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Comparative studies on lipogenic enzyme activities in the liver of human and some animal species
Comp. Biochem. PhysioL Vol. 95B, No. 3, pp. 469-472, 1990
0305-0491/90 $3.00 + 0.00 Pergamon Press plc
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COMPARATIVE STUDIES ON LIPOGENIC ENZYME ACTIVITIES IN THE LIVER OF H U M A N A N D SOME ANIMAL SPECIES MATEUSZ Z,ELEWSKI and JULIAN SWIERCZYI~SKI Department of Biochemistry, Academic Medical School, ul.Dc;binki 1, 80-211 Gdafisk, Poland (Received 29 March 1989)
Abstract--1. The activities of enzymes involved in fatty acid synthesis in the human liver (sample taken during abdominal surgery) and in the livers of some animals were studied. 2. Fatty acid synthase, ATP-citrate lyase and malic enzyme activities were found to be from 4 to 70-fold lower in human liver than in rat or bird livers. 3. The activities of hexose monophosphate shunt dehydrogenases in human liver were from half to almost equal to the corresponding activities in birds, but much lower than in rat liver. 4. The activities of all enzymes listed above in human and beef liver were very similar (except fatty acid synthase which was undetectable in the beef liver). 5. Very high activity of NADP-linked isocitrate dehydrogenase was found in livers of all species tested. 6. These results are discussed in relation to the role of the human liver in lipogenesis. 7. The activities of the enzymes generating NADPH in human liver taken during abdominal surgery were similar to the activities observed in the tissue obtained post mortem. 8. This suggested that post mortem tissue may be used as a reliable human material for some enzyme assays. 9. Thus we also examined the activity of malic enzyme in post mortem human kidney cortex, heart, skeletal muscle and brain. I0 Relatively high activity of NADP-linked malic enzyme has been observed in human brain.
of these data and the results published previously by Bray (1972) indicating that human adipose tissue may play a considerable role in lipogenesis, it remains to be elucidated how important is the contribution of liver in human lipogenesis.
INTRODUCTION
Although significant amounts of fatty acids are known to be synthesized both in the liver and white adipose tissue of several animal species, until recently it has been difficult to establish the contribution to the total synthesis in vivo of each of these tissues. Previous studies by Goodridge and Ball (1966) and Leville et al. (1968) have shown that birds do not synthesize fatty acids de novo in white adipose tissue. They also indicated that fatty acids are synthesized in the liver and then transported to the adipose tissue for storage as triacylglycerols. Shrago and coworkers (1966, 1967, 1971) have shown that human adipose tissue also does not synthesize fatty acids at an appreciable rate and that ATP-citrate lyase, a key enzyme in the pathway of fatty acid synthesis, is present in negligible amounts in this tissue. They concluded that triacylglycerols in human adipose tissue are formed from free fatty acids synthesized in liver and carried to the adipose tissue with blood. This indicates that birds and humans are similar to each other but different from rats as far as fatty acid synthesis in the liver and adipose tissue is concerned. Since the activity of lipogenic enzymes in avian liver is relatively high (Goodridge and Ball, 1966; Brdiczka and Pette, 1970), much higher than in rats (Brdiczka and Pette, 1970) one can expect that human liver would also display high activity of lipogenic enzymes. Surprisingly, previous studies by Shrago et al. (1966) have shown that the activities of glucose 6-phosphate dehydrogenase, malic enzyme and ATP-citrate lyase are essentially the same in humans and rats. In view
MATERIALS AND METHODS
Chemicals
Substrates for malic enzyme, hexose monophosphate shunt dehydrogenases, NADP-linked isocitrate dehydrogenase, ATP-citrate lyase and fatty acid synthase were obtained from Sigma Chemical Co. (St Louis, USA). Triton X-100 was from Serva Feinbiochemica Heidelberg (FRG). All other chemicals were of highest purity available commercially from P.O.Ch. Gliwice (Poland). Human samples
Samples of human liver were taken during abdominal surgery from patients of both sexes aged 3 to 61 years. In all cases liver sampling was the routine practice performed for the diagnostic purposes. Experiments in which liver samples from patients were used have been approved by the local Ethic Commission for the Research on Human Subjects and are in accordance with Polish law. The patients were fasted 12 hr before the surgery. Neither the type of anasthesia nor preoperative medication were controlled. None of the patients received treatment with hormones, which would be likely to influence the enzyme activity levels. An attempt was also made to exclude patients who may have been in severe nutritional imbalance. Samples of human liver, skeletal muscle, heart, kidney and brain were taken from the autopsy made in the Department of Forensic Medicine, Medical School in Gdafisk not later than I0 hr after death.
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Morphologically no pathological changes were observed in the tissues the samples were taken from. Animals Female Wistar rats, domestic chickens and pigeons were fed an ad libitum standard laboratory diet (LSM, Centralne Laboratorium Przemystu Paszowego, Wytw6rnia Pasz, Motycz, Poland) and had free access to water. The animals were sacrificed by decapitation and the livers from all species and rat skeletal muscle, heart, kidney and brain were taken immediately. The samples of beef liver were obtained from the local slaughter-house. Preparation of the tissue extracts Liver samples (0.15~0.3g) were placed in 5ml of 0.25 M sucrose, 10 mM Tris HC1 buffer (pH 7.8), homogenised manually with a Teflon pestle homogeniser size A (A. Thomas Co. Philadelphia, PA, USA) and then centrifuged at 20,000 g for 30 min. The resulting supernatant was centrifuged at 105,000 g for 1 hr. Supernatant from the last centrifugation was used for enzyme assay. For the comparison of malic enzyme activities in rat and human tissues 1 g samples of human tissues taken during autopsy and rat tissues were homogenized in 8 ml of 20 mM Tris-HC1 buffer (pH 7.8) containing 0.1% Triton X-100 and centrifuged at 20,000g for 30min. Supernatant was decanted and the pellet was rehomogenized in 5 ml of the same medium and centrifuged again at 20,000 × g for 30min. Combined supernatants were used for the enzyme assays. Enzyme assays All enzyme activities were followed spectrophotometrically with a Specord M-40 spectrophotometer by monitoring the absorbancy at 340 nm changing as the result of either the appearance of NADPH or disappearance of NADPH and NADH. Malic enzyme [L-malate:NADP + oxidoreductase (oxaloacetate decarboxylating) EC 1.l.1.40] and NADP-linked isocitrate dehydrogenase [threo-oisocitrate:NADP + oxidoreductase (decarboxylating) EC 1.1.1.42] activities were measured as described by Zelewski and Swierczyfiski (1983). Glucose 6-phosphate dehydrogenase [D-glucose 6-phosphate:NADP + l oxidoreductase, EC 1.1.1.49] and 6-phosphogluconate dehydrogenase [6-phospho-o-gluconate:NADP + 2-oxidoreductase (decarboxylating), EC 1.1.1.44] were asayed according to Glock and McLean (1953). The incubation medium for the assay of ATP-citrate lyase [ATP-citrate(pro-3S)-lyase, EC 4.1.3.8] activity was as described previously (2elewski and Swierczyfiski, 1986). Fatty acid synthase [EC 2.3.1.85] activity was followed according to Goodridge (1972) in the medium containing 0.1 M potassium phosphate buffer (pH7.0), 3mM EDTA, 1 mM dithiothreitol, 0.3mM NADPH, 25/~M acetylCoA, 100/~M malonylCoA and the amount of tissue extract causing the decrease of absorbancy in the range from 0.014).02 per min. Protein was measured by the standard biuret method.
RESULTS AND DISCUSSION The question of h o w h u m a n s meet actual energy requirements while utilizing food sources t h a t vary in a b u n d a n c e a n d chemical nature has not been solved satisfactory. One aspect of this p r o b l e m involves the regulation of conversion of dietary c a r b o h y d r a t e to tissue fatty acids. This problem c a n n o t be unequivocally solved, unless the m a j o r tissue(s) that synthesize fatty acids are identified. In m a m m a l s , adipose tissue is considered to be the most i m p o r t a n t site o f fatty acids synthesis with the liver playing a less i m p o r t a n t role (Feller, 1954; H a u s b e r g e r et al., 1954; H u t c h e n s et al., 1954; J a n s e n et al., 1966). O n the other h a n d in birds liver is the main site of fatty acids synthesis, adipose tissue being less active in the de novo lipogenesis (Goodridge and Ball, 1966, 1967). In contrast to the studies with m a m m a l i a n a n d avian species, hum a n liver has received little a t t e n t i o n (Shrago et al., 1966; Z a k i m et al., 1969) and only few papers report on fatty acid synthesis in h u m a n adipose tissue (Shrago et al., 1966, 1967, 1971; Shrago et al., 1969; Patel et al., 1975). A l t h o u g h , no direct evidence has been presented so far, the i n f o r m a t i o n available indicates that h u m a n liver may play a crucial role in lipogenesis. Experimentally it is not easy to determine the role of h u m a n liver in lipogenesis. One possible way to assess the capacity of the metabolic p a t h w a y responsible for fatty acid synthesis in h u m a n liver is to measure the lipogenic enzyme activities in this organ. Thus c o m p a r a t i v e studies of lipogenic enzyme activities in the liver of h u m a n beings and in animals in which the role of liver in lipogenesis is well d o c u m e n t e d were undertaken. The activities of malic enzyme, glucose 6 - p h o s p h a t e dehydrogenase a n d ATP-citrate lyase in h u m a n liver were 6.0, 6.3 and 1.5 n m o l m i n ~m g prot ~ respectively (not shown). These values are esentially similar to those o b t a i n e d by Shrago et al. (1966). Shrago et al. (1966) reported that the activities of above m e n t i o n e d enzymes in h u m a n liver were c o m p a r a b l e to the activities found in rat liver. In c o n t r a s t our data (Table 1) indicate t h a t malic enzyme, glucose 6 - p h o s p h a t e dehydrogenase, 6 - p h o s p h o g l u c o n a t e dehydrogenase and fatty acid synthase activities in rat liver were approximately 6-7 times higher t h a n those f o u n d in h u m a n liver. ATP-citrate lyase was even a b o u t 16 times more active in rat t h a n in h u m a n liver. It should be n o t e d t h a t the activity of this enzyme in h u m a n liver was a b o u t two times lower t h a n fatty acid synthase. In contrast the activity of ATP-citrate lyase in rat, domestic chicken a n d pigeon liver was c o m p a r a b l e to the activity of fatty acid synthase. Domestic chicken a n d pigeon liver showed 40 a n d 8 times as m u c h
Table I. The activity of lipogenic enzymes in human, rat, beef, pigeon and chicken liver Enzyme Human Rat Beef Pigeon Chicken Malic enzyme 0.89_+0.12 5.49±0.25 0.42+_0.08 46.3_+8.4 50.5+3.58 Glucose 6-phosphate dehydrogenase 0.83 _+0.08 4.64 _+0.33 0.44 + 0.08 1.23 _+0.35 0.59 ± 0.07 6-Phosphogluconate dehydrogenase 4.40 + 0.28 32.3 +_1.33 3~08+_0.43 2.62 _+0.40 3.16 _+0.08 NADP-isocitrate dehydrogenase 49.7 + 3.05 55.9 + 1.73 29.6 ± 3.95 44.0 _-+5.98 59.7 ± 3.26 ATP-citrate lyase 0.19 + 0.01 3.07 ± 0.14 0.21 +-0.05 1.50 -+ 0.40 7.63 _+0.34 Fatty acid synthase 0.40 +_0.0l 2.66 _+0.25 ND 1.67 _+0.51 9.21 ± 0.36 Enzyme activities are expressed as #molmin-t g ~ wet tissue. The results represent the means + SEM from eight experiments. For other experimental conditions see '~Materials and Methods." NI), not detectable.
Lipogenic enzyme activities ATP-citrate lyase activity as found in human liver, whereas the activities in beef liver and human liver were similar. Fatty acid synthase displayed a similar pattern of variation as ATP-citrate lyase, with the highest activity in domestic chicken and undetectable in beef liver. The activities of the two dehydrogenases of hexose monophosphate pathway and malic enzyme are quite variable among the species tested. It is interesting that in the species displaying very high activity of malic enzyme (birds) relatively low activities of glucose 6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase were found. In human, rat and beef liver the activity ratio of malic enzyme to glucose 6-phosphate dehydrogenase was approximately 1. In these species the activity of 6-phosphogluconate dehydrogenase was 5--8 times higher than malic enzyme activity. If one assumes that the activity of glucose 6-phosphate dehydrogenase is limiting the total N A D P H production by hexose monophosphate shunt dehydrogenases then human, chicken, pigeon and beef liver are approximately equal, whereas rat liver higher in their capacity to produce NADPH. The capacity for N A D P H production by malic enzyme would be about 20 and 40 times greater than by hexose monophosphate shunt in pigeon and domestic chicken respectively. Such findings suggest that malic enzyme could play a more prominent role than hexose monophosphate shunt dehydrogenases in furnishing N A D P H for lipogenesis in birds' liver, whereas in human, rat and beef livers malic enzyme and hexose monophosphate shunt dehydrogenases would be almost equal in furnishing N A D P H for fatty acid synthesis. The activity of liver NADP-linked isocitrate dehydrogenase was found to be very high even in human and beef liver in which the activities of other N A D P H producing enzymes were relatively low. In the rat, isocitrate dehydrogenase does not appear to be of major importance in the generation of reducing equivalents, as its activity is not adaptable to dietary changes affecting lipogenesis (Leville and Hanson, 1966; Young et al., 1964). However in ruminants mammary gland the activity of NADP-linked isocitrate dehydrogenase plays an important role in generation of N A D P H for fatty acid synthesis (Bauman et al., 1970). One can assume that this enzyme plays a similar role in the liver. Considering the very low activity of fatty acid synthase in beef liver (practically undetectable) one can suppose that the rate of lipogenesis in this organ is extremely low. Thus it is not excluded that N A D P H production by NADP-linked malic enzyme and hexose monophosphate shunt dehydrogenases is high enough to support lipogenesis in beef liver. Data presented above indicate that the activities of key enzymes involved in lipogenesis are very low in human liver, much lower than in rat and birds and comparable to beef liver (except of fatty acid synthase, which is undetectable in beef liver). However, the human situation is complicated, since the dietary state as well as the age of the individual at the time of tissue biopsy must be considered. It is well known that in the rat starvation or high fat diet can depress the rate of lipogenesis in the liver. Since the human diet is normally rich in fat and the biopsies were taken after an overnight starvation, it is difficult to differenCBP(B) 95/3--D
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tiate between the depressive effect of diet or starvation on lipogenic enzyme activities and a possible true low activity of these enzymes. The age and sex of the animal can also alter markedly the activity of lipogenic enzyme in the liver. Older rats and males have been shown to display a lower activity of lipogenic enzymes than younger rats and females (Iritani et al., 1981; Boll et al., 1982; Lee et al., 1986). However Shrago et al. (1967) did not observe any variations in some lipogenic enzyme activities in human adipose tissue depending on either sex or age. Despite the much lower activity of lipogenic enzymes in human liver than in rat the relation between these enzymes are very similar if not identical. This may indicate that the role of the human liver in lipogenesis may be comparable to that of rat liver. An estimate of the relative importance of human liver in fatty acid synthesis may be attempted by using the data on ATP-citrate lyase activity as the enzyme exhibiting the lowest activity (Table 1). Let us assume we are dealing with a 70 kg human possessing 1500 g liver. The liver of this subject contains enough of the enzyme activity under the conditions of the in vitro test to produce approximately 2 mmol of palmitate per hr. However, on the basis of in vitro estimation this calculation must be overestimated and should be interpreted with caution. Usually it is difficult to obtain a sample of human liver from the subject under the same dietary regime, age, sex and environmental exposure. To overcome this problem we have been trying to use the post mortem tissue for human enzyme study. Data presented in Table 2 clearly indicate that the activity of some enzymes generating N A D P H in the post mortem obtained tissue were very similar to that found in the liver biopsies taken during abdominal surgery. These results suggest that the tissue obtained at post mortem may be used for some enzyme assays provided the test is being carried out within a few hours after death. It should also be pointed out that we did not observe significant effects of either sex or age on malic enzyme and hexose monophosphate shunt dehydrogenases activities while using postmortem liver. These experiments support the previously published data (Shrago et aL, 1967) indicating that sex, age and some hormones do not affect lipogenic enzyme activities in human tissue. Using the tissue obtained post mortem we also checked the malic enzyme activity in kidney cortex, heart, skeletal muscle and brain. Unexpectedly relatively high activity of this enzyme in human brain was found (Table 3). The values obtained were approximately 2-fold higher than in other human organs tested and are comparable to those found in rat brain. Table 2. NADP-linkeddehydrogenasesactivitiesmeasuredin postmortem samplesof human liver Enzyme /~molmin ~g ~wet tissue Malic enzyme 0.77 _+0.25 Glucose 6-phosphate dehydrogenase 0.42 ± 0.03 6-Phosphogluconate dehydrogenase 3.48 ± 0.34 NADP-isocitrate dehydrogenase 44.0 __+2.15 The results are means+ SEM from eight experiments. For other experimentalconditionssee Materialsand Methods.
MATEUSZZELEWSKIand JULIANSWIERCZYI~SKI
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Table 3. Malic enzyme activities in human and rat tissues Enzyme activity (~mol min -~ g ~ wet tissue) Ratio Human Rat Rat/Human Liver Kidney cortex Heart Skeletal muscle Brain
0.78 + 0.10 0.76 _ 0.06 0.99 + 0.04 0.60 ± 0.06 1.51 ± 0.11
5.33 ± 0.24 1.68 + 0.09 2.99 ± 0.14 0.80 ± 0.04 1.83 ± 0.05
6.8 2.2 3.0 1.3 1.2
The results represent the means ± SEM from five experiments. For other experimental conditions see Materials and Methods. Acknowledgements--We are indebted to Professor M. M. :~ydowo and Professor L. Zelewski for valuable discussion. The work was supported by the Polish Academy of Science within the project CPBR 3.13.6.1.7.
REFERENCES Bauman D. E., Brown R. E. and Davis C. L. (1970) Pathways of fatty acid synthesis and reducing equivalents generation in mammary glands of rat, sow and cow. Arch. Bioehem. Biophys. 140, 237-244. Boll M., Bruckner E. and Berndt J. (1982) Age-related differences in dietary regulation of lipogenic enzymes in rat liver. Hoppe-Seyler's Z. physiol. Chem. 363, 103-106. Bray G. A. (1972) Lipogenesis in human adipose tissue: Some effects of nibbling and gorging. J. din. Invest. 51, 537-548. Brdiczka D. and Pette D. (1970) Intra and extramitochondrial isozymes of (NADP) malate dehydrogenase. Eur. J. Biochem. 19, 546-551. Feller D. D. (1954) Metabolism of adipose tissue. I. Incorporation of acetate carbon into lipids by slices of adipose tissue. J. biol. Chem. 206, 171-180. Glock G. E. and McLean P. (1953) Further studies on the properties and assay of glucose 6-phosphate and 6-phosphogluconate dehydrogenase in rat liver. Biochem. J. 55, 400-408. Goodridge A. G. (1972) Regulation of the activity of acetyl coenzyme A carboxylase by palmitoyl coenzyme A and citrate. J. biol. Chem. 247, 694645952. Goodridge A. G. and Ball E. G. (1966) Lipogenesis in the pigeon: in vitro studies. Am. J. Physiol. 211, 803-808. Goodridge A. G. and Ball E. G. (1967) Lipogenesis in the pigeon: in vivo studies. Am. J. Physiol. 213, 245-249. Hausberger F. X., Hilstein S. W. and Rutman R. J. (1954) The influence of insulin on glucose utilization in adipose and hepatic tissue in vitro. J. biol. Chem. 208, 431-438. Hutchens T. T., Van Bruggen J. T., Cockburn R. M. and West E. S. (1954) The effect of fasting upon tissue lipogenesis in the intact rat. J. biol. Chem. 208, 115-122.
Iritani N., Fukuda H. and Fukuda E. (1981) Age-dependent modification of lipogenic enzymes. Bioehim. biophys. Acta 665, 636-639. Jansen G. R., Hutchinson C. F. and Zanetti M. E. (1966) Studies on lipogenesis in vivo. Effect of dietary fat or starvation on conversion of (~4C) glucose into fat and turnover of newly synthesised fat. Biochem. J. 99, 323-332. Lee V. M., Szepeszi B. and Hansen R. J. (1986) Genderlinked differences in dietary regulation of hepatic glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme in the rat. J. Nutr. 116, 1547 1554. Leville G. A. and Hanson R. W. (1966) Adaptative changes in enzyme activity and metabolic pathways in adipose tissue from meal-fed rats. J. Lipid. Res. 7, 56-55. Leville G. A., O'Hea E. K. and Chakrabarty K. (1968) In vitro 128, 398-401. Patel M. S., Owen O. E., Goldman L. J. and Hanson R. W. (1975) Fatty acid synthesis by human adipose tissue. Metabolism 24, 161 173. Shrago E., Glennon J. A. and Gordon E. S. (1966) Enzyme studies in human liver and adipose tissue. Nature (Lond.) 212, 1263. Shrago E., Glennon J. A. and Gordon E. S. (1971) Comparative aspects of lipogenesis in mammalian tissues. Metabolism 20, 54452. Shrago E., Spannetta T. and Gordon E. (1969) Fatty acid synthesis in human adipose tissue. J. biol. Chem. 224, 2761-2766. Young J. W., Shrago E. and Lardy H. A. (1964) Metabolic control of enzymes involved in tipogenesis and gluconeogenesis. Biochemistry 3, 1687-1692. Zakim D., Herman R. H. and Gordon W. C., Jr (1969) The conversion of glucose and fructose to fatty acids in the human liver. Biochem. Med. 2, 427-437. Zelewski M. and Swierczyfiski J. (1983) The effect of clofibrate feeding on the NADP-linked dehydrogenases activity in rat tissue. Biochim. biophys. Acta 758, 152-157. Zelewski M. and Swierczyfiski J. (1986) Effect of clofibrate feeding on some lipogenic enzyme activities induced by high carbohydrate diet. Biochem. Int. 13, 591 598.
0305-0491/90 $3.00 + 0.00 Pergamon Press plc
Printed in Great Britain
COMPARATIVE STUDIES ON LIPOGENIC ENZYME ACTIVITIES IN THE LIVER OF H U M A N A N D SOME ANIMAL SPECIES MATEUSZ Z,ELEWSKI and JULIAN SWIERCZYI~SKI Department of Biochemistry, Academic Medical School, ul.Dc;binki 1, 80-211 Gdafisk, Poland (Received 29 March 1989)
Abstract--1. The activities of enzymes involved in fatty acid synthesis in the human liver (sample taken during abdominal surgery) and in the livers of some animals were studied. 2. Fatty acid synthase, ATP-citrate lyase and malic enzyme activities were found to be from 4 to 70-fold lower in human liver than in rat or bird livers. 3. The activities of hexose monophosphate shunt dehydrogenases in human liver were from half to almost equal to the corresponding activities in birds, but much lower than in rat liver. 4. The activities of all enzymes listed above in human and beef liver were very similar (except fatty acid synthase which was undetectable in the beef liver). 5. Very high activity of NADP-linked isocitrate dehydrogenase was found in livers of all species tested. 6. These results are discussed in relation to the role of the human liver in lipogenesis. 7. The activities of the enzymes generating NADPH in human liver taken during abdominal surgery were similar to the activities observed in the tissue obtained post mortem. 8. This suggested that post mortem tissue may be used as a reliable human material for some enzyme assays. 9. Thus we also examined the activity of malic enzyme in post mortem human kidney cortex, heart, skeletal muscle and brain. I0 Relatively high activity of NADP-linked malic enzyme has been observed in human brain.
of these data and the results published previously by Bray (1972) indicating that human adipose tissue may play a considerable role in lipogenesis, it remains to be elucidated how important is the contribution of liver in human lipogenesis.
INTRODUCTION
Although significant amounts of fatty acids are known to be synthesized both in the liver and white adipose tissue of several animal species, until recently it has been difficult to establish the contribution to the total synthesis in vivo of each of these tissues. Previous studies by Goodridge and Ball (1966) and Leville et al. (1968) have shown that birds do not synthesize fatty acids de novo in white adipose tissue. They also indicated that fatty acids are synthesized in the liver and then transported to the adipose tissue for storage as triacylglycerols. Shrago and coworkers (1966, 1967, 1971) have shown that human adipose tissue also does not synthesize fatty acids at an appreciable rate and that ATP-citrate lyase, a key enzyme in the pathway of fatty acid synthesis, is present in negligible amounts in this tissue. They concluded that triacylglycerols in human adipose tissue are formed from free fatty acids synthesized in liver and carried to the adipose tissue with blood. This indicates that birds and humans are similar to each other but different from rats as far as fatty acid synthesis in the liver and adipose tissue is concerned. Since the activity of lipogenic enzymes in avian liver is relatively high (Goodridge and Ball, 1966; Brdiczka and Pette, 1970), much higher than in rats (Brdiczka and Pette, 1970) one can expect that human liver would also display high activity of lipogenic enzymes. Surprisingly, previous studies by Shrago et al. (1966) have shown that the activities of glucose 6-phosphate dehydrogenase, malic enzyme and ATP-citrate lyase are essentially the same in humans and rats. In view
MATERIALS AND METHODS
Chemicals
Substrates for malic enzyme, hexose monophosphate shunt dehydrogenases, NADP-linked isocitrate dehydrogenase, ATP-citrate lyase and fatty acid synthase were obtained from Sigma Chemical Co. (St Louis, USA). Triton X-100 was from Serva Feinbiochemica Heidelberg (FRG). All other chemicals were of highest purity available commercially from P.O.Ch. Gliwice (Poland). Human samples
Samples of human liver were taken during abdominal surgery from patients of both sexes aged 3 to 61 years. In all cases liver sampling was the routine practice performed for the diagnostic purposes. Experiments in which liver samples from patients were used have been approved by the local Ethic Commission for the Research on Human Subjects and are in accordance with Polish law. The patients were fasted 12 hr before the surgery. Neither the type of anasthesia nor preoperative medication were controlled. None of the patients received treatment with hormones, which would be likely to influence the enzyme activity levels. An attempt was also made to exclude patients who may have been in severe nutritional imbalance. Samples of human liver, skeletal muscle, heart, kidney and brain were taken from the autopsy made in the Department of Forensic Medicine, Medical School in Gdafisk not later than I0 hr after death.
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MATEUSZ ~ELEWSKI and JULIANSWIERCZY~4SK1
Morphologically no pathological changes were observed in the tissues the samples were taken from. Animals Female Wistar rats, domestic chickens and pigeons were fed an ad libitum standard laboratory diet (LSM, Centralne Laboratorium Przemystu Paszowego, Wytw6rnia Pasz, Motycz, Poland) and had free access to water. The animals were sacrificed by decapitation and the livers from all species and rat skeletal muscle, heart, kidney and brain were taken immediately. The samples of beef liver were obtained from the local slaughter-house. Preparation of the tissue extracts Liver samples (0.15~0.3g) were placed in 5ml of 0.25 M sucrose, 10 mM Tris HC1 buffer (pH 7.8), homogenised manually with a Teflon pestle homogeniser size A (A. Thomas Co. Philadelphia, PA, USA) and then centrifuged at 20,000 g for 30 min. The resulting supernatant was centrifuged at 105,000 g for 1 hr. Supernatant from the last centrifugation was used for enzyme assay. For the comparison of malic enzyme activities in rat and human tissues 1 g samples of human tissues taken during autopsy and rat tissues were homogenized in 8 ml of 20 mM Tris-HC1 buffer (pH 7.8) containing 0.1% Triton X-100 and centrifuged at 20,000g for 30min. Supernatant was decanted and the pellet was rehomogenized in 5 ml of the same medium and centrifuged again at 20,000 × g for 30min. Combined supernatants were used for the enzyme assays. Enzyme assays All enzyme activities were followed spectrophotometrically with a Specord M-40 spectrophotometer by monitoring the absorbancy at 340 nm changing as the result of either the appearance of NADPH or disappearance of NADPH and NADH. Malic enzyme [L-malate:NADP + oxidoreductase (oxaloacetate decarboxylating) EC 1.l.1.40] and NADP-linked isocitrate dehydrogenase [threo-oisocitrate:NADP + oxidoreductase (decarboxylating) EC 1.1.1.42] activities were measured as described by Zelewski and Swierczyfiski (1983). Glucose 6-phosphate dehydrogenase [D-glucose 6-phosphate:NADP + l oxidoreductase, EC 1.1.1.49] and 6-phosphogluconate dehydrogenase [6-phospho-o-gluconate:NADP + 2-oxidoreductase (decarboxylating), EC 1.1.1.44] were asayed according to Glock and McLean (1953). The incubation medium for the assay of ATP-citrate lyase [ATP-citrate(pro-3S)-lyase, EC 4.1.3.8] activity was as described previously (2elewski and Swierczyfiski, 1986). Fatty acid synthase [EC 2.3.1.85] activity was followed according to Goodridge (1972) in the medium containing 0.1 M potassium phosphate buffer (pH7.0), 3mM EDTA, 1 mM dithiothreitol, 0.3mM NADPH, 25/~M acetylCoA, 100/~M malonylCoA and the amount of tissue extract causing the decrease of absorbancy in the range from 0.014).02 per min. Protein was measured by the standard biuret method.
RESULTS AND DISCUSSION The question of h o w h u m a n s meet actual energy requirements while utilizing food sources t h a t vary in a b u n d a n c e a n d chemical nature has not been solved satisfactory. One aspect of this p r o b l e m involves the regulation of conversion of dietary c a r b o h y d r a t e to tissue fatty acids. This problem c a n n o t be unequivocally solved, unless the m a j o r tissue(s) that synthesize fatty acids are identified. In m a m m a l s , adipose tissue is considered to be the most i m p o r t a n t site o f fatty acids synthesis with the liver playing a less i m p o r t a n t role (Feller, 1954; H a u s b e r g e r et al., 1954; H u t c h e n s et al., 1954; J a n s e n et al., 1966). O n the other h a n d in birds liver is the main site of fatty acids synthesis, adipose tissue being less active in the de novo lipogenesis (Goodridge and Ball, 1966, 1967). In contrast to the studies with m a m m a l i a n a n d avian species, hum a n liver has received little a t t e n t i o n (Shrago et al., 1966; Z a k i m et al., 1969) and only few papers report on fatty acid synthesis in h u m a n adipose tissue (Shrago et al., 1966, 1967, 1971; Shrago et al., 1969; Patel et al., 1975). A l t h o u g h , no direct evidence has been presented so far, the i n f o r m a t i o n available indicates that h u m a n liver may play a crucial role in lipogenesis. Experimentally it is not easy to determine the role of h u m a n liver in lipogenesis. One possible way to assess the capacity of the metabolic p a t h w a y responsible for fatty acid synthesis in h u m a n liver is to measure the lipogenic enzyme activities in this organ. Thus c o m p a r a t i v e studies of lipogenic enzyme activities in the liver of h u m a n beings and in animals in which the role of liver in lipogenesis is well d o c u m e n t e d were undertaken. The activities of malic enzyme, glucose 6 - p h o s p h a t e dehydrogenase a n d ATP-citrate lyase in h u m a n liver were 6.0, 6.3 and 1.5 n m o l m i n ~m g prot ~ respectively (not shown). These values are esentially similar to those o b t a i n e d by Shrago et al. (1966). Shrago et al. (1966) reported that the activities of above m e n t i o n e d enzymes in h u m a n liver were c o m p a r a b l e to the activities found in rat liver. In c o n t r a s t our data (Table 1) indicate t h a t malic enzyme, glucose 6 - p h o s p h a t e dehydrogenase, 6 - p h o s p h o g l u c o n a t e dehydrogenase and fatty acid synthase activities in rat liver were approximately 6-7 times higher t h a n those f o u n d in h u m a n liver. ATP-citrate lyase was even a b o u t 16 times more active in rat t h a n in h u m a n liver. It should be n o t e d t h a t the activity of this enzyme in h u m a n liver was a b o u t two times lower t h a n fatty acid synthase. In contrast the activity of ATP-citrate lyase in rat, domestic chicken a n d pigeon liver was c o m p a r a b l e to the activity of fatty acid synthase. Domestic chicken a n d pigeon liver showed 40 a n d 8 times as m u c h
Table I. The activity of lipogenic enzymes in human, rat, beef, pigeon and chicken liver Enzyme Human Rat Beef Pigeon Chicken Malic enzyme 0.89_+0.12 5.49±0.25 0.42+_0.08 46.3_+8.4 50.5+3.58 Glucose 6-phosphate dehydrogenase 0.83 _+0.08 4.64 _+0.33 0.44 + 0.08 1.23 _+0.35 0.59 ± 0.07 6-Phosphogluconate dehydrogenase 4.40 + 0.28 32.3 +_1.33 3~08+_0.43 2.62 _+0.40 3.16 _+0.08 NADP-isocitrate dehydrogenase 49.7 + 3.05 55.9 + 1.73 29.6 ± 3.95 44.0 _-+5.98 59.7 ± 3.26 ATP-citrate lyase 0.19 + 0.01 3.07 ± 0.14 0.21 +-0.05 1.50 -+ 0.40 7.63 _+0.34 Fatty acid synthase 0.40 +_0.0l 2.66 _+0.25 ND 1.67 _+0.51 9.21 ± 0.36 Enzyme activities are expressed as #molmin-t g ~ wet tissue. The results represent the means + SEM from eight experiments. For other experimental conditions see '~Materials and Methods." NI), not detectable.
Lipogenic enzyme activities ATP-citrate lyase activity as found in human liver, whereas the activities in beef liver and human liver were similar. Fatty acid synthase displayed a similar pattern of variation as ATP-citrate lyase, with the highest activity in domestic chicken and undetectable in beef liver. The activities of the two dehydrogenases of hexose monophosphate pathway and malic enzyme are quite variable among the species tested. It is interesting that in the species displaying very high activity of malic enzyme (birds) relatively low activities of glucose 6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase were found. In human, rat and beef liver the activity ratio of malic enzyme to glucose 6-phosphate dehydrogenase was approximately 1. In these species the activity of 6-phosphogluconate dehydrogenase was 5--8 times higher than malic enzyme activity. If one assumes that the activity of glucose 6-phosphate dehydrogenase is limiting the total N A D P H production by hexose monophosphate shunt dehydrogenases then human, chicken, pigeon and beef liver are approximately equal, whereas rat liver higher in their capacity to produce NADPH. The capacity for N A D P H production by malic enzyme would be about 20 and 40 times greater than by hexose monophosphate shunt in pigeon and domestic chicken respectively. Such findings suggest that malic enzyme could play a more prominent role than hexose monophosphate shunt dehydrogenases in furnishing N A D P H for lipogenesis in birds' liver, whereas in human, rat and beef livers malic enzyme and hexose monophosphate shunt dehydrogenases would be almost equal in furnishing N A D P H for fatty acid synthesis. The activity of liver NADP-linked isocitrate dehydrogenase was found to be very high even in human and beef liver in which the activities of other N A D P H producing enzymes were relatively low. In the rat, isocitrate dehydrogenase does not appear to be of major importance in the generation of reducing equivalents, as its activity is not adaptable to dietary changes affecting lipogenesis (Leville and Hanson, 1966; Young et al., 1964). However in ruminants mammary gland the activity of NADP-linked isocitrate dehydrogenase plays an important role in generation of N A D P H for fatty acid synthesis (Bauman et al., 1970). One can assume that this enzyme plays a similar role in the liver. Considering the very low activity of fatty acid synthase in beef liver (practically undetectable) one can suppose that the rate of lipogenesis in this organ is extremely low. Thus it is not excluded that N A D P H production by NADP-linked malic enzyme and hexose monophosphate shunt dehydrogenases is high enough to support lipogenesis in beef liver. Data presented above indicate that the activities of key enzymes involved in lipogenesis are very low in human liver, much lower than in rat and birds and comparable to beef liver (except of fatty acid synthase, which is undetectable in beef liver). However, the human situation is complicated, since the dietary state as well as the age of the individual at the time of tissue biopsy must be considered. It is well known that in the rat starvation or high fat diet can depress the rate of lipogenesis in the liver. Since the human diet is normally rich in fat and the biopsies were taken after an overnight starvation, it is difficult to differenCBP(B) 95/3--D
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tiate between the depressive effect of diet or starvation on lipogenic enzyme activities and a possible true low activity of these enzymes. The age and sex of the animal can also alter markedly the activity of lipogenic enzyme in the liver. Older rats and males have been shown to display a lower activity of lipogenic enzymes than younger rats and females (Iritani et al., 1981; Boll et al., 1982; Lee et al., 1986). However Shrago et al. (1967) did not observe any variations in some lipogenic enzyme activities in human adipose tissue depending on either sex or age. Despite the much lower activity of lipogenic enzymes in human liver than in rat the relation between these enzymes are very similar if not identical. This may indicate that the role of the human liver in lipogenesis may be comparable to that of rat liver. An estimate of the relative importance of human liver in fatty acid synthesis may be attempted by using the data on ATP-citrate lyase activity as the enzyme exhibiting the lowest activity (Table 1). Let us assume we are dealing with a 70 kg human possessing 1500 g liver. The liver of this subject contains enough of the enzyme activity under the conditions of the in vitro test to produce approximately 2 mmol of palmitate per hr. However, on the basis of in vitro estimation this calculation must be overestimated and should be interpreted with caution. Usually it is difficult to obtain a sample of human liver from the subject under the same dietary regime, age, sex and environmental exposure. To overcome this problem we have been trying to use the post mortem tissue for human enzyme study. Data presented in Table 2 clearly indicate that the activity of some enzymes generating N A D P H in the post mortem obtained tissue were very similar to that found in the liver biopsies taken during abdominal surgery. These results suggest that the tissue obtained at post mortem may be used for some enzyme assays provided the test is being carried out within a few hours after death. It should also be pointed out that we did not observe significant effects of either sex or age on malic enzyme and hexose monophosphate shunt dehydrogenases activities while using postmortem liver. These experiments support the previously published data (Shrago et aL, 1967) indicating that sex, age and some hormones do not affect lipogenic enzyme activities in human tissue. Using the tissue obtained post mortem we also checked the malic enzyme activity in kidney cortex, heart, skeletal muscle and brain. Unexpectedly relatively high activity of this enzyme in human brain was found (Table 3). The values obtained were approximately 2-fold higher than in other human organs tested and are comparable to those found in rat brain. Table 2. NADP-linkeddehydrogenasesactivitiesmeasuredin postmortem samplesof human liver Enzyme /~molmin ~g ~wet tissue Malic enzyme 0.77 _+0.25 Glucose 6-phosphate dehydrogenase 0.42 ± 0.03 6-Phosphogluconate dehydrogenase 3.48 ± 0.34 NADP-isocitrate dehydrogenase 44.0 __+2.15 The results are means+ SEM from eight experiments. For other experimentalconditionssee Materialsand Methods.
MATEUSZZELEWSKIand JULIANSWIERCZYI~SKI
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Table 3. Malic enzyme activities in human and rat tissues Enzyme activity (~mol min -~ g ~ wet tissue) Ratio Human Rat Rat/Human Liver Kidney cortex Heart Skeletal muscle Brain
0.78 + 0.10 0.76 _ 0.06 0.99 + 0.04 0.60 ± 0.06 1.51 ± 0.11
5.33 ± 0.24 1.68 + 0.09 2.99 ± 0.14 0.80 ± 0.04 1.83 ± 0.05
6.8 2.2 3.0 1.3 1.2
The results represent the means ± SEM from five experiments. For other experimental conditions see Materials and Methods. Acknowledgements--We are indebted to Professor M. M. :~ydowo and Professor L. Zelewski for valuable discussion. The work was supported by the Polish Academy of Science within the project CPBR 3.13.6.1.7.
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