Dietary regulation of liver NADP-isocitrate dehydrogenase in the rat

Nuuition Research. Vol. 19, No. 2, pp. 247-255.1999 Copyright Q 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0271-5317/99/$-see front matter

ELSEVIER

PI1SO271-5317(98)00188-2

DIETARY REGULATION

OF LIVER NADP-ISOCITRATE INTHERAT

DEHYDROGENASE

Dr. Fanny Zirulnik and Dr. Maria Sofia Gimenez* Area Quimica Biologica, Facultad de Quimica, Bioquimica y Farmacia. Universidad National de San Luis 5700- San Luis, Argentina

ABSTRACT The objective of this work is to study the dietary regulation of cytosolic NADP- linked &citrate dehydrogenase (EC 1.1.1.4.2) in male rat liver. Seven different diets were prepared: l- AIN(control), 3501 kcal/g diet; 2- Soy , 3540 kcal/g diet; 3- Low protein, 3593 kcallg diet; 4- Dextrin, 4056 kcalfg diet; S- High- carbohydrate, 4101 kcaWg diet; 6- Fat, 4120 kcal/g diet and 7High- fat, 6521 kcallg diet. The enzymatic activities of isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase, as marker enzyme, were determined. The variation of some serum parameters as glucose, total cholesterol and triglycerides, was studied. The results showed that isocitrate dehydrogenase did not change significantly with different diets. while for glucose- 6- phosphate dehydrogenase, significant differences were observed for each diet respect to control. Glucose, cholesterol and triglycerides did not show any difference to diets 2 and 3, compared with diet 1. In hypercaloric diets, there was a significative increase (p< 0.001) in glucose and triglycerides, while with cholesterol a significative decrease was observed. Likewise, the response of isocitrate dehydrogenase to starvation during 48 hours and refeeding fat diet, was studied and the enzyme activity did not show any modification in relation to the control. We could conclude that isocitrate dehydrogenase should not be under nutritional regulation, whereas glucose-6-phosphate dehydrogenase. glucose, cholesterol and triglycerides, are regulated by the diet. B 1999 ELswier Science Inc. Key Words: Isocitrate Dehydrogenase, Diets, Enzymatic Activity

INTRODUCTION In the last decades there have seen many advances in the knowledge of the homeostatic

*Corresponding Author: Dra. Maria Sofia Gimenez Area Quimica Biolbgica, Universidad National de San Luis. Chacabuco y Pedernera. 5700- San Luis- Argentina. FAX: 54-652-30224- E-mail: [email protected] 247

248

F. ZIRULNIK and M.S. GIMENEZ

mechanisms in several types of mammalian cells, particularly, hepatic parenchymal cells, which can adapt to various environmental stimuli such as changes in the diet. These mechanisms which have been studied mainly in the adult animal, involve the control of enzyme activity, by dietary changes. The enzymatic activities and concentrations are influenced by diverse hormonal and nutritional conditions (1). For this reason diierent authors have studied the changes of enzymes with diverse treatments to identify which treatment have influences or control over the activities of enzymes. There are dietary proteins that modify the cholesterol metabolism in particular the soybean protein as compared to animal proteins such as casein, as Nagata et al. have observed the former produces hypocholesterolemia (2). On the other hand, dietary factors can indeed alter the rate of lipogenic enzymes synthesis, which make cells to produce fat Ii-om the hepatic carbohydrates (3). Tomlinson et al. have studied the effect of dietary fat and sucrose on the activities of several rat hepatic enzymes, suggesting that fat diets induce a lower enzyme activity while sucrose diets induce higher enzyme activities (4). They also suggested that dietary carbohydrates specifically affect enzymes involved in carbohydrate metabolism (piruvate kinase, malate dehydrogenase, glucose6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase), whereas dietary fat does not. Moreover, hyperlipogenesis is a response to refeeding aher starvation (5). Other adaptive changes during refeeding, as an increase in the activity of enzymes of the monophosphate hexose shunt , occur (6)(7). Our goal was to investigate if cytosolic NADP- isocitrate dehydrogenase (ICD) in the liver of male rat is affected by diet.

MATERIALS

AND METHODS

To have a complete view of ICD behavior under different diets, we prepared hypo and hyperproteic diets; high carbohydrate and high fat diets modifying the metabolizable energy (kcaVg diet). Glucose-6-phosphate dehydrogenase (G6PD) was taken as marker enzyme because there is a lot of information about its nutritional regulation. Seven diets were prepared by modifying the AINcontrol diet (The American Institute of Nutrition Rodent Diets) for maintenance of adult rats (8). They were formulated to contain three different levels of calories: normal (diet 1, 2 and 3); medium (diet 4, 5 and 6) and high (diet 7) (Table 1). In Soybean flour was determined nitrogen by KjeldahI- Gunning- Dyer (9), presenting: proteins: 38%, lipids by ethereal extract: 19.21%, humidity: 7.046%, fiber: 5.7, ashes: 4.69, total carbohydrates (by difference): 25.354. Filly six Wistar adult male rats weighing between 150- 200g were obtained from the Biotherio at the University of San Luis. Animals were divided into 7 groups of 8 rats in each group, in such a manner that the mean body weight per group was similar. All the animals received 1 week of the AINdiet and then the different diets were supplied during 10 weeks. Body weight and the amount of food consumed by the animals were

NADP-ISOCITRATE

DEHYDROGENASE

249

(12h light: 12h dark cycle), in an air conditioned room (22” to 23°C). Fresh diets were given and let? over food discarded on a daily basis, (20g diet were adequated to ensure ad lib feeding). Water was provided ad libitum.

TABLE 1 COMPOSITION

OF THE EXPERIMENTAL

I

Diets (kcal/ g diet) Ingredients Cornstarch Casein Soybean Dextrin sucrose soybean oil Fiber Mineral Mix Vitamin mix L- Cystine Choline bitartrate Ascorbic acid Fat

DIETS

320.572 245.120 190 100 45 50 35 10 1.8 2.5 0.008

204

(4101)

204

( (6521)

200 405.692

65 1.692 45 50 35 IO 1.8 2.5 0.008

1 (4120)

65 1.692 45 50 35 IO 1.8 2.5 0.008

45 50 35 IO 1.8 2.5 0.008 250

Another experiment on starvation and refeeding was performed. A group of 8 animals was starved 48 hours. Another group of 8 animals received fat diet (diet 6) during 48 hours after starvation. The initial and final weights were recorded in all animals, after 2 days, 4 days respectively, for starved rats and refed rats. The animals of both groups were sacrificed after treatment by decapitation at 8:00 AM. Livers were removed, placed in cold physiologic solution, blotted, weighted and 4 g pieces were homogenized with 9 ml of 0.5 M TRIS-HCl buffer, pH 7.4, containing 1mM dithiotreitol (DTT). Liver homogenates were centrifuged at 100,000 x g for 1 hour in a Beckman LS 65 ultracentrifuge and enzymatic activities were measured in the supernatant. ICD and G6PD activities were determined by the rate of NADPH formation at 340nm in a Shimadzu spectrophotometer, according to Farrell, H.M. (10) and Glock. G E (1 l), respectively. The results were expressed as pmol NADP/rnin/mg of protein. Protein concentration was measured by the Biuret reaction (12). Serum determinations of glucose. cholesterol and triglycerides were carried out according to enzymatic method (13). quantitative method of glucose oxidase/ peroxidase (14) and calorimetric method with lipase (15) respectively. Statistical analysis: values are expressed as mean f standard deviation. Significant differences were considered at p< 0.05, as determined by one-way ANOVA test, followed by Duncan’s multiple range test.

250

F. ZIRULNIK and M.S. GIMl?NEZ RESULTS Comparing weight gain among the 7 groups, it was observed that the animals treated with diets 3. 4, 5 and 6 had lower weight gain (~~0.05) and those with diets 1 and 2 were similar to group 7. Comparing liver weight, there was no statistical difference between groups but animals fed diet 7 showed a trend of decreasing (Table 2). TABLE 2 WEIGHT VARIATIONS

IN MALE RAT

Data are presented as mean *SD, for 8 rats in each group. *p< 0.050, ANOVA test- 1 way, compared to diet 1. None of the normal, high protein, low protein, high sucrose or high fat diets modified the ICD activity (Figure 1). In contrast, G6PD showed signifkant differences (p
NADP-ISOCITRATE

2.5

DEHYDROGENASE

B

m

Al N-93 Soy Low protein Dextrin

c---l Sucrose UIIII Fat EE&YHigh fat

ii

ICD

GGPD

FIG. 1: NADP-isocitrate dehydrogenase and glucosed-phosphate dehydrogenase specific activities in liver of male rat. n=8; *p
TABLE 3 EFFECT OF DIFFERENT

DIETS ON SERUM GLUCOSE. CHOLESTEROL AND TRIGLYCERIDES Diets 1 Glucose (g/l) 1 cholesterol (g/l) triglycerides (g/l) I- AINI 0.917+0.10 I 0.59s 0.17 0.350+ 0.09 0.867* 0.10 0.598+ 0.13 2- soy 0.261* 0.03 0.850+ 0.10 3- Low protein 0.650+ 0.11 0.285* 0.06 4- Dextrin 1.880+ O.ll* 0.440-+ 0.05* 0.994% 0.15* 5- High sucrose 1.43& 0.26*a 0.41(H 0.17* 1.775* 0.31*b 6- Fat 1.39W 0.26* a 0.38Ok 0.06* 1.04& 0.16* 7- High Fat 1 1.5oO=to.15*” ( 0.470+ 0.10* 1 1.174* 0.17* Data are presented as mean f SD, for 8 rats in each group. *p
F. ZIRULNIK and M.S. GIMENEZ

252

Starvation and refeeding moditied body weight (Table 4). In the group of starving rats a decrease of 13.26% was seen while in the starvation and refeeding with 6- fat diet group a 19.89% decrease was observed. In these rats the ICD activity was not changed, but the G6PD activity decreased atler refeeding (Table 4).

TABLE 4 VARIATION

OF BODY AND LIVER WEIGHT IN STARVED AND REFED ANIMALS. ISOCITRATE DEHYDROGENASE AND GLUCOSEd-PHOSPHATE DEHYDROGENASE ACTIVITIES

lStarved rats Body Weight (8) Initial 354.75+ 19.3 307.25+ 18.6 Final 9.87* 1.6 Liver Weight Ipmol NADP /mid mg prot) Enzyme Activity in liver 35.05+ 7.6 ICD G6PD 19.5& 1.4 ll.O& 1.05* 1ata are represented by mean f SD, for 6 rats in each case. *p
!

DISCUSSION Changes in enzyme activity are the result of either alterations in the total amount of enzyme present in the liver, or activation/ inactivation of preexisting enzyme molecules. Numerous organisms are able to alter the concentration of enzymes in response to changes in the diet (4). One example is the abiity of mammals to dramatically alter the concentration of enzymes which synthesize fat from carbohydrate in liver. It is not clear whether diet constituents or some of their metabolic products regulate the rate of the enzymes synthesis directly or indirectly via some intermediate such as hormones or cyclic adenosine monophosphate (CAMP). The concentration of lipogenic enzymes increases when rats are fed fat tree- high carbohydrate diet and decreases when unsaturated fatty acids are given, or in the presence of CAMP (3, 5). Katsurada et al. (16) studied the effects of diet nutrients on substrate and effector levels of lipogenic enzymes and they observed that hepatic fatty acid synthesis is reduced in animals fasted or fed with fat, and increased in those refed with a fat free- high carbohydrate diet. Although the activities of lipogenic enzymes rise or fall coordinately in response to the nutritional variations, our attention has been focused on ICD, which plays a role as a NADPH generator system in hepatocytes cytosol. Starvation and refeeding result in a decrease of the rats body weight, probably due to the time of treatment or the quality of the diet. Our results show that ICD does not change with different diets or alter starvation and refeeding with fat diet. We observed

NADP-ISOCITRATE

DEHYDROGENASE

significant changes between the 6 different groups of rats and the control group for G6PD. The results obtained for this enzyme are consistent with those obtained by Katsurada (16), who observed a lower enzymatic induction when diet was high in fat; Peavy (17), Gimenez (18), MC Donald (19), &soon (20) and Mazier (21), have also concluded that high sucrose diets increased G6PD specific activity. Iritani (22, 23) and Potter (24), showed that the activities of enzymes as G6PD, malic enzyme, acetyl- CoA carboxylase and fatty acid synthetase were markedly lower in animals fed soybean protein or gluten than those fed casein or fish protein. Hyperlipogenesis is a frequently reported response to realimentation following food deprivation. It has been reported that when starved adult rats were refed with either high carbohydrate or high protein diets, one third of the total weight gain was deposited as fat (6). It would seem that ICD did not supply the NADPH necessary for fasted state or for high carbohydrate/ fat diet, with different energy value, because enzyme activity did not alter. However, it could be suggested that ICD might supply NADPH for redox reactions in detoxification processes by means of the cytocrome P-450- dependent monooxygenase system (25). In relation to intoxication with metals such as cadmium, which increases the peroxydation process, an increase of ICD but not for G6PD activity was observed (26). And our results suggest that such a mechanism could be an indicator that ICD catalyzes NADPH production for reduction of reactive- oxygen substances (ROS), but not for lipid and carbohydrate metabolism. In relation to serum parameters, we can conclude that soy diet or low protein diet did not produce any changes in serum glucose, cholesterol or triglyceride levels. This lack of change may be due to the time of treatment. When hypercaloric diets containing either high sucrose, or fat were supplied, the increment of glucose and triglyceride values might be due to the excess of calorie horn food, metabolic transformation or fat deposition (19). Our experiments revealed that ICD is not under nutritional regulation, whereas G6PD, glucose, cholesterol and triglyceride are influenced by the level of fat- carbohydrate calories in the diet.

ACKNOWLEDGEMENTS Authors are grateful to Dra. Liliana Oliveros for her valuable advice and discussions. Our thanks to Lit. Silvia Fernandez, for make nitrogen determinations of soybean flour and to Lit. Ana C. Anzulovich, Lit. Maria Rosa Femandez and Mr. Dominguez for their technical assistance.This work was supported by a grant from CONICET- 0724 and National University of San Luis, Project 8 104.

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Accepted

for

publication

July

21,

1998.

following chronic

effect of