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Hormonal response of glycolytic key enzymes of erythrocytes in insulinoma
Hormonal Response of Glycolytic Key Enzymes of Erythrocytes in Insulinoma By
H. KIMURA,N. HORIUCHI,T. KITAMURA,ANDK. MORITA
Activities of glycolytic key enzymes in erythrocytes were measured in diabetics and in insulinoma prior to and after the extirpation of the tumor. In insulinoma the enzyme activities were found higher than those in normal subjects but returned to the normal levels after the op-
e&ion, while in diabetics the activities often lowered from the normal levels. The present data suggest that the glycolytic key enzymes in erythrocytic cells could be under hormonal control of insulht.
I
T IS WELL KNOWN that insulin regulates the glycolytic key enzymes in various animal tissues.1-4 On the other hand, erythrocytes have long been considered insensitive to insulin. Although it was recently reported that insulin injection resulted in the increase of pyruvate and lactate in erythrocytes with the simultaneous decrease of inorganic phosphate,5 little is known whether the glycolytic key enzymes in erythrocytic cells are regulated by insulin or not. The present paper deals with the levels of glycolytic key enzymes in insulinoma and diabetics, indicating that the enzymes in erythrocytic cells could be under hormonal control of insulin. MATERIALSAND METHODS Erythrocytes were separated by centrifugation at 900 g for 10 min at 0°C from heparinized blood. To minimize the contamination of leukocytes erythrocytes were twice washed by 0.15 M KCl, and the buffy coat obtained after the centrifugation was thoroughly removed. The washed erythrocytes were suspended for 10 min in an equal volume of cold distilled water. The resultant hemolysate was mixed with one-tenth volume of 1.5 M KC1 containing 5 mM ,8-mercaptoethanol and centrifuged at 23,000 g for 20 min at 0°C. The supernatant was used for assay of the enzyme activities. Hexokinase was measured by the method of Salas et al.6 Phosphofructokinase was assayed by the method of Mansour;’ pyruvate kinase by the method of Tanaka et al.;4 glucose-(j-phosphate dehydrogenase by the method of Kornberg and Horecker.8
RESULTS AND DISCUSSION The serum levels of immunoreactive insulin in insulinoma prior tion fluctuated from 16 to over 200 @/ml in a day, while blood fairly constant and was 50 mg/lOO ml. No blood transfusion was or after the operation. At the third day after the extirpation of the
to the sugar given tumor,
operastayed during blood
From the Department of Biochemistry and Section of Hepatic and Pancreatic Disease, Department of Internal Medicine, The Center for Adult Diseases, Osaka, Japan. Received for publication June 25, 1971. H. KIMURA, M.D.: Instructor, Department of Biochemistry, The Center for Adult Diseases, Osaka, Japan. N. HORIUCHI, M.D.: Chief, Section of Hepatic Disease, The Center for Adult Diseases, Osaka, Japan. T. KITAMURA, M.D.: Chief, Section of Pancreatic Disease, The Center for Adult Diseases, Osaka, Japan. K. MORITA, B.S.: Research Associate, Department of Biochemistry, The Center for Adult Diseases, Osaka, Japan. METABOLISM, VOL. 20, No.
12 (DECEMBER),
1971
1119
1120
KIMURA
Table 1 .-Changes
in Activities of Glycolytic Enzymes Insulmoma and Diabetic
Insulinoma Prior to 47th Day After
Operation
Hexokinase Phosphofructokinase Glucose&-phosphate dehydrogenase Pyruvate kinase Normal values parentheses.
of Erytbrocytes
Diabetics (Vmoles/min per ms protein)
ET AL.
in
Normal Subjects
Operation
Case. 1
Case2
Case3
Case4
0.66 1.58
0.27
0.00 0.13
0.00 0.00
0.19 0.32
0.17 0.20
O-19+-0.06 (6) 0.42rtO.16 (6)
6.33 28.0
1.00 1.40
0.26 0.65
0.22 0.07
1.35 1.29
0.14 2.22
1.68kO.71 2.69r0.92
are expressed
5 SD. Number
of normal
subjects examined
(6) (6)
is given in
sugar returned to the normal level, Table 1 illustrates activities of glycolytic key enzymes and glucosed-phosphate dehydrogenase of erythrocytes in insulinoma as well as in diabetics. The enzyme activities in insulinoma were higher than those of the normal subjects. At the 47th day after the operation they returned to the levels of normal subjects. On the contrary, in diabetics, the enzyme activities often showed lower than in the normal levels as demonstrated in the same table. These facts indicate that insulin acted on erythrocytic cells directly or indirectly and increased the activities of glycolytic key enzymes regulating the glucose metabolism in the cells. Since peripheral erythrocytes are destroyed in about 120 days and are newly produced in and supplied from the bone marrow,g the possible site of the insulin action may either be the peripheral erythrocytes or the bone marrow. If insulin acts on the erythrocytes in peripheral blood, insulin should activate the enzymes in order to increase the levels of enzymes, since no protein synthesis occurs in the cells. No such activation by transformation induced by insulin, however, of glycolytic key enzymes has clearly been demonstrated yet, except that of hexokinase in muscle and heart from IIb to 1Ia.l In an alternative possibility where insulin acts on erythropoietic cells, the increased content of the enzymes in peripheral erythrocytes could be the result of the increased synthesis of the enzymes in the bone marrow. Although we did not determine whether insulin specifically accelerated the synthesis of glycolytic enzymes under study or nonspecifically stimulated the general protein synthesis, the marked increase of the specific activity of glycolytic enzymes on the basis of the total amount of protein extracted as demonstrated in Table 1 may well indicate rather selective function of the hormone on the synthesis of glycolytic enzymes. If insulin could promote general protein synthesis at a similar rate, no considerable increase of specific activity of the enzymes would have been observed. We, at present, rather suppose that insulin could act on erythropoietic cells in the bone marrow resulting in induction of the enzymes rather than on peripheral erythrocytes causing activation of the enzymes. If insulin acts only on erythropoietic cells, the peripheral erythrocytes would have to be completely replaced to normalize the enzyme levels after the removal of the tumor. Thus the decay curve of the enzyme levels in erythrocytes after the operation should coincide with the turnover rate of peripheral erythrocytes. The present result that the enzyme levels returned to the normal levels at or before 47 days after the extirpation of the tumor seems earlier than that we can
1121
GLYCOLYTIC KEY ENZYMES OF ERYTHROCYTES
estimate from the half life of the peripheral erythrocytes. Since we failed to obtain more samples to get a precise decay curve of the enzyme levels within days after operation, this should be reexamined with the other cases of insulinoma. Since the enzymes determined in the present study may not simply be controlled only by insulin, but may be regulated by the other hormones or by ionic or metabolic factors that could be changed in response to levels of insulin, the increase of glycolytic enzymes in the erythrocytes here reported may have resulted by an indirect effect of insulin that remains to be solved. It is also now under study by using tissue-cultured erythrocytes whether insulin may act directly on peripheral erythrocytes to activate the glycolytic key enzymes. ACKNOWLEDGMENT The authors thank Dr. H. Akedo, Department of Biochemistry, Diseases, Osaka, for his valuable advice and encouragement.
The Center
for Adult
REFERENCES 1. Katzen, H. M.: The multiple forms of mammalian hexokinase and their significance to the action of insulin. In Weber, G. (Ed.) : Advances in Enzyme Regulation, Vol. 5. New York, Macmillan, 1967. 2. -, Soderman, D. D., and Wiley, C. E.: Multiple forms of hexokinase activities associated with subcellular particulate and soluble fractions of normal and streptozotocin diabetic rat tissues. J. Biol. Chem. 245:4081, 1970. 3. Underwood, A. H., and Newsholme, E. A.: Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis. Biochem. J. 95:868, 1965. 4. Tanaka, T., Harano, Y., and Morimura (Kimura), H.: Crystallization, characterization, and metabolic regulation of two types
of pyruvate kinase isolated from rat tissues. J. Biochem. (Tokyo) 62:71, 1967. 5. Zurukzoglu, W.: The where and how of insulin. Lancet 11:746, 1966. 6. Salas, M., Vinuela, E., and Sols, A.: Insulin-dependent synthesis of liver glucokinase in the rat. J. Biol. Chem. 238:3535, 1963. 7. Mansour, T. E.: Studies on heart phosphofructokinase: purification, inhibition, and activation. J. Biol. Chem. 238:2285, 1963. 8. Kornberg, A., and Horecker, B. L.: Glucose-6-phosphate dehydrogenase. In Colowick, S. P., and Kaplan, N. 0. (Eds.) : Methods in Enzymology, Vol. 1. New York, Academic, 1955. 9. Rittenberg, S. D.: Life span of human red blood cell. J. Biol. Chem. 166:627, 1946.
H. KIMURA,N. HORIUCHI,T. KITAMURA,ANDK. MORITA
Activities of glycolytic key enzymes in erythrocytes were measured in diabetics and in insulinoma prior to and after the extirpation of the tumor. In insulinoma the enzyme activities were found higher than those in normal subjects but returned to the normal levels after the op-
e&ion, while in diabetics the activities often lowered from the normal levels. The present data suggest that the glycolytic key enzymes in erythrocytic cells could be under hormonal control of insulht.
I
T IS WELL KNOWN that insulin regulates the glycolytic key enzymes in various animal tissues.1-4 On the other hand, erythrocytes have long been considered insensitive to insulin. Although it was recently reported that insulin injection resulted in the increase of pyruvate and lactate in erythrocytes with the simultaneous decrease of inorganic phosphate,5 little is known whether the glycolytic key enzymes in erythrocytic cells are regulated by insulin or not. The present paper deals with the levels of glycolytic key enzymes in insulinoma and diabetics, indicating that the enzymes in erythrocytic cells could be under hormonal control of insulin. MATERIALSAND METHODS Erythrocytes were separated by centrifugation at 900 g for 10 min at 0°C from heparinized blood. To minimize the contamination of leukocytes erythrocytes were twice washed by 0.15 M KCl, and the buffy coat obtained after the centrifugation was thoroughly removed. The washed erythrocytes were suspended for 10 min in an equal volume of cold distilled water. The resultant hemolysate was mixed with one-tenth volume of 1.5 M KC1 containing 5 mM ,8-mercaptoethanol and centrifuged at 23,000 g for 20 min at 0°C. The supernatant was used for assay of the enzyme activities. Hexokinase was measured by the method of Salas et al.6 Phosphofructokinase was assayed by the method of Mansour;’ pyruvate kinase by the method of Tanaka et al.;4 glucose-(j-phosphate dehydrogenase by the method of Kornberg and Horecker.8
RESULTS AND DISCUSSION The serum levels of immunoreactive insulin in insulinoma prior tion fluctuated from 16 to over 200 @/ml in a day, while blood fairly constant and was 50 mg/lOO ml. No blood transfusion was or after the operation. At the third day after the extirpation of the
to the sugar given tumor,
operastayed during blood
From the Department of Biochemistry and Section of Hepatic and Pancreatic Disease, Department of Internal Medicine, The Center for Adult Diseases, Osaka, Japan. Received for publication June 25, 1971. H. KIMURA, M.D.: Instructor, Department of Biochemistry, The Center for Adult Diseases, Osaka, Japan. N. HORIUCHI, M.D.: Chief, Section of Hepatic Disease, The Center for Adult Diseases, Osaka, Japan. T. KITAMURA, M.D.: Chief, Section of Pancreatic Disease, The Center for Adult Diseases, Osaka, Japan. K. MORITA, B.S.: Research Associate, Department of Biochemistry, The Center for Adult Diseases, Osaka, Japan. METABOLISM, VOL. 20, No.
12 (DECEMBER),
1971
1119
1120
KIMURA
Table 1 .-Changes
in Activities of Glycolytic Enzymes Insulmoma and Diabetic
Insulinoma Prior to 47th Day After
Operation
Hexokinase Phosphofructokinase Glucose&-phosphate dehydrogenase Pyruvate kinase Normal values parentheses.
of Erytbrocytes
Diabetics (Vmoles/min per ms protein)
ET AL.
in
Normal Subjects
Operation
Case. 1
Case2
Case3
Case4
0.66 1.58
0.27
0.00 0.13
0.00 0.00
0.19 0.32
0.17 0.20
O-19+-0.06 (6) 0.42rtO.16 (6)
6.33 28.0
1.00 1.40
0.26 0.65
0.22 0.07
1.35 1.29
0.14 2.22
1.68kO.71 2.69r0.92
are expressed
5 SD. Number
of normal
subjects examined
(6) (6)
is given in
sugar returned to the normal level, Table 1 illustrates activities of glycolytic key enzymes and glucosed-phosphate dehydrogenase of erythrocytes in insulinoma as well as in diabetics. The enzyme activities in insulinoma were higher than those of the normal subjects. At the 47th day after the operation they returned to the levels of normal subjects. On the contrary, in diabetics, the enzyme activities often showed lower than in the normal levels as demonstrated in the same table. These facts indicate that insulin acted on erythrocytic cells directly or indirectly and increased the activities of glycolytic key enzymes regulating the glucose metabolism in the cells. Since peripheral erythrocytes are destroyed in about 120 days and are newly produced in and supplied from the bone marrow,g the possible site of the insulin action may either be the peripheral erythrocytes or the bone marrow. If insulin acts on the erythrocytes in peripheral blood, insulin should activate the enzymes in order to increase the levels of enzymes, since no protein synthesis occurs in the cells. No such activation by transformation induced by insulin, however, of glycolytic key enzymes has clearly been demonstrated yet, except that of hexokinase in muscle and heart from IIb to 1Ia.l In an alternative possibility where insulin acts on erythropoietic cells, the increased content of the enzymes in peripheral erythrocytes could be the result of the increased synthesis of the enzymes in the bone marrow. Although we did not determine whether insulin specifically accelerated the synthesis of glycolytic enzymes under study or nonspecifically stimulated the general protein synthesis, the marked increase of the specific activity of glycolytic enzymes on the basis of the total amount of protein extracted as demonstrated in Table 1 may well indicate rather selective function of the hormone on the synthesis of glycolytic enzymes. If insulin could promote general protein synthesis at a similar rate, no considerable increase of specific activity of the enzymes would have been observed. We, at present, rather suppose that insulin could act on erythropoietic cells in the bone marrow resulting in induction of the enzymes rather than on peripheral erythrocytes causing activation of the enzymes. If insulin acts only on erythropoietic cells, the peripheral erythrocytes would have to be completely replaced to normalize the enzyme levels after the removal of the tumor. Thus the decay curve of the enzyme levels in erythrocytes after the operation should coincide with the turnover rate of peripheral erythrocytes. The present result that the enzyme levels returned to the normal levels at or before 47 days after the extirpation of the tumor seems earlier than that we can
1121
GLYCOLYTIC KEY ENZYMES OF ERYTHROCYTES
estimate from the half life of the peripheral erythrocytes. Since we failed to obtain more samples to get a precise decay curve of the enzyme levels within days after operation, this should be reexamined with the other cases of insulinoma. Since the enzymes determined in the present study may not simply be controlled only by insulin, but may be regulated by the other hormones or by ionic or metabolic factors that could be changed in response to levels of insulin, the increase of glycolytic enzymes in the erythrocytes here reported may have resulted by an indirect effect of insulin that remains to be solved. It is also now under study by using tissue-cultured erythrocytes whether insulin may act directly on peripheral erythrocytes to activate the glycolytic key enzymes. ACKNOWLEDGMENT The authors thank Dr. H. Akedo, Department of Biochemistry, Diseases, Osaka, for his valuable advice and encouragement.
The Center
for Adult
REFERENCES 1. Katzen, H. M.: The multiple forms of mammalian hexokinase and their significance to the action of insulin. In Weber, G. (Ed.) : Advances in Enzyme Regulation, Vol. 5. New York, Macmillan, 1967. 2. -, Soderman, D. D., and Wiley, C. E.: Multiple forms of hexokinase activities associated with subcellular particulate and soluble fractions of normal and streptozotocin diabetic rat tissues. J. Biol. Chem. 245:4081, 1970. 3. Underwood, A. H., and Newsholme, E. A.: Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis. Biochem. J. 95:868, 1965. 4. Tanaka, T., Harano, Y., and Morimura (Kimura), H.: Crystallization, characterization, and metabolic regulation of two types
of pyruvate kinase isolated from rat tissues. J. Biochem. (Tokyo) 62:71, 1967. 5. Zurukzoglu, W.: The where and how of insulin. Lancet 11:746, 1966. 6. Salas, M., Vinuela, E., and Sols, A.: Insulin-dependent synthesis of liver glucokinase in the rat. J. Biol. Chem. 238:3535, 1963. 7. Mansour, T. E.: Studies on heart phosphofructokinase: purification, inhibition, and activation. J. Biol. Chem. 238:2285, 1963. 8. Kornberg, A., and Horecker, B. L.: Glucose-6-phosphate dehydrogenase. In Colowick, S. P., and Kaplan, N. 0. (Eds.) : Methods in Enzymology, Vol. 1. New York, Academic, 1955. 9. Rittenberg, S. D.: Life span of human red blood cell. J. Biol. Chem. 166:627, 1946.