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Changes in hepatic lipogenic enzyme activities in voles and mice treated with monosodium aspartate
Research in Veterinary Science 1992, 53, 247-249
Changes in hepatic lipogenic enzyme activities in voles and mice treated with monosodium aspartate T. ARAI, Y. H A S E G A W A , Y. OKI, Department of Veterinary Biochemistry, Nippon Veterinary and Animal Science University, 1-7-1 Kyonaneho, Musashino, Tokyo 180, Japan
Changes in activities of hepatic lipogenic enzymes, ATP citrate lyase and acetyl-CoA carboxylase, were measured in voles and C57BL mice following neonatal administration of monosodium aspartate (MSA). Hepatic lipogenic enzyme activities in voles were considerably lower than those in mice; these low activities were considered to be one of the characteristics of voles as a herbivore. In the MSA-treated voles and mice, the plasma insulin concentrations increased significantly. The MSAtreated mice showed remarkable obesity and increased lipogenic enzyme activities. In the MSA-treated voles, signs of obesity were not observed and hepatic ATP citrate lyase activity increased significantly; acetyl-CoA carboxylase activity did not increase. HERBIVOROUS voles, Mierotus arvalis Pallas, have the unusual nutritional characteristic of gaining energy from volatile fatty acids or other organic acids produced in the digestive tract (Kudo and Oki 1982, 1984). Like ruminants, M arvalis also has low activities of hepatic glycolytic enzymes and a low renal threshold for glucose in parallel with a low blood glucose concentration (about 4 mM) unlike that of mice (Arai and Oki 1986, Arai et al 1989). Administration of monosodium glutamate (MSG), monosodium aspartate (MSA) or related substances to neonatal rodents has been reported to induce lesions of the ventromedial hypothalamus, resulting in a syndrome of obesity without hyperphagia (Olney 1970, Cameron et al 1978). Administration of MSA to neonatal voles induced glycosuria at a high percentage (Sasaki et al 1989). In the present study, activities of hepatic lipogenic enzymes, ATP citrate lyase and acetyl-CoA carboxylase, were investigated in voles and C57BL mice treated with MSA. M arvalis and C57BL/6J mice were maintained in the authors' laboratory. Male animals were used in the experiment. The voles were fed on commercial pellets for herbivores (zc; Oriental Yeast, Tokyo) and cubed hay and the mice were fed on commercial pellets (CMF; Oriental Yeast). In the experimental group, at one day old, the neonates were given monosodiumL-aspartate (Wako Pure Chemical Industries, Osaka) at 4 mg g-1 bodyweight. The untreated animals were
used as controls. Bodyweights and food intake were measured after four weeks of age. The animals were killed at 20 weeks by decapitation under ether anaesthesia. Blood samples were collected from the jugular veins into heparinised tubes and the livers removed immediately after decapitation. The glucose concentration in whole blood was measured by a glucoseoxidase method (Huggett and Nixon 1957) and the plasma insulin concentration was measured by a micro ELISA sandwich method (Arai et al 1989). The liver was homogenised in four volumes of homogenisation buffer containing 0.25 M sucrose and 20 m M TrisHC1 (pH 8.0) with a glass homogeniser. The homogenate was centrifuged at 15,000g for 30 minutes and then 100,000 g for 30 minutes at 4°C. The resulting supernatant was used as enzyme extract. ATP citrate lyase was assayed by coupling to malate dehydrogenase and following the decrease in absorbance at 340 nm (Takeda et al 1969). The activity was expressed as nmol Of substrate degraded per minute per milligram of protein in the extract. Acetyl CoA carboxylase activity was determined by the H14CO3 fixation assay (Nakanishi and N u m a 1970). The activity was expressed as nmol ofmalonyl-CoA formed per minute per milligram of protein. The protein concentration of the enzyme extract was determined by the method of Bradford (1976) using bovine serum albumin as the standard. The bodyweights (mean + SD) of six MSA-treated voles at 20 weeks old were 38.1 _+ 2.7 g, almost the same as those of controls (33.2 + 3.4 g, n=8). The bodyweights of six MSA-treated mice (42.9 + 5.4 g) significantly increased (P<0-01) compared to those of controls (32.8 + 1.8 g, n=8) at 20 weeks old. The MSAtreated mice showed remarkable obesity at 20 weeks of age. However, a significant difference in food intake was not observed between the MSA-treated animals and the control groups. The blood glucose and plasma insulin concentrations and hepatic lipogenic enzyme activities in voles and mice are shown in Table 1. The mean blood glucose concentrations and hepatic lipogenic enzyme activities in the control voles were very low compared to those in the control mice. In the MSA-treated voles, the blood glucose concentration
247
248
T. AraL Y. Hasegawa, Y. O k i TABLE 1 : Blood glucose and plasma insulin concentrations and hepatic lipogenic enzyme activities in voles and C57BL mice ~les
Blood glucose (mM) Plasma insulin (I.tU ml-1)
C57BLmice
Cont~l(8)
MSA-treated(6)
Control(8)
MSA-tre~ed(6)
4.2 ± 0.5 26.4 ± 6.0
5"6±0-7* 120"0±40'0"
8"3±0'6 21"3±3"1
9"9±1"3 98"7±23"5*
9.3±0-8 2.4±0-5
32.9±5.7* 4.6± 0.7*
Hepatic enzymes (nmol min -1 mg-1 protein) ATP citrate lyase 1-7 ± 0"2 AcetyI-CoA carbexylase 1-0 + 0.3
3.7±1.6" 1.2±0.4
* Significantly increased (P<0.01) compared with the control group (Student's t test) Values are expressed as means + SD. The numbers in brackets indicate the number of animals studied
increased significantly and the plasma insulin concentration increased to over four times that in the control group. The hepatic ATP citrate lyase activity increased to over twice that in the control group. The acetylC o A carboxylase activity did not increase significantly. In the MSA-treated mice, the blood glucose concentration did not increase, b u t the plasma insulin concentration increased to over four times that in the control group. The hepatic ATP citrate lyase activity increased approximately 3.5 times and acetyl-CoA carboxylase approximately 1.9 times higher than those in the control mice. It has been reported that MSA or MSG induced neuronal necrosis in several brain regions, including the arcuate and the ventromedial hypothalamic nuclei, and affected the secretion of hormones by increasing insulin release or decreasing growth hormone release. This resulted in hypothalamic obesity without hyperphagia in rodents (Cameron et al 1978, D a t a et al 1984, Higuchi et al 1989). The hypothesis that neural mediation of the rise in insulin is the primary factor in the development of hypothalamic obesity has been suggested (Inoue et al 1977, 1978). Activated secretion of insulin may cause development of obesity through the induction of hepatic enzymes involved in glycolysis, the pentose pathway and lipogenesis, which may result in active lipogenesis from glucose (Taketomi et al 1973). In MSA-treated voles and mice, hyperphagia was not observed, but there was a marked increase in plasma insulin concentration. It was interesting that the obese mice showed a greater increase of hepatic lipogenic enzyme activities than non-obese voles. The hepatic lipogenic enzyme activities in voles were very low as is the case in ruminants. Such low activities of hepatic lipogenic enzymes are considered to be one of the characteristics of voles as herbivores. In a previous study by the present authors, diabetes without obesity was induced after 30 weeks of age in over 50 per cent of voles treated with MSA (Sasaki et al 1989). In the present study, the b l o o d glucose c o n c e n t r a t i o n increased significantly even at 20 weeks old in the MSA-treated voles. In the MSA-treated mice, a marked obesity was observed but glycosuria was not. The dif-
ference in response to MSA-treatment between voles and mice was considered to be due to the greater difference in hepatic glycolytic and lipogenic enzyme activities. In ruminant species, the liver is much less important than the adipose tissue in lipogenesis, and the rate of fatty acid synthesis from glucose is very low although the rate from lactate is very high in ruminant adipose tissue (Prior 1978, Robertson et al 1982). The changes of lipogenic enzyme activities in adipose tissue and the rate of fatty acid synthesis from metabolites other than glucose should be further studied in voles. References ARAI, T. & OKI, Y (1986) Changes of glucokinase and hexokinase activities in the diabetic Microtus arvalis Pallas. Japanese Journal of Veterinary Science 48, 833-836 ARAI, T., MACHIDA, Y., SASAKI, M. & OKI, Y. (1989) Hepatic enzymeactivitiesand plasma insulin concentrations in diabeticherbivorous voles. Veterinary Research Communications 13, 421-426 BRADFORD, M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254 CAMERON, D. P., CUTBUSH, L. & OPAT, F. (1978) Effects of monosodium glutamate-induced obesity in mice on carbohydrate metabolism in insulin secretion. Clinical and Experimental Pharmacology and Physiology 5, 41-51 DATA, M. O., CAMPBELL, G. T. & BLAKE, C. A. (1984) Effects of neonatal administration of monosodium glutamate on somatotroph and growth hormone secretion in prepubertal male and female rats. Endocrinology 115, 996-1003 HIGUCHI, N., SASAKI, M., ARAI, T & OKI, Y. (1989) Effects of androgen on the onset of diabetes in the KK mice treated with monosodium aspartate. Experimental Animals (Tokyo) 38, 25-29 HUGGETT, A. G. & NIXON, D. A. (1957) Use of glucoseoxidase, peroxidase and o-dianisidinein determination of blood and urinary glucose. Lancet ii, 368-370 INOUE, S., BRAY, G. A. & MULLEN, Y. S. (1977) Effect of transplantation of pancreas on development of hypothalamic obesity. Nature 266, 742-744 INOUE, S., BRAY,G. A. & MULLEN, Y. S. (1978)Transplantation of pancreatic B-cellsprevents developmentofhypothalamic obesity in rats. American Journal of Physiology 235, E266-E271 KUDO, H. & OKI, Y. (1982) Breedingand rearing of Japanese field voles (Microtus montebelli Milne-Edwards) and Hungarian voles (Microtus arvalis Pallas) as new herbivorous laboratory animal species. Experimental Animals (Tokyo) 31, 175-183
L i p o g e n i c e n z y m e s in voles a n d m i c e KUDO, H. & OKI, Y. (1984) Microtus species as new herbivorous laboratory animals: Reproduction, bacterial flora and fermentation in the digestive tracts and nutritional physiology. Veterinary Research Communications 8, 77-91 NAKANISHI, S. & NUMA, S. (1970) Purification of rat liver acetyl Coenzyme A carboxylase and immunochemical studies on its synthesis and degradation. European Journal of Biochemistry 16, 161173 OLNEY, J. W. (1970) Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine. Nature 227, 609-610 PRIOR, R.L. (1978) Effect of level of feed intake on lactate and acetate metabolism and lipogenesis in vivo in sheep. Journal of Animal Nutrition 108, 926-935 ROBERTSON, J. P., FAULKNER, A. & VERNON, R. G. (1982) Regulation of glycolysis and fatty acid synthesis from glucose in
249
sheep adipose tissue. Biochemical Journal 206, 577-586 SASAKI, M., ARAI, T., MACHIDA, Y. & OKI, Y. (1989) Diabetic syndi'ome induced by monosodium aspartate administration in Microtus arvalis Pallas. Japanese Journal of Veterinary Science 51, 669-675 TAKEDA, Y., SUZUKI, F. & INOUE, H. (1969) ATP citrate lyase (citrate-cleavage enzyme). Methods in Enzymology. Volume 13. New York, Academic Press. pp 153-160 TAKETOMI, S., TSUDA, M., MATSUO, T., IWATSUKA, H. & SUZUOKI, Z. (1973) Alterations of hepatic enzyme activities in KK and yellow KK mice with various diabetic states. Hormone and Metabolic Research 5, 333-339
Received May 13, 1991 Accepted March 17, 1992
Changes in hepatic lipogenic enzyme activities in voles and mice treated with monosodium aspartate T. ARAI, Y. H A S E G A W A , Y. OKI, Department of Veterinary Biochemistry, Nippon Veterinary and Animal Science University, 1-7-1 Kyonaneho, Musashino, Tokyo 180, Japan
Changes in activities of hepatic lipogenic enzymes, ATP citrate lyase and acetyl-CoA carboxylase, were measured in voles and C57BL mice following neonatal administration of monosodium aspartate (MSA). Hepatic lipogenic enzyme activities in voles were considerably lower than those in mice; these low activities were considered to be one of the characteristics of voles as a herbivore. In the MSA-treated voles and mice, the plasma insulin concentrations increased significantly. The MSAtreated mice showed remarkable obesity and increased lipogenic enzyme activities. In the MSA-treated voles, signs of obesity were not observed and hepatic ATP citrate lyase activity increased significantly; acetyl-CoA carboxylase activity did not increase. HERBIVOROUS voles, Mierotus arvalis Pallas, have the unusual nutritional characteristic of gaining energy from volatile fatty acids or other organic acids produced in the digestive tract (Kudo and Oki 1982, 1984). Like ruminants, M arvalis also has low activities of hepatic glycolytic enzymes and a low renal threshold for glucose in parallel with a low blood glucose concentration (about 4 mM) unlike that of mice (Arai and Oki 1986, Arai et al 1989). Administration of monosodium glutamate (MSG), monosodium aspartate (MSA) or related substances to neonatal rodents has been reported to induce lesions of the ventromedial hypothalamus, resulting in a syndrome of obesity without hyperphagia (Olney 1970, Cameron et al 1978). Administration of MSA to neonatal voles induced glycosuria at a high percentage (Sasaki et al 1989). In the present study, activities of hepatic lipogenic enzymes, ATP citrate lyase and acetyl-CoA carboxylase, were investigated in voles and C57BL mice treated with MSA. M arvalis and C57BL/6J mice were maintained in the authors' laboratory. Male animals were used in the experiment. The voles were fed on commercial pellets for herbivores (zc; Oriental Yeast, Tokyo) and cubed hay and the mice were fed on commercial pellets (CMF; Oriental Yeast). In the experimental group, at one day old, the neonates were given monosodiumL-aspartate (Wako Pure Chemical Industries, Osaka) at 4 mg g-1 bodyweight. The untreated animals were
used as controls. Bodyweights and food intake were measured after four weeks of age. The animals were killed at 20 weeks by decapitation under ether anaesthesia. Blood samples were collected from the jugular veins into heparinised tubes and the livers removed immediately after decapitation. The glucose concentration in whole blood was measured by a glucoseoxidase method (Huggett and Nixon 1957) and the plasma insulin concentration was measured by a micro ELISA sandwich method (Arai et al 1989). The liver was homogenised in four volumes of homogenisation buffer containing 0.25 M sucrose and 20 m M TrisHC1 (pH 8.0) with a glass homogeniser. The homogenate was centrifuged at 15,000g for 30 minutes and then 100,000 g for 30 minutes at 4°C. The resulting supernatant was used as enzyme extract. ATP citrate lyase was assayed by coupling to malate dehydrogenase and following the decrease in absorbance at 340 nm (Takeda et al 1969). The activity was expressed as nmol Of substrate degraded per minute per milligram of protein in the extract. Acetyl CoA carboxylase activity was determined by the H14CO3 fixation assay (Nakanishi and N u m a 1970). The activity was expressed as nmol ofmalonyl-CoA formed per minute per milligram of protein. The protein concentration of the enzyme extract was determined by the method of Bradford (1976) using bovine serum albumin as the standard. The bodyweights (mean + SD) of six MSA-treated voles at 20 weeks old were 38.1 _+ 2.7 g, almost the same as those of controls (33.2 + 3.4 g, n=8). The bodyweights of six MSA-treated mice (42.9 + 5.4 g) significantly increased (P<0-01) compared to those of controls (32.8 + 1.8 g, n=8) at 20 weeks old. The MSAtreated mice showed remarkable obesity at 20 weeks of age. However, a significant difference in food intake was not observed between the MSA-treated animals and the control groups. The blood glucose and plasma insulin concentrations and hepatic lipogenic enzyme activities in voles and mice are shown in Table 1. The mean blood glucose concentrations and hepatic lipogenic enzyme activities in the control voles were very low compared to those in the control mice. In the MSA-treated voles, the blood glucose concentration
247
248
T. AraL Y. Hasegawa, Y. O k i TABLE 1 : Blood glucose and plasma insulin concentrations and hepatic lipogenic enzyme activities in voles and C57BL mice ~les
Blood glucose (mM) Plasma insulin (I.tU ml-1)
C57BLmice
Cont~l(8)
MSA-treated(6)
Control(8)
MSA-tre~ed(6)
4.2 ± 0.5 26.4 ± 6.0
5"6±0-7* 120"0±40'0"
8"3±0'6 21"3±3"1
9"9±1"3 98"7±23"5*
9.3±0-8 2.4±0-5
32.9±5.7* 4.6± 0.7*
Hepatic enzymes (nmol min -1 mg-1 protein) ATP citrate lyase 1-7 ± 0"2 AcetyI-CoA carbexylase 1-0 + 0.3
3.7±1.6" 1.2±0.4
* Significantly increased (P<0.01) compared with the control group (Student's t test) Values are expressed as means + SD. The numbers in brackets indicate the number of animals studied
increased significantly and the plasma insulin concentration increased to over four times that in the control group. The hepatic ATP citrate lyase activity increased to over twice that in the control group. The acetylC o A carboxylase activity did not increase significantly. In the MSA-treated mice, the blood glucose concentration did not increase, b u t the plasma insulin concentration increased to over four times that in the control group. The hepatic ATP citrate lyase activity increased approximately 3.5 times and acetyl-CoA carboxylase approximately 1.9 times higher than those in the control mice. It has been reported that MSA or MSG induced neuronal necrosis in several brain regions, including the arcuate and the ventromedial hypothalamic nuclei, and affected the secretion of hormones by increasing insulin release or decreasing growth hormone release. This resulted in hypothalamic obesity without hyperphagia in rodents (Cameron et al 1978, D a t a et al 1984, Higuchi et al 1989). The hypothesis that neural mediation of the rise in insulin is the primary factor in the development of hypothalamic obesity has been suggested (Inoue et al 1977, 1978). Activated secretion of insulin may cause development of obesity through the induction of hepatic enzymes involved in glycolysis, the pentose pathway and lipogenesis, which may result in active lipogenesis from glucose (Taketomi et al 1973). In MSA-treated voles and mice, hyperphagia was not observed, but there was a marked increase in plasma insulin concentration. It was interesting that the obese mice showed a greater increase of hepatic lipogenic enzyme activities than non-obese voles. The hepatic lipogenic enzyme activities in voles were very low as is the case in ruminants. Such low activities of hepatic lipogenic enzymes are considered to be one of the characteristics of voles as herbivores. In a previous study by the present authors, diabetes without obesity was induced after 30 weeks of age in over 50 per cent of voles treated with MSA (Sasaki et al 1989). In the present study, the b l o o d glucose c o n c e n t r a t i o n increased significantly even at 20 weeks old in the MSA-treated voles. In the MSA-treated mice, a marked obesity was observed but glycosuria was not. The dif-
ference in response to MSA-treatment between voles and mice was considered to be due to the greater difference in hepatic glycolytic and lipogenic enzyme activities. In ruminant species, the liver is much less important than the adipose tissue in lipogenesis, and the rate of fatty acid synthesis from glucose is very low although the rate from lactate is very high in ruminant adipose tissue (Prior 1978, Robertson et al 1982). The changes of lipogenic enzyme activities in adipose tissue and the rate of fatty acid synthesis from metabolites other than glucose should be further studied in voles. References ARAI, T. & OKI, Y (1986) Changes of glucokinase and hexokinase activities in the diabetic Microtus arvalis Pallas. Japanese Journal of Veterinary Science 48, 833-836 ARAI, T., MACHIDA, Y., SASAKI, M. & OKI, Y. (1989) Hepatic enzymeactivitiesand plasma insulin concentrations in diabeticherbivorous voles. Veterinary Research Communications 13, 421-426 BRADFORD, M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254 CAMERON, D. P., CUTBUSH, L. & OPAT, F. (1978) Effects of monosodium glutamate-induced obesity in mice on carbohydrate metabolism in insulin secretion. Clinical and Experimental Pharmacology and Physiology 5, 41-51 DATA, M. O., CAMPBELL, G. T. & BLAKE, C. A. (1984) Effects of neonatal administration of monosodium glutamate on somatotroph and growth hormone secretion in prepubertal male and female rats. Endocrinology 115, 996-1003 HIGUCHI, N., SASAKI, M., ARAI, T & OKI, Y. (1989) Effects of androgen on the onset of diabetes in the KK mice treated with monosodium aspartate. Experimental Animals (Tokyo) 38, 25-29 HUGGETT, A. G. & NIXON, D. A. (1957) Use of glucoseoxidase, peroxidase and o-dianisidinein determination of blood and urinary glucose. Lancet ii, 368-370 INOUE, S., BRAY, G. A. & MULLEN, Y. S. (1977) Effect of transplantation of pancreas on development of hypothalamic obesity. Nature 266, 742-744 INOUE, S., BRAY,G. A. & MULLEN, Y. S. (1978)Transplantation of pancreatic B-cellsprevents developmentofhypothalamic obesity in rats. American Journal of Physiology 235, E266-E271 KUDO, H. & OKI, Y. (1982) Breedingand rearing of Japanese field voles (Microtus montebelli Milne-Edwards) and Hungarian voles (Microtus arvalis Pallas) as new herbivorous laboratory animal species. Experimental Animals (Tokyo) 31, 175-183
L i p o g e n i c e n z y m e s in voles a n d m i c e KUDO, H. & OKI, Y. (1984) Microtus species as new herbivorous laboratory animals: Reproduction, bacterial flora and fermentation in the digestive tracts and nutritional physiology. Veterinary Research Communications 8, 77-91 NAKANISHI, S. & NUMA, S. (1970) Purification of rat liver acetyl Coenzyme A carboxylase and immunochemical studies on its synthesis and degradation. European Journal of Biochemistry 16, 161173 OLNEY, J. W. (1970) Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine. Nature 227, 609-610 PRIOR, R.L. (1978) Effect of level of feed intake on lactate and acetate metabolism and lipogenesis in vivo in sheep. Journal of Animal Nutrition 108, 926-935 ROBERTSON, J. P., FAULKNER, A. & VERNON, R. G. (1982) Regulation of glycolysis and fatty acid synthesis from glucose in
249
sheep adipose tissue. Biochemical Journal 206, 577-586 SASAKI, M., ARAI, T., MACHIDA, Y. & OKI, Y. (1989) Diabetic syndi'ome induced by monosodium aspartate administration in Microtus arvalis Pallas. Japanese Journal of Veterinary Science 51, 669-675 TAKEDA, Y., SUZUKI, F. & INOUE, H. (1969) ATP citrate lyase (citrate-cleavage enzyme). Methods in Enzymology. Volume 13. New York, Academic Press. pp 153-160 TAKETOMI, S., TSUDA, M., MATSUO, T., IWATSUKA, H. & SUZUOKI, Z. (1973) Alterations of hepatic enzyme activities in KK and yellow KK mice with various diabetic states. Hormone and Metabolic Research 5, 333-339
Received May 13, 1991 Accepted March 17, 1992