Comp. Biochern. PhysioL Vol. 101B, No. 1/2, pp. 283-288, 1992 Printed in Great Britain
0305-0491/92 $5.00+0.00 © 1992 Pergamon Press pie
DEVELOPMENTAL CHANGES OF LIPOGENIC ENZYME ACTIVITIES A N D LIPOGENESIS IN BROWN ADIPOSE TISSUE A N D LIVER OF THE RAT ZDZISLAW KOCHAN and JULIAN SWlERCZYIglSKI Department of Biochemistry, Academic Medical School, ul. Dfbinki 1, 80-211 Gdafisk, Poland (Received 2 May 1991)
Abstract--1. Brown adipose tissue (BAT) and liver lipogenesis in vivo estimated by using 3H20 as tracer was very low and did not change significantly between 10 and 20 days after birth. Lipogenesis increased dramatically in both tissues by weaning at 20 days, peaking between 25 and 30 days of age. Since that time the rate of fatty acid synthesis in BAT decreased gradually to reach adult level after 2 months, whereas in the liver there was a sharp decrease of lipogenesis. 2. The activities of fatty acid synthase, citrate cleavage enzyme, malic enzyme and glucose 6-phosphate dehydrogenase essentially followed a similar course of developmental changes as lipogenesis. 3. In contrast to the enzymes listed above NADP-linked isocitrate dehydrogenase remained unaltered over the period studied, whereas lactate and malate dehydrogenases exhibited very high activity at 10 days after birth and from then decreased to reach adult level at the age of about 20 days. 4. The data obtained indicate that no substantial differences could be detected in the developmental pattern of lipogenesis and lipogenic enzyme activities between BAT and liver up to 30 days of age but after this time these processes were not co-ordinated in both tissues. Beyond this time the BAT was characterized by a much higher rate of lipogenesis than the liver. 5. The results are discussed in terms of the nutrient changes and the relationship between thermogenesis and lipogenesis in BAT.
INTRODUCTION
Brown adipose tissue (BAT) is known to be an important site of non-shivering thermogenesis. This process is fuelled mainly by fatty acids Osier et al., 1987) and requires a continual supply of oxidizable substrate which must either be provided directly by circulation or be mobilized from triacylglycerols stored within the tissue (Mercer and Williamson, 1988). If triacylglycerol stores are the source of energy, the mechanism to maintain or replenish the intracellular pool of triacylglycerols is required, functioning either by exogenous supply of triacylglycerols and free fatty acids, or by de novo of fatty acid synthesis. In adult rats and mice the capacity of BAT for fatty acid biosynthesis is very high (McCormack and Denton, 1977; Trayhurn, 1979, 1981; Agius and Williamson, 1980; Lavau et al., 1982; Goubern and Portet, 1986; Buckley and Rath, 1987; i~elewski and $wierczyfiski, 1990) suggesting that this process is playing an essential role in replenishing fatty acids. Relatively little work has been undertaken to investigate full developmental changes in BAT lipogenesis and previous studies concerning this subject gave some conflicting results. Pillay and Bailey (1982) showed that BAT lipogenesis in normally weaned rats is very low between days 15 and 20 of life, and rises slowly from day 20 and then rapidly after day 23, reaching a maximum at around 25 days of life. Moreover, Pillay and Bailey (1982) showed that at this period of life lipogenic flux is closely linked to the activity of acetyl-CoA carboxylase, malic enzyme and glucose 6-phosphate dehydrogenase. It has also been shown that the activities of citrate cleavage enzyme 283
(Hahn and Drahota, 1966) and acetyl-CoA carboxylase (Hahn, 1970) activity in BAT of rat increase within the first 30 days of life. In contrast to the data cited above Hahn and Skala (1972) showed that the activities of enzymes presumably supplying N A D P H for fatty acid synthesis remain unaltered (malic enzyme) or substantially decreases (glucose 6-phosphate dehydrogenase) at this period of life. Considering that malic enzyme and glucose 6-phosphate dehydrogenase activities are very well correlated with other lipogenic enzymes and with the rate of lipogenesis in the liver (Taylor et al., 1967), these results are somewhat surprising. No changes of malic enzyme activity at weaning and considerable decrease in glucose 6-phosphate dehydrogenase activity in BAT after birth (Hahn and Skala, 1972) might suggest that these enzymes play a minor role in fatty acid biosynthesis in BAT. To our knowledge the developmental changes of the fatty acid synthase has not been studied in BAT so far. The discrepancies mentioned and the fact that there is no information available about the timecourse of changes in BAT lipogenic enzymes occurring after day 30 of the rat life led us to carry out the present study. The main aim of this study is to compare the full developmental changes (between day 10 and 100 of life) of lipogenic enzyme activities and the rate of lipogenesis in vivo both in BAT and the liver of the rat. MATERIALS AND METHODS
All substrates for enzyme activity measurements were obtained from Sigma (St Louis, MO). Tritiated water was
ZDZISLAWKOCHANand JULIANSWIERCZYIqSKI
284
obtained from the Institute of Atomic Energy (~wierk, Poland). All other chemicalswere of highest purity available from POCh (Gliwice, Poland). Young rats (only males from litters sizes of 8-10) were separated from their mothers at day 20 after birth, and fed ad libitum on a standard diet. At a required day the animals were killed by decapitation, the total interscapular BAT and approximately 1 g of liver were removed, weighed and homogenized in 8 ml of ice-cold 20 mM Tris-HC1 buffer (pH 7.8). Special care was taken to dissect away any white adipose tissue adhering to the interscapular BAT. The homogenates were centrifuged at 20,000g for 30 min. The resulting supernatant was decanted, pellet was rehomogenized in 5 ml of the isolation medium and centrifuged again. The combined supernatants were used for the enzymes assay. Fatty acid synthase (EC 2.3.1.85), citrate cleavage enzyme (EC 4.1.3.8), malic enzyme (EC 1.1.1.40), NADPlinked isocitrate dehydrogenase (EC 1.1.1.42) and glucose 6-phosphate dehydrogenase (EC 1. I. 1.49) were assayed as described previously (2elewski and ~;wierczyfiski, 1990); lactate dehydrogenase (EC 1.1.1.27) and malate dehydrogenase (EC 1.1.1.37) as described in ~;wierczyfiski et al. (1980). All assays were performed on the Specord M40 spectrophotometer at 37°C. The fatty acid synthesis in vivo was measured by using the incorporation of tritium from tritiated water into fatty acids. Rats were injected intraperitoneally with 2-5mCi 3H20. One hour after injection animals were killed, the total interscapular BAT and approximately 1g of liver were dissected, weighed, and incorporation of tritium into fatty acids was assayed according to the method of Stansbie et al. (1976). Protein assays were performed according to Petersen (1977). DNA content was measured as described by Burton (1956).
RESULTS Figure 1 shows that at 10 days of age the fatty acid synthesis in BAT (Fig. la) and liver (Fig. 2b) was very low and did not change significantly between the 10th and 20thday after birth. The synthesis increased dramatically in both tissues by weaning at 20 days, reaching the maximum between 25 and 35 day of age. Since that time the rate of lipogenesis in BAT decreased gradually to reach adult level after 2 months. In contrast, in the liver there was a sharp decrease in the synthesis rate between day 30 and 50, and then, as in the BAT, the rate did not change significantly.
60 (a)
i
20-(b)
l
10
.2
20
[
~/+~±~T
5 +/+ I
I
I
%0 40 610 80 I~) 0 20 40 60 80 100 Age(days) Fig. 1. Developmental changes of in vivo lipogenesis (expressed as #g of 3H incorporated per hr per g wet tissue) in (a) interscapular BAT and (b) liver. Each point represents the mean of three to six experiments. The vertical bars indicate SD (the lack of vertical bar indicates that SD was too small to put on the figure).
6 -(a)
0.7[(b)
5 4
_
0305-0491/92 $5.00+0.00 © 1992 Pergamon Press pie
DEVELOPMENTAL CHANGES OF LIPOGENIC ENZYME ACTIVITIES A N D LIPOGENESIS IN BROWN ADIPOSE TISSUE A N D LIVER OF THE RAT ZDZISLAW KOCHAN and JULIAN SWlERCZYIglSKI Department of Biochemistry, Academic Medical School, ul. Dfbinki 1, 80-211 Gdafisk, Poland (Received 2 May 1991)
Abstract--1. Brown adipose tissue (BAT) and liver lipogenesis in vivo estimated by using 3H20 as tracer was very low and did not change significantly between 10 and 20 days after birth. Lipogenesis increased dramatically in both tissues by weaning at 20 days, peaking between 25 and 30 days of age. Since that time the rate of fatty acid synthesis in BAT decreased gradually to reach adult level after 2 months, whereas in the liver there was a sharp decrease of lipogenesis. 2. The activities of fatty acid synthase, citrate cleavage enzyme, malic enzyme and glucose 6-phosphate dehydrogenase essentially followed a similar course of developmental changes as lipogenesis. 3. In contrast to the enzymes listed above NADP-linked isocitrate dehydrogenase remained unaltered over the period studied, whereas lactate and malate dehydrogenases exhibited very high activity at 10 days after birth and from then decreased to reach adult level at the age of about 20 days. 4. The data obtained indicate that no substantial differences could be detected in the developmental pattern of lipogenesis and lipogenic enzyme activities between BAT and liver up to 30 days of age but after this time these processes were not co-ordinated in both tissues. Beyond this time the BAT was characterized by a much higher rate of lipogenesis than the liver. 5. The results are discussed in terms of the nutrient changes and the relationship between thermogenesis and lipogenesis in BAT.
INTRODUCTION
Brown adipose tissue (BAT) is known to be an important site of non-shivering thermogenesis. This process is fuelled mainly by fatty acids Osier et al., 1987) and requires a continual supply of oxidizable substrate which must either be provided directly by circulation or be mobilized from triacylglycerols stored within the tissue (Mercer and Williamson, 1988). If triacylglycerol stores are the source of energy, the mechanism to maintain or replenish the intracellular pool of triacylglycerols is required, functioning either by exogenous supply of triacylglycerols and free fatty acids, or by de novo of fatty acid synthesis. In adult rats and mice the capacity of BAT for fatty acid biosynthesis is very high (McCormack and Denton, 1977; Trayhurn, 1979, 1981; Agius and Williamson, 1980; Lavau et al., 1982; Goubern and Portet, 1986; Buckley and Rath, 1987; i~elewski and $wierczyfiski, 1990) suggesting that this process is playing an essential role in replenishing fatty acids. Relatively little work has been undertaken to investigate full developmental changes in BAT lipogenesis and previous studies concerning this subject gave some conflicting results. Pillay and Bailey (1982) showed that BAT lipogenesis in normally weaned rats is very low between days 15 and 20 of life, and rises slowly from day 20 and then rapidly after day 23, reaching a maximum at around 25 days of life. Moreover, Pillay and Bailey (1982) showed that at this period of life lipogenic flux is closely linked to the activity of acetyl-CoA carboxylase, malic enzyme and glucose 6-phosphate dehydrogenase. It has also been shown that the activities of citrate cleavage enzyme 283
(Hahn and Drahota, 1966) and acetyl-CoA carboxylase (Hahn, 1970) activity in BAT of rat increase within the first 30 days of life. In contrast to the data cited above Hahn and Skala (1972) showed that the activities of enzymes presumably supplying N A D P H for fatty acid synthesis remain unaltered (malic enzyme) or substantially decreases (glucose 6-phosphate dehydrogenase) at this period of life. Considering that malic enzyme and glucose 6-phosphate dehydrogenase activities are very well correlated with other lipogenic enzymes and with the rate of lipogenesis in the liver (Taylor et al., 1967), these results are somewhat surprising. No changes of malic enzyme activity at weaning and considerable decrease in glucose 6-phosphate dehydrogenase activity in BAT after birth (Hahn and Skala, 1972) might suggest that these enzymes play a minor role in fatty acid biosynthesis in BAT. To our knowledge the developmental changes of the fatty acid synthase has not been studied in BAT so far. The discrepancies mentioned and the fact that there is no information available about the timecourse of changes in BAT lipogenic enzymes occurring after day 30 of the rat life led us to carry out the present study. The main aim of this study is to compare the full developmental changes (between day 10 and 100 of life) of lipogenic enzyme activities and the rate of lipogenesis in vivo both in BAT and the liver of the rat. MATERIALS AND METHODS
All substrates for enzyme activity measurements were obtained from Sigma (St Louis, MO). Tritiated water was
ZDZISLAWKOCHANand JULIANSWIERCZYIqSKI
284
obtained from the Institute of Atomic Energy (~wierk, Poland). All other chemicalswere of highest purity available from POCh (Gliwice, Poland). Young rats (only males from litters sizes of 8-10) were separated from their mothers at day 20 after birth, and fed ad libitum on a standard diet. At a required day the animals were killed by decapitation, the total interscapular BAT and approximately 1 g of liver were removed, weighed and homogenized in 8 ml of ice-cold 20 mM Tris-HC1 buffer (pH 7.8). Special care was taken to dissect away any white adipose tissue adhering to the interscapular BAT. The homogenates were centrifuged at 20,000g for 30 min. The resulting supernatant was decanted, pellet was rehomogenized in 5 ml of the isolation medium and centrifuged again. The combined supernatants were used for the enzymes assay. Fatty acid synthase (EC 2.3.1.85), citrate cleavage enzyme (EC 4.1.3.8), malic enzyme (EC 1.1.1.40), NADPlinked isocitrate dehydrogenase (EC 1.1.1.42) and glucose 6-phosphate dehydrogenase (EC 1. I. 1.49) were assayed as described previously (2elewski and ~;wierczyfiski, 1990); lactate dehydrogenase (EC 1.1.1.27) and malate dehydrogenase (EC 1.1.1.37) as described in ~;wierczyfiski et al. (1980). All assays were performed on the Specord M40 spectrophotometer at 37°C. The fatty acid synthesis in vivo was measured by using the incorporation of tritium from tritiated water into fatty acids. Rats were injected intraperitoneally with 2-5mCi 3H20. One hour after injection animals were killed, the total interscapular BAT and approximately 1g of liver were dissected, weighed, and incorporation of tritium into fatty acids was assayed according to the method of Stansbie et al. (1976). Protein assays were performed according to Petersen (1977). DNA content was measured as described by Burton (1956).
RESULTS Figure 1 shows that at 10 days of age the fatty acid synthesis in BAT (Fig. la) and liver (Fig. 2b) was very low and did not change significantly between the 10th and 20thday after birth. The synthesis increased dramatically in both tissues by weaning at 20 days, reaching the maximum between 25 and 35 day of age. Since that time the rate of lipogenesis in BAT decreased gradually to reach adult level after 2 months. In contrast, in the liver there was a sharp decrease in the synthesis rate between day 30 and 50, and then, as in the BAT, the rate did not change significantly.
60 (a)
i
20-(b)
l
10
.2
20
[
~/+~±~T
5 +/+ I
I
I
%0 40 610 80 I~) 0 20 40 60 80 100 Age(days) Fig. 1. Developmental changes of in vivo lipogenesis (expressed as #g of 3H incorporated per hr per g wet tissue) in (a) interscapular BAT and (b) liver. Each point represents the mean of three to six experiments. The vertical bars indicate SD (the lack of vertical bar indicates that SD was too small to put on the figure).
6 -(a)
0.7[(b)
5 4
_
