Significance of increase in glucose 6-phosphatase activity in brown adipose cells of cold-exposed and starved mice.код для вставкиСкачать
THE ANATOMICAL RECORD 219:39-44 (1987) Significance of Increase in Glucose 6-Phosphatase Activity in Brown Adipose Cells of Cold-Exposed and Starved Mice J U N WATANABE, SHINSUKE KANAMURA, HIROHIKO TOKUNAGA, MINORU SAKAIDA, AND KAZUO KANA1 Department of Anatomy, Kansai Medical University, Fumizonecho 1, Moriguchi, Osaka, 570 Japan ABSTRACT Cytochemical and biochemical glucose 6-phosphatase (G6Pase) activity was examined in brown adipose tissues of normal, cold-exposed, or starved mice. In addition, G6Pase activity in white adipose tissue and hexokinase activity in brown and white adipose tissues were biochemically measured. In normal animals, the reaction product for G6Pase activity was localized in the endoplasmic reticulum and nuclear envelope of brown adipose cells. The amount of the reaction product increased in cold-exposed or starved animals. Biochemical G6Pase activity (259.7 f 48.5 ng Pi/min/mg protein) in brown adipose tissues of normal animals was higher when the value was compared with values of other organs. Biochemical G6Pase and hexokinase activities increased rapidly in brown adipose tissues of coldexposed animals, and a close relation was found between activities of the two enzymes. In brown adipose tissues of animals starved for 3 days, biochemical G6Pase activity increased, but hexokinase activity did not change. In white adipose tissues of normal, cold-exposed,or starved animals, G6Pase activity was very low, although the enzyme activity increased slightly in animals starved for 3 days. The results show that the high G6Pase activity in brown adipose cells probably relates to thermogenesis in cold-exposed animals and may be concerned with glucose release into the blood in starved animals. Brown adipose cells of rodents produce heat from stored lipids by oxidation of fatty acids (Rothwell and Stock, 1979; Nedergaard and Lindberg, 1982). On the other hand, livers of mammals and flight muscles of insects make heat by a substrate (futile) cycle; blood glucose is converted into glucose 6-phosphate (G6P) by hexokinase activity and G6P is reconverted into glucose by glucose 6-phosphatase (G6Pase) activity, producing heat by consumption of ATP (Newsholme and Crabtree, 1973, 1976; Hue and Hers, 1974; Surholt and Newsholme, 1981). Cooney and Newsholme (1982) have observed high hexokinase activity in brown adipose tissue of rats, which increases in cold-acclimated animals. Therefore, if brown adipose cells possess G6Pase activity, the substrate cycle, another mechanism for thermogenesis, may be available in the cells. On the other hand, brown adipose cells are able to produce G6P from stored lipids via gluconeogenesis (Cannon and Nedergaard, 1979; Frohlich et al., 1977; Seccombe et al., 1977). If high G6Pase activity is present in brown adipose cells, the cells may be capable of supplying glucose from stored lipids into the blood. Thus, information on the presence of G6Pase activity is of interest in brown adipose cells. However, there have been no reports on G6Pase activity in brown adipose cells. In the present study, therefore, cytochemical and biochemical G6Pase activity was examined in the brown adipose tissue of normal, cold-exposed, or starved mice. 0 1987 ALAN R. LISS, INC. In addition, G6Pase activity in the white adipose tissue and hexakinase activity in brown and white adipose tissues were biochemically measured. The aim of examining cold-exposed or starved animals was to test the following working hypotheses: (1)if G6Pase in brown adipose cells relates to thermogenesis, the enzyme activity will increase with hexokinase activity in the cells from cold-exposed animals, and (2) if G6Pase in brown adipose cells is concerned with gluconeogenesis, the enzyme activity will increase in the cells from starved animals. MATERIALS AND METHODS One hundred and eight male ddY mice, 10-12 weeks old, were used. They had free access to food (NMF, Oriental) and water before the experiments. The animals were divided into 3 groups: one group was kept at 4°C with free access to food and water (cold-exposed animals), the second group was kept a t 25°C and given only water (starved animals), and the third group animals were kept at 25°C with free access to food and water (normal animals). These animals were maintained for 3 days (cytochemical experiments) and for 1, 2, or 3 days Received January 16, 1987; accepted March 24, 1987. Address reprint requests to Dr. Shinsuke Kanamura, Department of Anatomy, Kansai Medical University, Fumizonocho 1, Moriguchi, Osaka, 570 Japan. J. WATANABE ET AL. 40 (biochemical experiments) in individual cages, and sacAll biochemical data were subjected to statistical analrificed by cervical dislocation except for those used for ysis (analysis of variance and Student's t-test). Spearthe cytochemical experiments. man's rank order correlation coefficient was used for ascertaining the relationship between G6Pase and hexCytochernical Method okinase activities. Interscapular brown adipose tissues of the animals RESULTS under sodium pentobarbital anesthesia were perfused via the left ventricle, first with ice-cold saline for 15 The average body weight of normal mice was 35.6 seconds and then with 2% glutaraldehyde in 0.1 M so- 2.2 gm (means f S.D., n = 6). The weight of cold-exposed dium cacodylate (pH 7.4,4"C)for 5 min (6 ml/min). The animals for 1 day (35.3 f 2.3 gm), for 2 days (36.3 f 2.5 fixed tissues were sectioned a t 40 pm with a freezing gm), and for 3 days (35.5 k 2.3 gm), and of animals microtome. The sections were washed with 5% sucrose starved for 1day (33.7 f 2.8 gm) did not differ from the in 0.1 M sodium cacodylate (pH 7.4) at 4°C for 1 hour value of normal animals. The weight decreased signifi(Kanamura, 19731, and incubated at room temperature cantly at 2 days (29.1 k 1.9 gm) and 3 days (27.3 f 2.6 for 30 minutes in a reaction medium (3.7 mM G6P, 3.6 gm) of starvation (P < 0.05). mM lead nitrate, 80 mM sodium cacodylate, 230 mM Cytochernical Results sucrose and 10 mM levamisole, pH 6.7; Watanabe et al., 1983,1986;Kanai et al., 1983,1986; Shugyo et al., 1986). The reaction product for G6Pase activity was observed Levamisole was used to inhibit alkaline phosphatase in the endoplasmic reticulum and nuclear envelope of activity (Van Belle, 1972). The sections were washed brown adipose cells from normal, cold-exposed,or starved with 5% sucrose in 0.1 M sodium cacodylate (pH 7.4) at animals (Figs. 1, 2). However, the reaction product be4°C for 30 minutes, postfixed in 1% buffered osmium came abundant in cold-exposed or starved animals (see tetroxide (pH 7.4) a t 4°C for 1 hour, dehydrated in a Figs. 4-6, 8). No deposition of the reaction product was graded series of ethanol, and embedded in Spurr's resin. seen in mitochondria, Golgi apparatus, and cell matrix Thin sections were stained with uranyl acetate and lead of brown adipose cells. However, small amounts of final citrate and examined in a JEM-100s electron microscope. product were occasionally present in lysosomes, lipid Control experiments for ascertaining the specificity of droplets, and the plasma membrane of the cells. Small the reaction product (Kanamura, 1971a,b71975a,b)con- amounts of reaction product for G6Pase activity was also sisted of: (1) incubation of the sections in the reaction observed in the endoplasmic reticulum and nuclear enmedium lacking G6P, (2) incubation in the reaction medium containing 3.7 mM 0-glycerophosphate in place of G6P, (3) preincubation in 0.25 M sucrose containing 10 mM NaF at 37°C for 15 minutes, and then incubation in the reaction medium containing 10 mM Nal?. Biochemical Methods Interscapular brown adipose tissues or epididymal white adipose tissues were homogenized at 4°C with 50 volumes of 0.25 M sucrose in a Potter-Elvehejem homogenizer for 2 minutes at 2,000 rpm. The homogenate was centrifuged at 4°C for 10 minutes a t 3,000 g. G6Pase activity in a n aliquot (0.1 ml) of the supernatant was assayed by the method of Leskes et al. (1971). The assay medium contained 30 mM G6P, 30 mM sodium cacodylate, and 10 mM levamisole (pH 6.7). Incubation was done a t 37°C for 30 minutes. The inorganic phosphorus released was determined by Phosphor C-test (Wako Pure Chemicals, Osaka, Japan). Protein was estimated by the method of Lowry et al. (1951). The enzyme activity was expressed as nanograms of phosphorus liberated per minute per milligram of protein. Hexokinase activity was measured according to the method of Joshi and Jagannathan (1966).An aliquot (0.1 ml) of the supernatant was mixed with 2.8 ml of the medium containing 15 mM glucose, 10 mM MgC12, 40 mM Tris-HC1 buffer, 1.4 IU glucose 6-phosphate dehydrogenase, 0.01 mM ethylenediaminetetraacetate, and 0.3 mM 0-nicotinamide adenine dinucleotide phosphate (NADP), pH 7.4. Then, 0.1 ml of 3.3 mM adenosine triphosphate (ATP) was added to the mixture and the formation of NADP-reduced form (NADPH) was estimated at 25°C from the second to the tenth minute after adding ATP. The enzyme activity was expressed as nanomoles of NADPH formed per min per milligram of protein. Figs. 1-8. Cytochemical demonstration of G6Pase activity in brown adipose tissues from mice. Sections (40 pm) cut from perfusion-fixed tissues were incubated in the reaction medium at room temperature for 30 minutes. Figs. 1, 2. Portions of brown adipose cells from normal animals. The reaction product for G6Pase activity is seen in the endoplasmic reticulum (arrows) and nuclear envelope (arrowheads)of brown adipose cells. Deposition of final products is also seen in lysosomes (Ly) and lipid droplets (L).Fig. 1:(a) X8,OOO; 6)~28,000;Fig. 2, X20,OOO. Fig. 3. Control experiment; portion of a brown adipose cell from normal animal. Fixed section was preincubated in 0.25 M sucrose containing 10 mM NaF for 15 minutes and incubated in the reaction medium containing 10 mM NaF for 30 minutes. No reaction product is seen in the endoplasmic reticulum and nuclear envelope. L, lipid drop let. ~8,000. Figs. 4,5. Portions of brown adipose cells and endothelial cells from animals kept at 4°C for 3 days. The reaction product is abundant in the endoplasmic reticulum and nuclear envelope of brown adipose cells. Deposition of final products is present in lysosomes Cy), lipid droplets (L), and pits of plasma membrane (P).The reaction product is also observed in the endoplasmic reticulum of an endothelial cell (El. Fig. 4, x 8,000; Fig. 5, X 12,000. Fig. 6. High-power magnification of a portion (lower right) in Figure 5. Abundant reaction product is localized in the cisternae of smooth endoplasmic reticulum surrounding lipid droplet (L). ~80,000. Fig. 7. Small amount of the reaction product (see Figs. 4,5) is seen in nuclear envelope of a n endothelial cell (arrowheads). The reaction product in the nuclear envelope of a brown adipose cell @) is abundant. Deposition of final products is also seen in the plasma membrane of both cells. x 10,000. Fig. 8. Portion of a brown adipose cell from an animal starved for 3 days. The reaction product is abundant in the endoplasmic reticulum and nuclear envelope. ~8,000. GLUCOSE 6-PHOSPHATASE ACTIVITY IN MICE 41 42 - J. WATANABE ET AL. 1 BROWN E 600 0) 4- 9 n 0 E 400\ F E \ < 200m C 200 z g t 2 0- L WHITE 2 DAYS OF 3 STARVATION o \ 3 (0 s 0, 'D 1 4 NORMAL I L 3 m 3 Fig. 10. Changes of G6Pase and hexokinase activities in brown (upper) and white (lower) adipose tissues of normal (fed) and starved mice. Normal animals and animals given only water for 1,2, or 3 days Fig. 9. Changes of G6Pase and hexokinase activities in brown (upper are sacrificed at 10 A.M. See legend of Figure 9 for explanation. figure) and white (lower figure) adipose tissues of normal and coldexposed mice. Normal animals (kept at 25°C) and animals kept at 4°C for 1, 2, or 3 days are sacrificed at 10 A.M. Dotted column: G6Pase activity. Open column: hexokinase activity. Values are means k S.D. However, the two enzyme activities did not change in for 6 animals. *P < 0.05, **P < 0.01: significantly different from the white adipose tissues if animals were exposed to cold. value of normal animals (Student's t-test). (AT25"C) DAYSOF I EXPERIMENT(AT4Y) velope in endothelial cells of capillaries in brown adipose tissues (see Figs. 5,7). Deposition of a little final product was occasionally seen in lysosomes, cytoplasmic vesicles, and the plasma membrane of the endothelial cells. Omission of G6P from the incubation medium resulted in a n absence of the reaction product except for that in lipid droplets. Use of 0-glycerophosphate in place of G6P in the reaction medium caused a loss of the reaction product except for that in lysosomes, lipid droplets, and the plasma membrane. Preincubation of the sections in 0.25 M sucrose containing 10 mM NaF, followed by incubation in the reaction medium containing 10 mM NaF, abolished the total reaction (Fig. 31, but the final product was occasionally visible in lipid droplets and the plasma membrane. These results indicate that the reaction product in the endoplasmic reticulum and nuclear envelope in brown adipose cells and endothelial cells is due to G6Pase activity. The deposition of final product in lysosomes and the plasma membrane is probably related to nonspecific phosphatase or acid phosphatase activity, and the final product in lipid droplets is nonspecific deposition of lead. Biochemical Results In normal, cold-exposed, and starved animals, G6Pase and hexokinase activities in brown adipose tissues were always higher than those in white adipose tissues (Figs. 9,101. G6Pase and hexokinase activities in brown adipose tissues of cold-exposed animals became higher than those of normal animals at all time points examined (Fig. 9). G6Pase activity in brown and white adipose tissues of animals starved for 1 or 2 days was not different from that of normal animals (Fig. lo), but the activity increased in animals starved for 3 days. Hexokinase activity in brown and white adipose tissues was unchanged in starved animals. A strong positive correlation between hexokinase and G6Pase activities was observed in brown adipose tissues of cold-exposed animals (correlation coefficient, 0.937; Fig. 11). Correlation coefficients between the two enzyme activities in white adipose tissues of cold-exposed animals, brown adipose tissues of starved animals, and white adipose tissues of starved animals were 0.735, - 0.473, and -0.322, respectively. DISCUSSION As revealed in the present study, G6Pase activity was present in brown adipose cells from mice. The reaction product for the activity was localized in the endoplasmic reticulum and nuclear envelope. The biochemical activity in the brown adipose tissue of normal animals (259.7 f 48.5 ng Pi/min/mg protein) was higher than that of the white adipose tissue (14.3 f 3.81, submandibular gland and pancreas (76.3 f 3.2 and 94.4 f 4.4, Watanabe et al., 1983), and leg muscles (139 & 20 to 217 28, Watanabe et al., 1986; Hirose et al., 1986), and was similar to that of the seminal vesicle, which is considered to be high (270.0 & 28.6, Kanai et al., 1986). Further, the amount of the reaction product in brown adipose cells and biochemical activity in the brown adipose tissue increased in cold-exposedor starved animals. These results probably show the presence of thermogenesis by a substrate (futile) cycle and suggest the ability to supply glucose into the blood in brown adipose cells of mice, supporting our working hypothesis. GLUCOSE 6-PHOSPHATASEACTIVITY IN MICE c > I 600 400 E a 0 0% m -> 200 C r = 0.937 00 43 brown adipose cells from starved rats. The increased G6Pase activity possibly hydrolizes G6P produced from gluconeogenesis. Glucose thus produced may be released into the blood. In starved animals, however, the greater part of blood glucose is produced in the liver from lactate and alanine derived from the skeletal muscle (Felig et al., 1970; Kusaka and Ui, 1977), and from fatty acids derived from white and brown adipose tissues (ZaragozaHermans, 1974; Nedergaard and Lindberg, 19821, and is from liver glycogen (Kanamura et al., 1980). The role of glucose released directly from brown adipose cells, even if present in small amounts, is possibly to elevate the glucose level, which inclined to be low in the blood of mice starved for 3 days. In brown adipose cells, lipolysis and thermogenesis are stimulated rapidly by P-adrenergic effect of norepinephrine (Nedergaard and Lindberg, 1982) and high concentration of glucagon (Kuroshima and Yahata, 1979). Glucocorticoids stimulate slowly the synthesis of gluconeogenic enzymes in brown adipose cells (Nedergaard and Lindberg, 1982). Therefore, rapid increase in G6Pase activity in the cells of cold-exposedanimals may relate to actions of norepinephrine and glucagon, and slow increase in G6Pase activity of starved animals might be concerned with glucocorticoids. In white adipose cells, G6Pase may not be concerned with thermogenesis, because the activity did not increase in the cells of cold-exposed animals. Further, the activity was low if it increased in animals starved for 3 days. Therefore, the significance of G6Pase in white adipose cells appears unclear from these results. On the other hand, the white adipose tissue maintained relatively high hexokinase activity during starvation. Pilkis (1970) showed t h a t the major source of G6P in white adipose cells is neither glycogen nor gluconeogenic precursors but blood glucose. Further, the rate of lipid synthesis is low in the white adipose tissue of starved rats (Zaragoza-Hermans, 1974). Therefore, the intracellular concentration of G6P is probably relatively high in white adipose cells from starved animals. A possible role of G6Pase in white adipose cells is to regulate the intracellular concentration of G6P, hydrolyzing any excess. :L 3 3 a 0 0 ( 200 400 HEXOKINASEACTIVITY (D n mot NADPH/min/mg protein) Fig. 11. Relationship between GGPase and hexokinase activities in brown adipose tissues of normal and cold-exposed mice. G6Pase activity in brown adipose tissue of each animal is plotted as a function of hexokinase activity in the tissue of the same animal. Open circle: normal animal. Closed symbols: animals kept at 4°C for 1 (circle), 2 (triangle) and 3 (square) days. r: coefficient of correlation. Generally, sources of G6P are gluconeogenic precursors, blood glucose, and glycogen. In brown adipose cells from normal nonfetal animals, glycogen is not detected (Nedergaard and Lindberg, 1982). Cooney and Newsholme (1982) showed that brown adipose cells of rats have a potent ability to produce G6P from blood glucose by high hexokinase activity. Further, Cannon and Nedergaard (1979), Frohlich et al. (1977), and Seccombe e t al. (1977) suggest that brown adipose cells can convert gluconeogenic precursors produced from stored lipids into G6P. Thus, the sources of G6P in brown adipose cells are probably the blood glucose and gluconeogenic precursors. In the brown adipose tissue of cold-exposed animals, G6Pase and hexokinase activities increased rapidly with a strong positive correlation. G6Pase is a multifunctional enzyme (Nordlie, 1971). However, in vivo, the hydrolysis of G6P is probably its sole function (Arion et al., 1972). Therefore, the results show that G6P is produced steadily from the blood glucose by the increased hexokinase activity and then G6P is converted into glucose by the increased G6Pase activity in brown adipose cells of cold-exposed animals. Therefore, a substrate cycle between glucose and G6P, which produces large amounts of heat (Newsholme and Crabtree, 1973, 1976; Hue and Hers, 1974; Surholt and Newsholme, 19811, probably exists in addition to the established heat production mechanism, P-oxidation of fatty acids (Rothwell and Stock, 1979; Nedergaard and Lindberg, 19821, in brown adipose cells from cold-exposed mice. In the brown adipose tissue of animals starved for 3 days, G6Pase activity increased, but hexokinase activity did not change. Gluconeogenic precursors produced from stored lipid can be changed into G6P by brown adipose cells (Frohlich et al., 1977; Seccombe et al., 1977; Cannon and Nedergaard, 1979). 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