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“Stable” effects of insulin and isoproterenol on adipocyte pyruvate dehydrogenase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 256, No. 2, August 1, pp. 699-702, 1987
COMMUNICATION “Stable”
Effects
of Insulin and lsoproterenol KANG
*Merck Rahway,
CHENG**’
AND
on Adipocyte JOSEPH
Pyruvate
Dehydrogenase
LARNERt
Sharp & Dohme Research Laboratories, Department of Animal Drug Discovery, New Jersey 07065, and fllniversity of Virginia School of Medicine, Department Charlottesville, Virginia 22908 Received
March
6,1987,
and in revised
form
April
P.O. Box 2oO0, of Pharmacology,
22, 1987
Insulin, at a concentration of 1 mu/ml, stimulated glycogen synthase and pyruvate dehydrogenase about threefold in isolated rat adipocytes. Upon the removal of insulin, glycogen synthase activity remained in the activated state for 10 min and thereafter rapidly returned to basal level. On the other hand, insulin-stimulated pyruvate dehydrogenase activity remained elevated for at least 30 min. Isoproterenol (low8 M) stimulated phosphorylase and inhibited pyruvate dehydrogenase through the activation of @-adrenergic receptors. Addition of the P-antagonist, propranolol (lop5 M), after isoproterenol reversed the action of isoproterenol on phosphorylase but not its action on pyruvate dehydrogenase. Dibutyryl cyclic AMP, when added to intact adipocytes, produced an effect on pyruvate dehydrogenase similar to that induced by isoproterenol. Our results indicate that both insulin and the ,&agonist have a unique action on pyruvate dehydrogenase which is different from their effects on other enzymes such as glycogen synthase and phosphorylase. o 1987 Academic PM, IIK. _.
Adipocyte pyruvate dehydrogenase is rapidly activated by insulin through a dephosphorylation mechanism (1). Studies with purified components derived from bovine kidney indicate that phosphorylation occurs at three separate serine sites on the asubunit which result in inactivation of the enzyme (2, 3). Careful kinetic studies indicate that the phosphorylation of site 1 is faster than the phosphorylation of sites 2 and 3 and is closely correlated with the inactivation of the enzyme. Thus, the phosphorylation state of site 1 is important in regulating the pyruvate dehydrogenase activity. Insulin decreases the phosphorylation of all three sites to the same extent via the stimulation of a Mg+-dependent Ca’+-stimulated phosphatase (4). Recently, a number of laboratories have reported that a low-molecular-weight mediator which is generated by insulin from plasma membrane stimulates pyruvate dehydrogenase in a cell-free system (5). Catecholamines have dual actions ruvate dehydrogenase: stimulation
1 To whom
correspondence
should
receptors (6) and inhibition via P-adrenergie receptors (6-9). In this study, we demonstrate that both insulin and isoproterenol have a stable effect on pyruvate dehydrogenase even after the termination of hormone action while their effects on glycogen synthase and phosphorylase are rapidly reversed. MATERIALS
AND
METHODS
Sprague-Dawley rats (150-200 g) were purchased from Hilltop Lab Animals, Inc. Bovine serum albumin (BSA’; Fraction V), bovine insulin, 1-(-)-isoproterenol bitartrate, 1-(f)-propranolol hydrochloride, dibutyryl cyclic AMP (CAMP), @-NAD+, thiamine pyrophosphate, and coenzyme A were from Sigma. Crude bacterial collagenase (type 1) was from Cooper Biochemical. Phenethylamine was from Chemical Dynamics Corp. Dinonyl phthalate (d = 0.98 g/cc) was from K and K Laboratories. [1-‘%]Pyruvic acid was from Amersham Corp. Adipocyte preparation. Adipocytes were prepared from rat epididymal fat pads by collagenase digestion
on adipocyte pyvia a-adrenergic
’ Abbreviations used: CAMP, vine serum albumin.
be addressed. 699
0003-9861/87 Copyright All rights
cyclic
AMP;
BSA,
bo-
$3.00
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
700
CHENG
AND
LARNER
in Krebs-Ringer bicarbonate buffer as previously described (6). Enzyme assays Pyruvate debydrogenase activity was determined by the rate of the release of i4C02 from [1-i4C]pyruvate as previously described in detail (6). Glycogen synthase and phosphorylase activities were assayed as described by Guinovart et aL (10) and Gilboe et al. (ll), respectively. Protein concentration was measured by the method of Lowry et al. (12) using bovine serum albumin as the standard. RESULTS
AND
DISCUSSION
Incubation of rat adipocytes with 1 mu/ml of insulin at 37°C for 20 min resulted in an increase of glycogen synthase activity ratio from 0.06 to 0.20 (Fig. 1A). After the removal of insulin by washing the cells
5
10
20
x)
Time(min)
FIG. 1. Differential effects of insulin on glycogen synthase and pyruvate dehydrogenase after the termination of insulin action. Adipocytes (2O%)were incubated with (0) or without (0) insulin (1 mu/ml) at 37°C for 20 min. After incubation, the cells were washed three times with fresh Krebs-Ringer bicarbonate buffer containing 2 mM glucose and 3% BSA, pH 7.4, and resuspended in the same buffer and incubated at 3’7’C. One-milliliter aliquots were removed at various times indicated and the enzyme activities were measured as described. Data represent means + SE from three experiments performed on different days.
IL---IO
20
30
60
Time (mid
FIG. 2. Effect of isoproterenol on pyruvate dehydrogenase activity in the presence and absence of propranolol. Adipocytes were incubated without any addition (0), with lo-* M isoproterenol (O), or with 10m8 M isoproterenol + lo-’ M propranolol (A). When present, propranolol was added 1 min prior to the addition of isoproterenol. At the times indicated, lml aliquots were removed and pyruvate dehydrogenase activities were assayed. Data represent means + SE from three experiments performed on different days.
with hormone-free buffer, the insulin-stimulated glycogen synthase activity decreased with time and returned to control level after about 20 min. On the contrary, under the same conditions, the insulinstimulated pyruvate dehydrogenase activity stayed elevated for at least 30 min. (Fig. 1B). More recent results indicate that the insulin-stimulated pyruvate dehydrogenase activity persisted up to 2 h after the removal of hormone (data not shown). Throughout the entire period, the basal activities of glycogen synthase and pyruvate dehydrogenase were unchanged. Previously, we and others have shown that isoproterenol inhibited pyruvate dehydrogenase in isolated rat adipocytes (6-9). The inhibition was completely abolished by propranolol provided the antagonist was added prior to the addition of the agonist (Fig. 2). In contrast, if propranolol was added 20 min after the addition of isoproterenol, the isoproterenol-induced inhibition of pyruvate dehydrogenase was not affected by the antagonist (Fig. 3A), whereas the isoproterenolstimulated phosphorylase activation was reversed by propranolol in 10 min (Fig. 3B). Since the inhibitory action of isoproterenol on pyruvate dehydrogenase can be blocked by propranolol added before the isoproterenol, suggesting the involvement of B-adrenergic receptors in isoproterenol action on pyruvate dehydrogenase, the effect of dibutyryl CAMP on this enzyme was examined. Dibutyryl CAMP, at a concentration of 2 mM, inhibited adipocyte pyruvate dehydrogenase by about 50% in 20 min. After the removal
HORMONAL
REGULATION
OF
ADIPOCYTE
PYRUVATE
701
DEHYDROGENASE
“stable” effects of insulin and isoproterenol on pyruvate dehydrogenase are rather unlikely artifacts. Pyruvate dehydrogenase, like glycogen synthase and phosphorylase, is regulated by hormones through a phosphorylation-dephosphorylation mechanism. However, the present results clearly demonstrate the different time courses of the responses of pyruvate dehydrogenase and of the other two enzymes following the termination of extracellular signals. Namely, the stimulatory effect of insulin and the inhibitory effect of isoproterenol on pyruvate dehydrogenase were stable for at least 30 min after the removal of hormones, while their effects on glycogen synthase and phosphorylase were rapidly reversed. One possible explanation at the physiological level is that both glycogen synthase and phosphorylase are involved in glycogen metabolism which is subjected to minute-to-minute regulation while pyruvate dehydrogenase is involved
I 5
I IO
I 20
I 30
Time (mid
FIG. 3. Effect of propranolol on pyruvate dehydrogenase and phosphorylase activities in rat adipocytes previously incubated with isoproterenol. Adipocytes were incubated with (0, A) or without (0) lo-sM isoprOterenO1 at 37’c for 20 IrkI. PrOpranOlOl (lo-’ M) was then added at time 0 to the isoproterenol-treated cells (a). The cells were further incubated at 37°C for 30 min. At the times indicated, l-ml aliquots were removed and enzyme activities were determined. Data represent means + SE from three experiments performed on different days.
of dibutyryl CAMP by washing the cells with fresh medium, the pyruvate dehydrogenase activity remained in the depressed state for at least 30 min (Fig. ‘l-A), whereas the phosphorylase activity gradually returned to control level in 30 min (Fig. 4B). The reason that dibutyryl CAMP-stimulated phosphorylase activity recovered more slowly than that activated by isoproterenol might be due to the fact that dibutyryl cAMP is more stable than CAMP. It is interesting to point out that the pyruvate dehydrogenase activity stimulated either by Car+-dependent hormones such as phenylephrine and vasopressin or by extracellular Ca’+ returned rapidly (within 10 min) to control level after the removal of stimuli (data not shown). This result indicated that adipocytes used in this study had functionally intact mitochondria. Therefore, the
1
/
I
I
I 5
10
1 20
1 30
El
I
Tune (m!n)
FIG. 4. Reversibilities of pyruvate dehydrogenase and phosphorylase activities in rat adipocytes pretreated with dibutyryl CAMP. Adipocytes were incubated with (0, A) or without (0) 2 mM dibutyryl CAMP at 3’7°C for 20 min. One-half of dibutyryl CAMPtreated cells (A) were washed three times with KrebsRinger bicarbonate buffer to remove extracellular dibutyryl CAMP. All the cells were then incubated at 37°C for an additional 30 min. At the times indicated, l-ml aliquots were removed and enzyme activities were determined. Data represent means + SE from three experiments performed on different days.
702
CHENG
AND
in lipogenesis which is a rather long-term process. Therefore, the interconversion of glycogen synthase and phosphorylase between active and inactive forms in response to extracellular stimuli should he fast while the response of pyruvate dehydrogenase to the same stimuli would be slower. Furthermore, our results suggest that the effects of insulin on glycogen synthase and pyruvate dehydrogenase might be mediated by two different second messengers with different stabilities. Alternatively, the effects might be mediated by the same second messenger but with the regulatory enzymes such as protein kinases and phosphatases for pyruvate dehydrogenase and glycogen synthase responding differently to this second messenger. Interestingly, it has been demonstrated that insulin activated pyruvate dehydrogenase but had no effect on glycogen synthase in B&H-l myocytes in the absence of exogenous glucose (13). However, both glycogen synthase phosphatase and kinase activities were present in BCsH-1 cell extracts and were regulated in the expected manner by glucose 6-phosphate and CAMP, respectively. The author concluded that two classes of messengers are generated by insulin in these cells: one for pyruvate dehydrogenase and the other for glycogen synthase. A similar stable effect of insulin on adipocyte pyruvate dehydrogenase has been reported by Denton et al. (14) in isolated mitochondria derived from insulin-treated adipocytes. In the present study, we further demonstrate that the inhibitory effect of isoproterenol on pyruvate dehydrogenase is also much more stable than its stimulatory effect on phosphorylase. The differential stability effects of isoproterenol on these two enzymes could also be explained by the aforementioned explanations for insulin. The correlation between the “stable” effects of insulin and isoproterenol on pyruvate dehydrogenase and their effects on the phosphorylation state of the enzyme is currently under investigation.
LARNER ACKNOWLEDGMENTS We thank for preparing
Karen G. Rosa the manuscript.
and Anna-Louise
Wallis
REFERENCES 1. HUGHES, W. A., BROWNSEY, R. W., AND DENTON, R. M. (1980) Biochem. J. 192,469-481. 2. DAVIS, P. F., PETIT, F. H., AND REED, L. J. (1977) Bio&em Biophys. Res. Commun 75,541-549. 3. YEAMAN, S. J., HUTCHESON, E. T., ROCHE, T. E., PETTIT, F. H., BROWN, J. R., REED, L. J., WATSON, D. C., AND DIXON, G. H. (1978) Bidwmistrg 17,2364-2369. 4. HUGHES, W. A., AND DENTON, R. M. (1976) Nature (London) 264,471-473. 5. CHENG, K., AND LARNER, J. (1985) Annu. Rev. Physiol 47,405-424. 6. CHENG, K., AND LARNER, J. (1985) J. Biol. Chem. 260,5279-5285. 7. COORE, H. G., DENTON, R. M., MARTIN, B. R., AND RANDLE, P. J. (1971) Biochem J. 125,115-127. 8. SICA, V., AND CUATRECASAS, P. (1973) Biochemistry 12,2282-2291. 9. SMITH, S. J., AND SAGGERSON, E. D. (1978) Biochem J. 174,119-130. 10. GUINOVART, J. J., SALAVERT, A., MASSAGUE, J., CIUDAD, C. J., SALSAS, E., AND ITARTJZ, E. (1979) FEBS I&t. 106,284288. 11. GILBOE, D. P., LARSON, K. L., AND NUTTALL, F. Q. (1972) Anal. Biochem 47,20-27. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDELL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. LUTTRELL, L. M., AND ROGOL, A. D. (1986) Mol. Pharmacol. 30,624630. 14. DENTON, R. M., MCCORMACK, J. G., AND MARSHALL, S. E. (1984) B&hem J 217,441-452.
COMMUNICATION “Stable”
Effects
of Insulin and lsoproterenol KANG
*Merck Rahway,
CHENG**’
AND
on Adipocyte JOSEPH
Pyruvate
Dehydrogenase
LARNERt
Sharp & Dohme Research Laboratories, Department of Animal Drug Discovery, New Jersey 07065, and fllniversity of Virginia School of Medicine, Department Charlottesville, Virginia 22908 Received
March
6,1987,
and in revised
form
April
P.O. Box 2oO0, of Pharmacology,
22, 1987
Insulin, at a concentration of 1 mu/ml, stimulated glycogen synthase and pyruvate dehydrogenase about threefold in isolated rat adipocytes. Upon the removal of insulin, glycogen synthase activity remained in the activated state for 10 min and thereafter rapidly returned to basal level. On the other hand, insulin-stimulated pyruvate dehydrogenase activity remained elevated for at least 30 min. Isoproterenol (low8 M) stimulated phosphorylase and inhibited pyruvate dehydrogenase through the activation of @-adrenergic receptors. Addition of the P-antagonist, propranolol (lop5 M), after isoproterenol reversed the action of isoproterenol on phosphorylase but not its action on pyruvate dehydrogenase. Dibutyryl cyclic AMP, when added to intact adipocytes, produced an effect on pyruvate dehydrogenase similar to that induced by isoproterenol. Our results indicate that both insulin and the ,&agonist have a unique action on pyruvate dehydrogenase which is different from their effects on other enzymes such as glycogen synthase and phosphorylase. o 1987 Academic PM, IIK. _.
Adipocyte pyruvate dehydrogenase is rapidly activated by insulin through a dephosphorylation mechanism (1). Studies with purified components derived from bovine kidney indicate that phosphorylation occurs at three separate serine sites on the asubunit which result in inactivation of the enzyme (2, 3). Careful kinetic studies indicate that the phosphorylation of site 1 is faster than the phosphorylation of sites 2 and 3 and is closely correlated with the inactivation of the enzyme. Thus, the phosphorylation state of site 1 is important in regulating the pyruvate dehydrogenase activity. Insulin decreases the phosphorylation of all three sites to the same extent via the stimulation of a Mg+-dependent Ca’+-stimulated phosphatase (4). Recently, a number of laboratories have reported that a low-molecular-weight mediator which is generated by insulin from plasma membrane stimulates pyruvate dehydrogenase in a cell-free system (5). Catecholamines have dual actions ruvate dehydrogenase: stimulation
1 To whom
correspondence
should
receptors (6) and inhibition via P-adrenergie receptors (6-9). In this study, we demonstrate that both insulin and isoproterenol have a stable effect on pyruvate dehydrogenase even after the termination of hormone action while their effects on glycogen synthase and phosphorylase are rapidly reversed. MATERIALS
AND
METHODS
Sprague-Dawley rats (150-200 g) were purchased from Hilltop Lab Animals, Inc. Bovine serum albumin (BSA’; Fraction V), bovine insulin, 1-(-)-isoproterenol bitartrate, 1-(f)-propranolol hydrochloride, dibutyryl cyclic AMP (CAMP), @-NAD+, thiamine pyrophosphate, and coenzyme A were from Sigma. Crude bacterial collagenase (type 1) was from Cooper Biochemical. Phenethylamine was from Chemical Dynamics Corp. Dinonyl phthalate (d = 0.98 g/cc) was from K and K Laboratories. [1-‘%]Pyruvic acid was from Amersham Corp. Adipocyte preparation. Adipocytes were prepared from rat epididymal fat pads by collagenase digestion
on adipocyte pyvia a-adrenergic
’ Abbreviations used: CAMP, vine serum albumin.
be addressed. 699
0003-9861/87 Copyright All rights
cyclic
AMP;
BSA,
bo-
$3.00
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
700
CHENG
AND
LARNER
in Krebs-Ringer bicarbonate buffer as previously described (6). Enzyme assays Pyruvate debydrogenase activity was determined by the rate of the release of i4C02 from [1-i4C]pyruvate as previously described in detail (6). Glycogen synthase and phosphorylase activities were assayed as described by Guinovart et aL (10) and Gilboe et al. (ll), respectively. Protein concentration was measured by the method of Lowry et al. (12) using bovine serum albumin as the standard. RESULTS
AND
DISCUSSION
Incubation of rat adipocytes with 1 mu/ml of insulin at 37°C for 20 min resulted in an increase of glycogen synthase activity ratio from 0.06 to 0.20 (Fig. 1A). After the removal of insulin by washing the cells
5
10
20
x)
Time(min)
FIG. 1. Differential effects of insulin on glycogen synthase and pyruvate dehydrogenase after the termination of insulin action. Adipocytes (2O%)were incubated with (0) or without (0) insulin (1 mu/ml) at 37°C for 20 min. After incubation, the cells were washed three times with fresh Krebs-Ringer bicarbonate buffer containing 2 mM glucose and 3% BSA, pH 7.4, and resuspended in the same buffer and incubated at 3’7’C. One-milliliter aliquots were removed at various times indicated and the enzyme activities were measured as described. Data represent means + SE from three experiments performed on different days.
IL---IO
20
30
60
Time (mid
FIG. 2. Effect of isoproterenol on pyruvate dehydrogenase activity in the presence and absence of propranolol. Adipocytes were incubated without any addition (0), with lo-* M isoproterenol (O), or with 10m8 M isoproterenol + lo-’ M propranolol (A). When present, propranolol was added 1 min prior to the addition of isoproterenol. At the times indicated, lml aliquots were removed and pyruvate dehydrogenase activities were assayed. Data represent means + SE from three experiments performed on different days.
with hormone-free buffer, the insulin-stimulated glycogen synthase activity decreased with time and returned to control level after about 20 min. On the contrary, under the same conditions, the insulinstimulated pyruvate dehydrogenase activity stayed elevated for at least 30 min. (Fig. 1B). More recent results indicate that the insulin-stimulated pyruvate dehydrogenase activity persisted up to 2 h after the removal of hormone (data not shown). Throughout the entire period, the basal activities of glycogen synthase and pyruvate dehydrogenase were unchanged. Previously, we and others have shown that isoproterenol inhibited pyruvate dehydrogenase in isolated rat adipocytes (6-9). The inhibition was completely abolished by propranolol provided the antagonist was added prior to the addition of the agonist (Fig. 2). In contrast, if propranolol was added 20 min after the addition of isoproterenol, the isoproterenol-induced inhibition of pyruvate dehydrogenase was not affected by the antagonist (Fig. 3A), whereas the isoproterenolstimulated phosphorylase activation was reversed by propranolol in 10 min (Fig. 3B). Since the inhibitory action of isoproterenol on pyruvate dehydrogenase can be blocked by propranolol added before the isoproterenol, suggesting the involvement of B-adrenergic receptors in isoproterenol action on pyruvate dehydrogenase, the effect of dibutyryl CAMP on this enzyme was examined. Dibutyryl CAMP, at a concentration of 2 mM, inhibited adipocyte pyruvate dehydrogenase by about 50% in 20 min. After the removal
HORMONAL
REGULATION
OF
ADIPOCYTE
PYRUVATE
701
DEHYDROGENASE
“stable” effects of insulin and isoproterenol on pyruvate dehydrogenase are rather unlikely artifacts. Pyruvate dehydrogenase, like glycogen synthase and phosphorylase, is regulated by hormones through a phosphorylation-dephosphorylation mechanism. However, the present results clearly demonstrate the different time courses of the responses of pyruvate dehydrogenase and of the other two enzymes following the termination of extracellular signals. Namely, the stimulatory effect of insulin and the inhibitory effect of isoproterenol on pyruvate dehydrogenase were stable for at least 30 min after the removal of hormones, while their effects on glycogen synthase and phosphorylase were rapidly reversed. One possible explanation at the physiological level is that both glycogen synthase and phosphorylase are involved in glycogen metabolism which is subjected to minute-to-minute regulation while pyruvate dehydrogenase is involved
I 5
I IO
I 20
I 30
Time (mid
FIG. 3. Effect of propranolol on pyruvate dehydrogenase and phosphorylase activities in rat adipocytes previously incubated with isoproterenol. Adipocytes were incubated with (0, A) or without (0) lo-sM isoprOterenO1 at 37’c for 20 IrkI. PrOpranOlOl (lo-’ M) was then added at time 0 to the isoproterenol-treated cells (a). The cells were further incubated at 37°C for 30 min. At the times indicated, l-ml aliquots were removed and enzyme activities were determined. Data represent means + SE from three experiments performed on different days.
of dibutyryl CAMP by washing the cells with fresh medium, the pyruvate dehydrogenase activity remained in the depressed state for at least 30 min (Fig. ‘l-A), whereas the phosphorylase activity gradually returned to control level in 30 min (Fig. 4B). The reason that dibutyryl CAMP-stimulated phosphorylase activity recovered more slowly than that activated by isoproterenol might be due to the fact that dibutyryl cAMP is more stable than CAMP. It is interesting to point out that the pyruvate dehydrogenase activity stimulated either by Car+-dependent hormones such as phenylephrine and vasopressin or by extracellular Ca’+ returned rapidly (within 10 min) to control level after the removal of stimuli (data not shown). This result indicated that adipocytes used in this study had functionally intact mitochondria. Therefore, the
1
/
I
I
I 5
10
1 20
1 30
El
I
Tune (m!n)
FIG. 4. Reversibilities of pyruvate dehydrogenase and phosphorylase activities in rat adipocytes pretreated with dibutyryl CAMP. Adipocytes were incubated with (0, A) or without (0) 2 mM dibutyryl CAMP at 3’7°C for 20 min. One-half of dibutyryl CAMPtreated cells (A) were washed three times with KrebsRinger bicarbonate buffer to remove extracellular dibutyryl CAMP. All the cells were then incubated at 37°C for an additional 30 min. At the times indicated, l-ml aliquots were removed and enzyme activities were determined. Data represent means + SE from three experiments performed on different days.
702
CHENG
AND
in lipogenesis which is a rather long-term process. Therefore, the interconversion of glycogen synthase and phosphorylase between active and inactive forms in response to extracellular stimuli should he fast while the response of pyruvate dehydrogenase to the same stimuli would be slower. Furthermore, our results suggest that the effects of insulin on glycogen synthase and pyruvate dehydrogenase might be mediated by two different second messengers with different stabilities. Alternatively, the effects might be mediated by the same second messenger but with the regulatory enzymes such as protein kinases and phosphatases for pyruvate dehydrogenase and glycogen synthase responding differently to this second messenger. Interestingly, it has been demonstrated that insulin activated pyruvate dehydrogenase but had no effect on glycogen synthase in B&H-l myocytes in the absence of exogenous glucose (13). However, both glycogen synthase phosphatase and kinase activities were present in BCsH-1 cell extracts and were regulated in the expected manner by glucose 6-phosphate and CAMP, respectively. The author concluded that two classes of messengers are generated by insulin in these cells: one for pyruvate dehydrogenase and the other for glycogen synthase. A similar stable effect of insulin on adipocyte pyruvate dehydrogenase has been reported by Denton et al. (14) in isolated mitochondria derived from insulin-treated adipocytes. In the present study, we further demonstrate that the inhibitory effect of isoproterenol on pyruvate dehydrogenase is also much more stable than its stimulatory effect on phosphorylase. The differential stability effects of isoproterenol on these two enzymes could also be explained by the aforementioned explanations for insulin. The correlation between the “stable” effects of insulin and isoproterenol on pyruvate dehydrogenase and their effects on the phosphorylation state of the enzyme is currently under investigation.
LARNER ACKNOWLEDGMENTS We thank for preparing
Karen G. Rosa the manuscript.
and Anna-Louise
Wallis
REFERENCES 1. HUGHES, W. A., BROWNSEY, R. W., AND DENTON, R. M. (1980) Biochem. J. 192,469-481. 2. DAVIS, P. F., PETIT, F. H., AND REED, L. J. (1977) Bio&em Biophys. Res. Commun 75,541-549. 3. YEAMAN, S. J., HUTCHESON, E. T., ROCHE, T. E., PETTIT, F. H., BROWN, J. R., REED, L. J., WATSON, D. C., AND DIXON, G. H. (1978) Bidwmistrg 17,2364-2369. 4. HUGHES, W. A., AND DENTON, R. M. (1976) Nature (London) 264,471-473. 5. CHENG, K., AND LARNER, J. (1985) Annu. Rev. Physiol 47,405-424. 6. CHENG, K., AND LARNER, J. (1985) J. Biol. Chem. 260,5279-5285. 7. COORE, H. G., DENTON, R. M., MARTIN, B. R., AND RANDLE, P. J. (1971) Biochem J. 125,115-127. 8. SICA, V., AND CUATRECASAS, P. (1973) Biochemistry 12,2282-2291. 9. SMITH, S. J., AND SAGGERSON, E. D. (1978) Biochem J. 174,119-130. 10. GUINOVART, J. J., SALAVERT, A., MASSAGUE, J., CIUDAD, C. J., SALSAS, E., AND ITARTJZ, E. (1979) FEBS I&t. 106,284288. 11. GILBOE, D. P., LARSON, K. L., AND NUTTALL, F. Q. (1972) Anal. Biochem 47,20-27. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDELL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. LUTTRELL, L. M., AND ROGOL, A. D. (1986) Mol. Pharmacol. 30,624630. 14. DENTON, R. M., MCCORMACK, J. G., AND MARSHALL, S. E. (1984) B&hem J 217,441-452.