by stimulating prostaglandin synthesis. If, as Dietze and Wicklmayr (2, 3) postulate, bradyki0161-7567/86
$1.50
Copyright
nin is the “muscular activity hypoglycemia factor,” this would be of considerable physiological interest and importance. However, in studies of glucose uptake by the human forearm it is not possible to quantify glucose transport into muscle, to measure blood flow accurately, to measure the extracellular space accurately (bradykinin is known to cause edema) (7), to control other humoral factors, or even to determine how much muscle is being drained by the cannulated deep vein. We have therefore used the isolated rat epitrochlearis muscle to reevaluate the hypothesis that bradykinin mediates the activation of glucose transport that occurs in response to muscle contractile activity, under better controlled conditions. Our results provide no support for the concept that bradykinin is the muscular activity hypoglycemia factor. METHODS
Animals. Male specific pathogen-free Wistar rats (loo125 g body wt) were obtained from Hilltop Laboratory Animals and fed a diet of Purina chow and water. They were anesthetized with pentobarbital sodium (5 mg/lOO g body wt), and the epitrochlearis muscles were dissected out. The muscle weights ranged from 15 to 25 mg. Because these muscles are ~0.2 mm thick, they are suitable for studies of glucose transport (17-19,24). Rat epitrochlearis muscles contain approximately lo-15% slowtwitch red fibers, 65% fast-twitch white fibers, and 2025% fast-twitch red fibers (17). Incubation of muscles with aprotinin, indomethacin, or bradykinin. Muscles were incubated in 1.5 ml of oxygenated Krebs-Henseleit bicarbonate buffer (14) containing 8 mM glucose and 32 mM mannitol (KHB medium) in 25-ml Erlenmeyer flasks. The flasks were gassedcontinuously with 95% 02-5% CO2 and incubated at 37°C in a shaking incubator. To evaluate the effect of aprotinin (Trasylol, FBA Pharmaceuticals), a polypeptide inhibitor of kallikrein that inhibits formation of kinins (7), muscles were incubated in KHB medium containing either 0, 100, or 1,000 kallikrein-inactivating units (KIU) of Trasylol per ml for 1 h. For the period from 45 to 60 min, one muscle of each pair was immersed in KHB medium in a test tube and stimulated to contract as described below. In the study to evaluate the effect of indomethacin, rats were given indomethacin by stomach tube: 5 mg 16 h before they were killed and 10 mg 2 h either before anesthesia, in the case of the animals used for the muscle
0 1986 the American
Physiological
Society
881
882
BRADYKININ
DOES
NOT
INCREASE
stimulation experiment and as controls, or before being exercised. The animals were exercised by swimming for -4 h as described previously (22); they were given an additional 5 mg of indomethacin after the first 2 h of swimming. These amounts of indomethacin are considerably above the dose (2.4 mg/kg) required to inhibit prostaglandin synthesis in the rat (20). The indomethatin doses were dissolved in 1 ml of whole milk. The muscles to be stimulated and the contralateral control muscles were incubated for 30 min in KHB medium containing 5 pg indomethacin (Indocin iv, Merck, Sharp and Dohme) per ml at 37°C; this is well above the concentration required to inhibit prostaglandin synthesis in muscle (15). Muscles were stimulated to contract during the last IO min of the 30-min incubation (see below). To evaluate the effect of bradykinin on sugar transport, muscles were first incubated for 60 min with various concentrations of bradykinin. The incubation medium was replaced with fresh medium every 10 min to minimize possible changes in bradykinin concentration due to the action of muscle kininases. The contralateral control muscles were incubated without bradykinin. Before measurement of sugar transport, the muscles were, in all experiments, subjected to two 5-min washes with the appropriate KHB medium containing no glucose, at 29OC. Musck stimulation. Before stimulation the muscle’s distal tendon was attached to a vertical Lucite rod containing two platinum electrodes (11). The proximal tendon was clipped to a jeweller’s chain,, which connected it to a Grass model FTOC isometric force transducer, and resting tension was adjusted to 0.4 g. The mounted muscle was immersed in 10 ml KHB medium in a test tube. The medium was gassed continuously with 95% Oz5% CO2 and maintained at 37OC. The muscles were stimulated with supramaximal square-wave pulses of 0.5 ms duration with a Grass S48 stimulator. After a 5-min recovery at resting tension, muscle contractions were produced by stimulation at 50 Hz for 10 s at 1 contraction/min for 10 min. The isometric tension developed by the muscles was recorded with a dual-channel HewlettPackard recorder. After stimulation the muscles were blotted and used for measurement of 3-methylglucose uptake. The contralateral muscle, which served as a control, was subjected to the same treatment, except it was not stimulated. Measurement of 3- O- methylglucose transport into muscle. 3-0-methylglucose, a glucose analogue, is transported into muscle by the same mechanism as glucose but is not further metabolized (16). Uptake of 3-methylglucose by epitrochlearis muscles was measured by a modification (24) of the method developed by Narahara and Ozand for use in frog sartorius muscle (16). Briefly the muscles were blotted on filter paper dampened with KHB and then incubated in 1.5 ml of oxygenated KHB containing 8 mM 3-0-[3H]methylglucose (3.5 &i/ml) and 32 mM [ 14C]mannitol (0.25 &i/ml) (New England Nuclear). In the studies involving either bradykinin or aprotinin, these substances were also included in the medium. The incubation period was 15 min. The muscles were incu-
SKELETAL
MUSCLE
SUGAR
UPTAKE
bated at. 29°C in stoppered 25-ml Erlenmeyer flasks with a gas phase of 95% Og-5% C02. VVe have found that intracellular accumulation of 3-0-methylglucose in rat epitrochlearis muscle under our conditions begins to deviate from linearity (i.e., slow down) when the intracellular concentration increases above -25% of the extracellular concentration (D. Young, S. H. Constable, and ‘J. 0. Holloszy, unpublished results). After incubation the muscles were blotted, frozen, weighed, homogenized in 10% trichloroacetic acid, and centrifuged at 1,000 g. Aliquots of the muscle extracts and of the incubation media were counted in a Packard liquid. scintillation counter with channels preset for simultaneous 3H and 14C counting. The amount of each isotope .in the samples was determined, and this information was used to calculate the extracellular space and the intracellular concentration of 3-methylglucose. Results are expressed in terms of micromoles 3-methylglucose accumulation per milliliter of intracellular wat.er. One-way analysis of variance was used to determine any significant differences of the main effects. Duncan’s post hoc test was used for testing subhypotheses. RESULTS
Stimulation of epitrachlearis muscles to contract resulted in approximately a fourfold increase in the rat of 3-methylglucose transport. Incubation of the muscles with high concentrations of aprotinin, a polypeptide inhibitor of kallikrein that blocks formation of kinins (7), did not inhibit the activation of sugar transport by contractile activity (Table 1). 1. Effects of muscle contractile activity and uprotinin on permeability of muscle to 3-O-methylglucose TABLE
Condition
n
Aprotinin, KIUJml
-Resting
control
Stimulated contract
to
9
0
6 9
100 1,000
9
0
6 9
100 1,000
3-O-Methylglucose Accumulation, pm01 Sml-l. 15 min-’ O”35kO.
10
0.32kO.13 0.22ztzO.07 1.42t0.16 1.64kO.31 1.4lItrO.18
Values are means -+ SE; n, no. of muscles. 3-O-methylglucose uptake is expressed per ml of intracellular water. KIU, kallikrein-inactivating units.
2. Activation of sugar transport in muscle by exercise or stimulation of contraction with, or without indomethacin treatment I______-TABLE
Condition
n
Indomethacin
control
10
-
Exercised (swimming) Stimulated to contract
12 12 12 8 12
+ + +
Resting
Values
are means
t SE; n, no. of muscles.
3OMethylglucose Accumulation, prnol. ml-‘. 15 min”
0.26t,O.O8 0.26kO.12 1.47t0.14 1.40t0.15 1.35kO.13
(10) (12) (12) (12) (8)
1.51,tO.ll
(12)
-
BRADYKININ
DOES
NOT
INCREASE
3. Effects of incubation with different concentrations of bradykinin on permeability of epitrochlearis muscle to 3-O-methylglucose
TABLE
Bradykinin
Concn, ndml
3-Q-Methylglucose Accumulation, pm01 . ml-l. 15 min-’
0 1 10 1,000 Values
are means
0.40t0.06 0.37tO.04 0.36t0.04 0.39*0.05
t SE for 6 animals/group.
Pretreatment of rats with large dosesof indomethacin, well above the amount needed to inhibit prostaglandin synthesis in the rat (ZO), failed to block the exerciseinduced increase in permeability of skeletal muscle to glucose (Table 2). Similarly, treatment with indomethatin did not prevent activation of sugar transport by stimulation of muscle contraction (Table 2). Since these results did not support the hypotheses of Dietze and Wicklmayr (2, 3), we evaluated the effect of bradykinin on the permeability of muscle to sugar. As shown in Table 3, incubation of epitrochlearis muscles with bradykinin did not have a stimulatory effect on uptake of 3-methylglucose either at a physiological concentration (1 rig/ml) or at a very high concentration. DISCUSSION
Dietze and Wicklmayr (2) have postulated that the increase in glucose uptake by muscle during exercise is mediated by kinins released from kininogens as the result of activation of kallikrein. The kinins, bradykinin and kallidin, have a high degree of biological activity and are potent vasodilators; they are involved in inflammation, production of edema, evoking pain, and stimulating prostaglandin synthesis (7). To our knowledge Dietze and Wicklmayr (2, 3) are the only investigators to claim that bradykinin or other kinins are involved in the response to exercise and/or in the activation of sugar transport in skeletal muscle. [They and their co-workers have also reported that the kallikrein-kinin system is involved in the “translation of insulin action on glucose metabolism in skeletal muscle” (4) and in bringing about the increase in glucose uptake caused by hypoxia (5).] The experimental basis for the claim by Dietze and Wicklmayr (2) that the increase in glucose uptake by muscle during exercise is mediated by bradykinin was their finding that infusion of the kallikrein inhibitor aprotinin (Trasylol) prevented the increase in glucose uptake during isometric forearm exercise. Our studies on isolated skeletal muscle provide no support for such an effect of aprotinin. Our inability to demonstrate an effect of aprotinin on the activation of glucose transport by muscle contractile activity cannot be due to use of an inadequate concentration of this kallikrein inhibitor. Our highest concentration (1,000 KIU/ml) was far above that which could have been attained by intravenous infusion in the subjects studied by Dietze and Wicklmayr (2). Dietze et al. (6) postulated that bradykinin mediates an increase in glucose uptake by stimulating prostaglandin synthesis. This concept was based on their finding
SKELETAL
MUSCLE
SUGAR
883
UPTAKE
that pretreatment with indomethacin, a potent inhibitor of prostaglandin synthesis, prevented the increase in glucose uptake by the forearm when bradykinin was infused. In the present study, treatment with large doses of indomethacin had no inhibitory effect on the activation’ of sugar transport in muscle either by exercise (swimming) or by stimulation of contraction in vitro. In addition to the indirect evidence reviewed above, Dietze et al. (3,6) supported their claim that the exerciseinduced increase in glucose uptake by muscle is mediated by the kallikrein-kinin system, by measurements of glucose uptake by the forearm during intrabrachial arterial infusion of bradykinin. They found that bradykinin infusion increased glucose efflux from the blood, as reflected in a greater arteriovenous glucose difference. However, our results provide no support for an insulinlike action of bradykinin on glucose uptake by skeletal muscle. We have no explanation for the differences between our results and those of Dietze et al. (2, 3, 6). Certainly the experimental models used, rat epitrochlearis muscle in our study and the human forearm in theirs, are very different. However, it seems unlikely that the mechanisms by which contractile activity brings about an increase in permeability of muscle to glucose are very different in human and rat skeletal muscle. In conclusion, the results of the present study provide no support for the claims that aprotinin prevents the activation of sugar transport in muscle by contractile activity or that bradykinin is the ‘“Goldstein” or “muscle activity hypoglycemia factor.” . This research was supported by Grant AM-18986 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. S. H. Constable was the recipient of National Research Service Award AM-07011 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. R. J. Favier was initially supported by a grant from the North Atlantic Treaty Organization and later by grants from Fondation pour la Recherche Medicale and Philippe Foundation. Present addresses: S. H. Constable, USAF School of Aerospace Medicine, VNC Building 160, Brooks Air Force Base, TX 78235; R. 9. Favier, Laboratoire de Physiologie A, UER Grange Blanche, 8 Ave. Rockefeller, 69373 Lyon Cedex 08, France. Received
12 November
1985; accepted
in final
form
21 April
X986.
REFERENCES 1. BERGER, M., S. HAGG, AND N. B. RUDERMAN. Glucose metabolism in perfused skeletal muscle: interaction of insulin and exercise on glucose uptake. Biochem. J. 146: 231-238,1975. 2. DIETZE, G., AND M. WICKLMAYR. Evidence for a participation of the kallikrein-kinin system in the regulation of muscle metabolism during muscular work. FE.%’ Lett. 74: 205-208,1977. 3. DIETZE, G., AND M. WICKLMAYR. Effekt von Bradykinin auf die Glukoseaufnahme durch die Muskulatur beim Menschen. Klipt. Wochenschr. 55: 357-358,1977. 4. DIETZE, G., M. WICKLMAYR, I. BOTTGER, AND L. MAYER. Inhibition of insulin action on glucose uptake into skeletal muscle by a kallikrein-trypsin inhibitor. Hoppe-Seylers 2. Physiol. Chem. 359: 1209-1215,1978. 5. DIETZE, G., M. WICKLMAYR, AND L. MAYER. Evidence for a participation of the kallikrein-kinin system in the regulation of muscle metabolism during hypoxia. Hoppe-Seylers Z. Physiol. Chem. 358: 633-638,1977. 6. DIETZE, G., M. WICKLMAYR, L. MAYER, I. BOTTGER, AND .H. V. FUNCKE. Bradykinin and human forearm metabolism: inhibition
BRADYKININ
884
DOES NOT INCREASE
synthesis. Hoppe-Seylers 2. Physiol. Chem. 359: 369-378,1978. 7. DOUGLAS, W. W. Plasma kinins. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics (6th ed.), edited by A. G.
SKELETAL
1953. 11. HOLLOSZY,
J. O., AND H. T. NARAHARA. Studies of tissue permeability. X. Changes in permeability to 3-methylglucose associated with contraction of isolated frog muscle. J. Biol. Chem. 240: 3493-
3500,1965. 12. INGLE, D.
J., J. E. NEZAMIS, AND K. L. RICE. Work output and blood glucose values in normal and in diabetic rats subjected to the stimulation of muscle. Endocrinology 46: 505-509, 1950. 13. IVY, J. L., AND J. 0. HOLLOSZY. Persistent increase in glucose uptake by rat skeletal muscle followng exercise. Am. J. Physiol. 241 (Cell Physiol. 10): C200-C203, 1981. Untersuchungen uber die 14. KREBS, H. A., AND K. HENSELEIT. Harnstoffbildung im Tierkorper. Hoppe-Seylers 2. Physiol. Chem. 210: 33-66,1932. 15. LEIGHTON, B., L. BUDOHOSKI, F. J. LOZEMAN, R. A. CHALLIS, AND E. A. NEWSHOLME. The effect of prostaglandins E1, Ez, and
SUGAR UPTAKE
Fga and indomethacin on the sensitivity of glycolysis and glycogen synthesis to insulin in stripped soleus muscles of the rat. Biochem.
of endogenous prostaglandin
Gilman, L. S. Goodman, and A. Gilman. New York: Macmillan, 1980, p. 659-667. 8. DULIN, W. E., AND J. J. CLARK. Studies concerning a possible humoral factor produced by working muscles. Its influence on glucose utilization. Diabetes 10: 289-297, 1961. 9. GOLDSTEIN, M. S. Humoral nature of the hypoglycemic factor of muscular work. Diabetes 10: 232-234, 1961. 10. GOLDSTEIN, M. S., V. MULLICK, B. HUDDLESTON, AND R. LEVINE. Action of muscular work on transfer of sugars across cell barriers: comparison with action of insulin. Am. J. Physiol. 173: 212-216,
MUSCLE
J. 227: 337-340,1985.
16. NARAHARA, H. T., AND P. OZAND. Studies of tissue permeability. IX. The effect of insulin on the penetration of 3-methylglucose-H3 in frog muscle. J. Biol. Chem. 238: 40-49, 1963. 17. NESHER, R., I. E. KARL, K. E. KAISER, AND D. M. KIPNIS. Epitrochlearis muscle. I. Mechanical performance, energetics and fiber composition. Am. J. Physiol. 239 (Endocrinol. Metab. 2): E454-E460,1980. 18. NESHER, R.,
II. Metabolic
I. E. KARL, AND D. M. KIPNIS. Epitrochlearis muscle. effects of contraction and catecholamines. Am. J.
Physiol. 239 (Endocrinol. NESHER, R., I. E. KARL,
Metab. 2): E461-E467, 1980. AND D. M. KIPNIS. Dissociation
Metab.
1969.
of effects of insulin and contraction on glucose transport in rat epitrochlearis 1985. 2. muscle. Am. J. Physiol. 249 (Cell Physiol. 18): C226-C232, SHEN, T-S., AND C. A. WINTER. Chemical and biological studies on indomethacin, sulindac and their analogs. In: Advances in Drug Research, edited by N. J. Harper and A. B. Simmonds. New York: Academic, 1977, p. 89-245. 21 SZABO, A. J., R. J. MAHLER, AND 0. SZABO. Glucose uptake by the isolated perfused rat hindlimb during rest and exercise. Horm.
lg.
l
l
Res. 1: 156-161,
22. TERJUNG,
R. L., G. H. KLINKERFUSS, K. M. BALDWIN, W. WINDER, AND J. 0. HOLLOSZY. Effect of exhausting exercise on rat heart mitochondria. Am. J. Physiol. 225: 300-305, 1973. 23. WAHREN, J. Glucose turnover during exercise in man. Ann. NY Acad. Sci. 301: 45-55, 24. WALLBERG-HENRIKSSON,
1977.
H., AND J. 0. HOLLOSZY. Activation of glucose transport in diabetic muscle: response to contraction and insulin. Am. J. Physiol. 249 (Cell Physiol. 18): C233-C237, 1985.
