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Induction of hepatic malic enzyme in response to insulin
Molecular and Cellular Endocrinology,
Elsevier/North-Holland
26 (1982)
309
309-314
Scientific Publishers, Ltd.
INDUCTION OF HEPATIC MALIC ENZYME IN RESPONSE TO INSULIN
Ed W. THOMPSON a and Richard L. DRAKE b a Department of Anatomy, The Medical College of Wisconsin, Milwaukee, WI 53226 and b Department (U.S.A.)
of Anatomy,
University of Cincinnati College of Medicine,
Cincinnati, OH 45267
Received 13 November 1981; revision received 12 January 1982; accepted 12 January 1982
The activity and quantity of hepatic malic enzyme were determined in diabetic rats at various times after insulin treatment. The increase in activity observed following insulin treatment was accompanied by a similar increase in the quantity of this enzyme, as determined by a specific radioimmunoassay. These results demonstrate that the insulin-mediated increase in malic enzyme activity was due to an increase in the quantity of enzyme and did not involve a modification of existing enzyme molecules. Keywords:
liver; diabetes; radioimmunoassay.
The insulin-mediated increase in the activity of hepatic malic enzyme [L-malate : NADP oxidoreductase (decarboxylating), EC 1 .l .1.40] in diabetic rats is well established (Shrago et al., 1963; Storey and Bailey, 1978;McCormick et al., 1978). However, none of these studies determined whether this insulin-mediated increase in activity was due to enzyme modification or increased enzyme content. In this study we present evidence from experiments using a radioimmunoassay specific for malic enzyme, that the increase in malic enzyme activity after treatment of diabetic rats with insulin is due to an increase in the quantity of enzyme.
MATERIALS
AND METHODS
Animal treatment and tissue preparation Diabetes was produced in male Sprague-Dawley. rats, having initial body weights of 150-175 g, by i.v. injection of 4% alloxan monohydrate (Eastman Organic Chemicals), at a dosage of 55 mg/kg body weight. Blood samples were obtained
Requests for reprints should be addressed to: Dr. Richard L. Drake, Department of Anatomy, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267 W.S.A.). 0303~7207/82/0000-0000/$02.75
0 Elsevier/North-Holland
Scientific Publishers, Ltd.
310
Ed W. Thompson, Richard L. Drake
weekly and plasma glucose levels were determined using hexokinase and glucose-6phosphate dehydrogenase (Bergmeyer et al., 1974). Rats having plasma glucose levels in excess of 300 mg percent and little or no weight gain were considered diabetic (Morgan and Lazarow, 1965). These rats were maintained for 10 days following administration of alloxan to allow stabilization of the diabetic condition. After 10 days, the diabetic rats were divided into 2 groups. One diabetic group was fed a high carbohydrate (5% sucrose), fat-free diet (AIN-76, Zeigler Brothers, Gardners, Pa.) for 60 h. The second diabetic group was fed the same diet and given subcutaneous injections of 4 units regular insulin (Regular Iletin) and 4 units NPH insulin (NPH Iletin) every 12 h for 60 h. At various times after the initiation of insulin treatment the rats were killed, a blood sample collected, the livers removed and weighed, a sample obtained for the determination of liver glycogen levels (Huijing, 1970), and the remainder homogenized (2% w/v) in 30 mM Tris-HCl, pH 7.7, containing 0.25 M sucrose, 1 mM dithiothreitol and 0.1 mM EDTA. The 100 000 X g supernatant was prepared from the liver homogenate and used to determine the activity of malic enzyme and malic enzyme protein levels using a malic enzyme specific radioimmunoassay. Enzyme assay Malic enzyme activity was assayed according to Wise and Ball (1964) as modified by Yeung and Carrico (1976). The reaction was initiated by the addition of malate. 1 unit of activity is defined as 1 pmole of NADP reduced/min. Enzyme specific radioimmunoassay The malic enzyme specific radioimmunoassay (RIA) was established in the following manner. Rat-liver malic enzyme was purified according to the procedure of Yeung and Carrico (1976) as modified by Towle et al. (1980). Antibody against this enzyme was prepared in a rabbit following the procedure of Kulkoski et al. (1979) and its specificity determined by the Ouchterlony procedure (Williams and Chase, 1971) using double-diffusion plates prepared from 0.8% agar in 0.1 M potassium phosphate, pH 7.2. The rabbit anti-malic enzyme antiserum was diluted 25 fold with 0.01 M Tris-HCl, pH 7.0, and 0.001 M CaCl? and used in the RIA. The following components were added to each RIA tube in the following order: (1) 0.2 ml IgG-free rabbit serum albumin fraction V (10 mg/ml). The tubes were vigorously vortexed to coat the inner surface, and the solution discarded, (2) appropriate aliquots of the 100000 Xg supernatant protein (0.1-l .2 mg), (3) a fixed amount of [‘*‘I]iodomalic enzyme (20000 cpm) iodinated according to the method of Winkelhake et al. (1979) (4) the volume of Tris-CaC12 buffer necessary to bring the final volume to 200 ~1 and the tubes gently mixed, (5) the amount of rabbit anti-malic enzyme antiserum, diluted 25fold, necessary to obtain maximum enzyme (75 c(l). This amount was determined by a binding of [ ‘*‘I]iodomalic quantitative precipitin test. After mixing, the tubes were incubated with constant shaking for 60 min in a 37°C water bath. Upon removal from the bath, 19 ~1 of a
Insulin induction of malic enzyme
311
10% suspension of heat-killed, formalin-fixed S. aureus (from Dr. J. Winkelhake, The Medical College of Wisconsin) was added, the tubes mixed, and allowed to stand at 4°C for 60 min. The tubes were then centrifuged at 10 000 X g, the supernatant transferred to new tubes, and both samples counted. A standard curve was of obtained by competing [ 1251]iodomalic enzyme against known concentrations unlabeled homogeneous malic enzyme. Protein determination
Protein determinations were done according to the method (195 1) using bovine serum albumin as the standard.
of Lowry et al.
RESULTS The induction of experimental diabetes in rats is indicated by plasma-glucose levels in excess of 300 mg percent and little or no weight gain. The alloxan-treated rats used in the studies presented here had plasma glucose levels of 459 + 67 mg percent (3 + S.D.) and lost 4.4 + 14.4 g/wk (x * S.D.), thus satisfying these 2 criteria for diabetic animals. Treatment of the diabetic rats with insulin had profound effects on liver size and liver glycogen levels. The liver weight increased gradually over the 60-h period of insulin treatment, resulting in a 2.3-fold increase in liver weight/l00 g body weight ratio by 60-h post insulin. By comparison, the increase in liver glycogen was more rapid, with maximum levels of approx. 7% reached by 12 h post insulin and maintained throughout the 60-h treatment period. Treatment with the high carbohydrate, fat-free diet alone for 60 h had a small effect on liver glycogen levels and no effect on body weight, liver weight or liver weight/100 g body weight ratio. In addition to the insulin-mediated increases in liver weight and liver glycogen, insulin treatment resulted in a substantial increase in malic enzyme activity (mUnits/mg soluble protein). However, this insulin-mediated increase in activity was delayed and extended over the entire treatment period of 60 h (Table 1). The malic enzyme activity showed no response in the first 12 h of insulin treatment, a moderate increase between 12 and 36 h, and a substantial increase between 36 and 60 h of insulin treatment. Diabetic rats maintained on the high carbohydrate, fatfree diet alone for the 60-h treatment period showed no increase in the activity of this enzyme. Additionally, an examination of time points between 0 and 12 h post insulin showed no initial rapid rise in enzyme activity (data not shown). A similar delayed and gradual response to insulin treatment was also observed when the activity of malic enzyme was expressed either as units/g liver or units/ 100 g body weight (Table 1). Expression of the malic enzyme activity in this manner demonstrates that a significant increase has occurred after insulin treatment irrespective of the increase in liver size. After 12 h of insulin treatment there was either little or no change in these parameters; however, by 36 h a moderate increase
312
Ed W. Thompson,
Richard L. Drake
Table 1 Activity of hepatic malic enzyme Results are presented as mean + SD. The number of rats in each group is given in parentheses. All rats were maintained on a high carbohydrate, fat-free diet for the duration of insulin treatment, Insulin treatment was 4 units each of regular and NPH insulin every 12 h.
-~___.-
Malic enzyme
Hours post insulin
_~...___ mU per mg soluble protein
U/g liver
U/100 g body wt.
(3) (4) (3) (7)
1.4 t 0.4 2.5 i 2.0 11.7+ 2.0 37.3 -+ 13.7
0.12 0.13 0.50 1.46
0.58 1.1 5.0 16.7
f; 0.2 f 0.2 + 2.3 f 4.1
60 (8) Diet aIone
2.4 c 0.6
0.6
c 0.2
0 12 36 60
f f f f
0.04 0.02 0.17 0.38
0.13 * 0.04
was observed, and a substantial increase occurred in both parameters by 60 h of insulin treatment. The high carbohydrate, fat-free diet alone had no effect. Antiserum was prepared against hepatic malic enzyme and its specificity tested by the Ouchter~ony double-diffusion procedure. A set of single sharp precipitin bands, fusing without any indication of spurs, was observed when the rabbit antiserum was tested against homogeneous malic enzyme and the 100 000 X g liver
2.0
4.0 kg
6.0
Malic enzyme
8.0 mg Soluble
Fig. 1. Standard curve for the malic enzyme specific radioimmunoassay. of 2 determinations.
Protein
Each point is the mean
Fig. 2. Radioimmunoassay comparing hepatic malic enzyme protein levels. [ ’ 25I]Iodomalic enzyme was displaced from anti-malic enzyme antibodies by adding increasing amounts of the 100 000 X g liver supernatant protein (soluble protein, 0.1-1.2 mg) from diabetic (0) and insulin-treated diabetic (o) rats. Values are means f S.D. of samples from 8 diabetic rats and 7 insuhn-treated diabetic rats. Beginning with the second value, all differences are statistically si~~icant, p < 0.001.
Insulin induction of malic enzyme
313
supernatant from an insulin-treated diabetic rat. This contrasts with the results obtained when malic enzyme was tested against normal rabbit serum, which showed no reactivity. These results indicate that a homogeneous antibody has been produced which is specific for hepatic malic enzyme. The sensitivity of the malic enzyme specific RIA developed for this study was determined by competing [ 1251]iodomalic enzyme against known concentrations of unlabeled homogeneous malic enzyme. The RIA was sensitive to as little as 400 ng unlabeled antigen, with 50% displacement of [ ’ 251] iodomalic enzyme obtained with 2.0 yg unlabeled malic enzyme (Fig. 1). This RIA was then used to compare the quantity of hepatic malic enzyme in the diabetic rats treated with diet alone and diet plus insulin for 60 h. These results (Fig. 2) indicate there was approx. a 5-fold increase in the quantity of hepatic malic enzyme following insulin treatment for 60 h. Additionally, a determination of malic enzyme levels after 12 and 36 h of insulin treatment showed that the increase in the quantity of enzyme paralleled the increase in enzyme activity (data not shown).
DISCUSSION The insulin-mediated increase in liver size observed in this experiment is consistent with the previous reports of Osborn et al. (1953). These investigators demonstrated that this increased liver size is partly due to an increase in fat accumulation caused by a rapid rate of lipogenesis during the first 3 days of insulin treatment of diabetic rats maintained on a high carbohydrate, fat-free diet. Therefore, this animal model is useful for studies on the influence of insulin on the lipogenic enzymes. The insulin-mediated increase in hepatic malic enzyme activity, which did not begin until at least 12 h of insulin treatment, is similar to previous results (Shrago et al., 1963) and suggests that the return from the depleted diabetic condition is not a rapid process. While the possibility exists that this delayed response is due to the inability of the liver from a diabetic rat to respond normally to insulin, the rapid rise in hepatic glycogen levels shown here and in other work (Thompson et al., 1981) argues against this, By 60’ h of treatment a substantial increase in malic enzyme activity had occurred, suggesting that the quantity of malic enzyme present had also increased. However, since assaying the activity of malic enzyme may measure only active enzyme molecules, a direct determination of the quantity of malic enzyme was necessary. Radioimmunoassay is used to directly measure changes in the number of enzyme molecules independent of enzyme activity. Using a malic enzyme specific RIA developed for this study, our results indicate that the insulin-mediated increase in malic enzyme activity was accompanied by a substantial increase in the quantity of enzyme, which paralleled the increase in enzyme activity. This clearly demonstrates that the modification of existing enzyme molecules is not involved in the control of malic enzyme activity by insulin.
314
h‘d W. Thompson, Richard L. Drake
These results suggest that insulin mediates an increase in malic enzyme synthesis in diabetic rats. However, since the level of an enzyme is dependent on the balance between synthesis and degradation, alterations in the rate of enzyme degradation can not be ruled out as contributing to the increased quantity of malic enzyme observed in this study. We did not determine turnover in this study; however previous investigations by Gibson et al. (1972) and Lyons and Gibson (1970, 1971), using refed rats which showed increased plasma insulin levels, demonstrated that initiation of enzyme synthesis, rather than decreased enzyme degradation, is the major contributing factor to the increased quantity of malic enzyme. Their findings support our conclusion that the increase in the quantity of malic enzyme observed in the present experiments is attributed to de novo synthesis of malic enzyme.
ACKNOWLEDGEMENTS The authors wish to thank Christine Kuepfer for her excellent technical assistance. This work was supported by grants from the American Diabetes Association and grant PCM-7822130 from the National Science Foundation.
REFERENCES Bergmeyer, H.U., Bernt, E., Schmidt, F., and Stork, H. (1974) in: Methods of Enzymatic Analysis (H.U. Bergmeyer, Ed.), Vol. 3, pp. 1196-1201. Gibson, D.M., Lyons, R.T., Scott, D.F., and Muto, Y. (1972) Adv. Enzyme Regulat. 10, 187204. Huijing, F. (1970) Clin. Chim. Acta 30,567-572. Kulkoski, J.A., Peterson, B.L., Elcombe, B., Winkelhake, J.L., and Ghazarian, J.G. (1979) FEBS Lett. 99, 183-188. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265275. Lyons, R.T., and Gibson, D.M. (1970) Fed. Proc. 29,901. Lyons, R.T., and Gibson, D.M. (1971) Fed. Proc. 30, 1080. McCormick, K.L., Widness, J.A., Susa, J.B., and Schwartz, R. (1978) Biochem. J. 172, 327331. Morgan, C.R., and Lazarow, A. (1965) Diabetes 14,669-671. Osborn, M.J., Felts, J.M., and Chaikoff, I.L. (1953) J. Biol. Chem. 203, 173-181. Shrago, E., Lardy, H.A., Nordlie, R.C., and Foster, D.O. (1963) J. Biol. Chem. 238, 31883192. Storey, J.M., and Bailey, E. (1978) Enzyme 23, 382-387. Thompson, E.W., Parks, W.C., and Drake, R.L. (1981) Am. J. Anat. 160,449-460. Towle, H.C., Maria& C.N., and Oppenheimer, J.H. (1980) Biochemistry 19, 579-585. Williams, C.A., and Chase, M.W. (1971) in: Methods in Immunology and Immunochemistry (C.A. Williams and M.W. Chase, Eds.), Vol. 3, p. 146. Winkelhake, J.L., Elcombe, B.M., and Hodach, A. (1979) Cancer Res. 39, 3058-3064. Wise, E.M., and Ball, E.G. (1964) Proc. Natl. Acad. Sci. (U.S.A.) 52, 1255-1263. Yeung, K.K., and Carrico, R.J. (1976) Anal. Biochem. 74, 369-375.
Elsevier/North-Holland
26 (1982)
309
309-314
Scientific Publishers, Ltd.
INDUCTION OF HEPATIC MALIC ENZYME IN RESPONSE TO INSULIN
Ed W. THOMPSON a and Richard L. DRAKE b a Department of Anatomy, The Medical College of Wisconsin, Milwaukee, WI 53226 and b Department (U.S.A.)
of Anatomy,
University of Cincinnati College of Medicine,
Cincinnati, OH 45267
Received 13 November 1981; revision received 12 January 1982; accepted 12 January 1982
The activity and quantity of hepatic malic enzyme were determined in diabetic rats at various times after insulin treatment. The increase in activity observed following insulin treatment was accompanied by a similar increase in the quantity of this enzyme, as determined by a specific radioimmunoassay. These results demonstrate that the insulin-mediated increase in malic enzyme activity was due to an increase in the quantity of enzyme and did not involve a modification of existing enzyme molecules. Keywords:
liver; diabetes; radioimmunoassay.
The insulin-mediated increase in the activity of hepatic malic enzyme [L-malate : NADP oxidoreductase (decarboxylating), EC 1 .l .1.40] in diabetic rats is well established (Shrago et al., 1963; Storey and Bailey, 1978;McCormick et al., 1978). However, none of these studies determined whether this insulin-mediated increase in activity was due to enzyme modification or increased enzyme content. In this study we present evidence from experiments using a radioimmunoassay specific for malic enzyme, that the increase in malic enzyme activity after treatment of diabetic rats with insulin is due to an increase in the quantity of enzyme.
MATERIALS
AND METHODS
Animal treatment and tissue preparation Diabetes was produced in male Sprague-Dawley. rats, having initial body weights of 150-175 g, by i.v. injection of 4% alloxan monohydrate (Eastman Organic Chemicals), at a dosage of 55 mg/kg body weight. Blood samples were obtained
Requests for reprints should be addressed to: Dr. Richard L. Drake, Department of Anatomy, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267 W.S.A.). 0303~7207/82/0000-0000/$02.75
0 Elsevier/North-Holland
Scientific Publishers, Ltd.
310
Ed W. Thompson, Richard L. Drake
weekly and plasma glucose levels were determined using hexokinase and glucose-6phosphate dehydrogenase (Bergmeyer et al., 1974). Rats having plasma glucose levels in excess of 300 mg percent and little or no weight gain were considered diabetic (Morgan and Lazarow, 1965). These rats were maintained for 10 days following administration of alloxan to allow stabilization of the diabetic condition. After 10 days, the diabetic rats were divided into 2 groups. One diabetic group was fed a high carbohydrate (5% sucrose), fat-free diet (AIN-76, Zeigler Brothers, Gardners, Pa.) for 60 h. The second diabetic group was fed the same diet and given subcutaneous injections of 4 units regular insulin (Regular Iletin) and 4 units NPH insulin (NPH Iletin) every 12 h for 60 h. At various times after the initiation of insulin treatment the rats were killed, a blood sample collected, the livers removed and weighed, a sample obtained for the determination of liver glycogen levels (Huijing, 1970), and the remainder homogenized (2% w/v) in 30 mM Tris-HCl, pH 7.7, containing 0.25 M sucrose, 1 mM dithiothreitol and 0.1 mM EDTA. The 100 000 X g supernatant was prepared from the liver homogenate and used to determine the activity of malic enzyme and malic enzyme protein levels using a malic enzyme specific radioimmunoassay. Enzyme assay Malic enzyme activity was assayed according to Wise and Ball (1964) as modified by Yeung and Carrico (1976). The reaction was initiated by the addition of malate. 1 unit of activity is defined as 1 pmole of NADP reduced/min. Enzyme specific radioimmunoassay The malic enzyme specific radioimmunoassay (RIA) was established in the following manner. Rat-liver malic enzyme was purified according to the procedure of Yeung and Carrico (1976) as modified by Towle et al. (1980). Antibody against this enzyme was prepared in a rabbit following the procedure of Kulkoski et al. (1979) and its specificity determined by the Ouchterlony procedure (Williams and Chase, 1971) using double-diffusion plates prepared from 0.8% agar in 0.1 M potassium phosphate, pH 7.2. The rabbit anti-malic enzyme antiserum was diluted 25 fold with 0.01 M Tris-HCl, pH 7.0, and 0.001 M CaCl? and used in the RIA. The following components were added to each RIA tube in the following order: (1) 0.2 ml IgG-free rabbit serum albumin fraction V (10 mg/ml). The tubes were vigorously vortexed to coat the inner surface, and the solution discarded, (2) appropriate aliquots of the 100000 Xg supernatant protein (0.1-l .2 mg), (3) a fixed amount of [‘*‘I]iodomalic enzyme (20000 cpm) iodinated according to the method of Winkelhake et al. (1979) (4) the volume of Tris-CaC12 buffer necessary to bring the final volume to 200 ~1 and the tubes gently mixed, (5) the amount of rabbit anti-malic enzyme antiserum, diluted 25fold, necessary to obtain maximum enzyme (75 c(l). This amount was determined by a binding of [ ‘*‘I]iodomalic quantitative precipitin test. After mixing, the tubes were incubated with constant shaking for 60 min in a 37°C water bath. Upon removal from the bath, 19 ~1 of a
Insulin induction of malic enzyme
311
10% suspension of heat-killed, formalin-fixed S. aureus (from Dr. J. Winkelhake, The Medical College of Wisconsin) was added, the tubes mixed, and allowed to stand at 4°C for 60 min. The tubes were then centrifuged at 10 000 X g, the supernatant transferred to new tubes, and both samples counted. A standard curve was of obtained by competing [ 1251]iodomalic enzyme against known concentrations unlabeled homogeneous malic enzyme. Protein determination
Protein determinations were done according to the method (195 1) using bovine serum albumin as the standard.
of Lowry et al.
RESULTS The induction of experimental diabetes in rats is indicated by plasma-glucose levels in excess of 300 mg percent and little or no weight gain. The alloxan-treated rats used in the studies presented here had plasma glucose levels of 459 + 67 mg percent (3 + S.D.) and lost 4.4 + 14.4 g/wk (x * S.D.), thus satisfying these 2 criteria for diabetic animals. Treatment of the diabetic rats with insulin had profound effects on liver size and liver glycogen levels. The liver weight increased gradually over the 60-h period of insulin treatment, resulting in a 2.3-fold increase in liver weight/l00 g body weight ratio by 60-h post insulin. By comparison, the increase in liver glycogen was more rapid, with maximum levels of approx. 7% reached by 12 h post insulin and maintained throughout the 60-h treatment period. Treatment with the high carbohydrate, fat-free diet alone for 60 h had a small effect on liver glycogen levels and no effect on body weight, liver weight or liver weight/100 g body weight ratio. In addition to the insulin-mediated increases in liver weight and liver glycogen, insulin treatment resulted in a substantial increase in malic enzyme activity (mUnits/mg soluble protein). However, this insulin-mediated increase in activity was delayed and extended over the entire treatment period of 60 h (Table 1). The malic enzyme activity showed no response in the first 12 h of insulin treatment, a moderate increase between 12 and 36 h, and a substantial increase between 36 and 60 h of insulin treatment. Diabetic rats maintained on the high carbohydrate, fatfree diet alone for the 60-h treatment period showed no increase in the activity of this enzyme. Additionally, an examination of time points between 0 and 12 h post insulin showed no initial rapid rise in enzyme activity (data not shown). A similar delayed and gradual response to insulin treatment was also observed when the activity of malic enzyme was expressed either as units/g liver or units/ 100 g body weight (Table 1). Expression of the malic enzyme activity in this manner demonstrates that a significant increase has occurred after insulin treatment irrespective of the increase in liver size. After 12 h of insulin treatment there was either little or no change in these parameters; however, by 36 h a moderate increase
312
Ed W. Thompson,
Richard L. Drake
Table 1 Activity of hepatic malic enzyme Results are presented as mean + SD. The number of rats in each group is given in parentheses. All rats were maintained on a high carbohydrate, fat-free diet for the duration of insulin treatment, Insulin treatment was 4 units each of regular and NPH insulin every 12 h.
-~___.-
Malic enzyme
Hours post insulin
_~...___ mU per mg soluble protein
U/g liver
U/100 g body wt.
(3) (4) (3) (7)
1.4 t 0.4 2.5 i 2.0 11.7+ 2.0 37.3 -+ 13.7
0.12 0.13 0.50 1.46
0.58 1.1 5.0 16.7
f; 0.2 f 0.2 + 2.3 f 4.1
60 (8) Diet aIone
2.4 c 0.6
0.6
c 0.2
0 12 36 60
f f f f
0.04 0.02 0.17 0.38
0.13 * 0.04
was observed, and a substantial increase occurred in both parameters by 60 h of insulin treatment. The high carbohydrate, fat-free diet alone had no effect. Antiserum was prepared against hepatic malic enzyme and its specificity tested by the Ouchter~ony double-diffusion procedure. A set of single sharp precipitin bands, fusing without any indication of spurs, was observed when the rabbit antiserum was tested against homogeneous malic enzyme and the 100 000 X g liver
2.0
4.0 kg
6.0
Malic enzyme
8.0 mg Soluble
Fig. 1. Standard curve for the malic enzyme specific radioimmunoassay. of 2 determinations.
Protein
Each point is the mean
Fig. 2. Radioimmunoassay comparing hepatic malic enzyme protein levels. [ ’ 25I]Iodomalic enzyme was displaced from anti-malic enzyme antibodies by adding increasing amounts of the 100 000 X g liver supernatant protein (soluble protein, 0.1-1.2 mg) from diabetic (0) and insulin-treated diabetic (o) rats. Values are means f S.D. of samples from 8 diabetic rats and 7 insuhn-treated diabetic rats. Beginning with the second value, all differences are statistically si~~icant, p < 0.001.
Insulin induction of malic enzyme
313
supernatant from an insulin-treated diabetic rat. This contrasts with the results obtained when malic enzyme was tested against normal rabbit serum, which showed no reactivity. These results indicate that a homogeneous antibody has been produced which is specific for hepatic malic enzyme. The sensitivity of the malic enzyme specific RIA developed for this study was determined by competing [ 1251]iodomalic enzyme against known concentrations of unlabeled homogeneous malic enzyme. The RIA was sensitive to as little as 400 ng unlabeled antigen, with 50% displacement of [ ’ 251] iodomalic enzyme obtained with 2.0 yg unlabeled malic enzyme (Fig. 1). This RIA was then used to compare the quantity of hepatic malic enzyme in the diabetic rats treated with diet alone and diet plus insulin for 60 h. These results (Fig. 2) indicate there was approx. a 5-fold increase in the quantity of hepatic malic enzyme following insulin treatment for 60 h. Additionally, a determination of malic enzyme levels after 12 and 36 h of insulin treatment showed that the increase in the quantity of enzyme paralleled the increase in enzyme activity (data not shown).
DISCUSSION The insulin-mediated increase in liver size observed in this experiment is consistent with the previous reports of Osborn et al. (1953). These investigators demonstrated that this increased liver size is partly due to an increase in fat accumulation caused by a rapid rate of lipogenesis during the first 3 days of insulin treatment of diabetic rats maintained on a high carbohydrate, fat-free diet. Therefore, this animal model is useful for studies on the influence of insulin on the lipogenic enzymes. The insulin-mediated increase in hepatic malic enzyme activity, which did not begin until at least 12 h of insulin treatment, is similar to previous results (Shrago et al., 1963) and suggests that the return from the depleted diabetic condition is not a rapid process. While the possibility exists that this delayed response is due to the inability of the liver from a diabetic rat to respond normally to insulin, the rapid rise in hepatic glycogen levels shown here and in other work (Thompson et al., 1981) argues against this, By 60’ h of treatment a substantial increase in malic enzyme activity had occurred, suggesting that the quantity of malic enzyme present had also increased. However, since assaying the activity of malic enzyme may measure only active enzyme molecules, a direct determination of the quantity of malic enzyme was necessary. Radioimmunoassay is used to directly measure changes in the number of enzyme molecules independent of enzyme activity. Using a malic enzyme specific RIA developed for this study, our results indicate that the insulin-mediated increase in malic enzyme activity was accompanied by a substantial increase in the quantity of enzyme, which paralleled the increase in enzyme activity. This clearly demonstrates that the modification of existing enzyme molecules is not involved in the control of malic enzyme activity by insulin.
314
h‘d W. Thompson, Richard L. Drake
These results suggest that insulin mediates an increase in malic enzyme synthesis in diabetic rats. However, since the level of an enzyme is dependent on the balance between synthesis and degradation, alterations in the rate of enzyme degradation can not be ruled out as contributing to the increased quantity of malic enzyme observed in this study. We did not determine turnover in this study; however previous investigations by Gibson et al. (1972) and Lyons and Gibson (1970, 1971), using refed rats which showed increased plasma insulin levels, demonstrated that initiation of enzyme synthesis, rather than decreased enzyme degradation, is the major contributing factor to the increased quantity of malic enzyme. Their findings support our conclusion that the increase in the quantity of malic enzyme observed in the present experiments is attributed to de novo synthesis of malic enzyme.
ACKNOWLEDGEMENTS The authors wish to thank Christine Kuepfer for her excellent technical assistance. This work was supported by grants from the American Diabetes Association and grant PCM-7822130 from the National Science Foundation.
REFERENCES Bergmeyer, H.U., Bernt, E., Schmidt, F., and Stork, H. (1974) in: Methods of Enzymatic Analysis (H.U. Bergmeyer, Ed.), Vol. 3, pp. 1196-1201. Gibson, D.M., Lyons, R.T., Scott, D.F., and Muto, Y. (1972) Adv. Enzyme Regulat. 10, 187204. Huijing, F. (1970) Clin. Chim. Acta 30,567-572. Kulkoski, J.A., Peterson, B.L., Elcombe, B., Winkelhake, J.L., and Ghazarian, J.G. (1979) FEBS Lett. 99, 183-188. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265275. Lyons, R.T., and Gibson, D.M. (1970) Fed. Proc. 29,901. Lyons, R.T., and Gibson, D.M. (1971) Fed. Proc. 30, 1080. McCormick, K.L., Widness, J.A., Susa, J.B., and Schwartz, R. (1978) Biochem. J. 172, 327331. Morgan, C.R., and Lazarow, A. (1965) Diabetes 14,669-671. Osborn, M.J., Felts, J.M., and Chaikoff, I.L. (1953) J. Biol. Chem. 203, 173-181. Shrago, E., Lardy, H.A., Nordlie, R.C., and Foster, D.O. (1963) J. Biol. Chem. 238, 31883192. Storey, J.M., and Bailey, E. (1978) Enzyme 23, 382-387. Thompson, E.W., Parks, W.C., and Drake, R.L. (1981) Am. J. Anat. 160,449-460. Towle, H.C., Maria& C.N., and Oppenheimer, J.H. (1980) Biochemistry 19, 579-585. Williams, C.A., and Chase, M.W. (1971) in: Methods in Immunology and Immunochemistry (C.A. Williams and M.W. Chase, Eds.), Vol. 3, p. 146. Winkelhake, J.L., Elcombe, B.M., and Hodach, A. (1979) Cancer Res. 39, 3058-3064. Wise, E.M., and Ball, E.G. (1964) Proc. Natl. Acad. Sci. (U.S.A.) 52, 1255-1263. Yeung, K.K., and Carrico, R.J. (1976) Anal. Biochem. 74, 369-375.