Carbohydrate metabolism in the rat peritoneal macrophages

Transcription

1 J. Biosci., Vol. 12, Number 4, December 1987, pp Printed in India. Carbohydrate metabolism in the rat peritoneal macrophages S. Κ. BANSAL* Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow , India * Present Address: Central Research Station, G.S.V.M. Medical College, Kanpur , India MS received 29 May 1986; revised 25 August 1987 Abstract. Rat peritoneal macrophages derive energy differently from other tissues. Resting rat peritoneal macrophages have been taken for the present investigation. Lactate produced by extracellular glycolysis in the peritoneal lavage fluid, is readily converted into pyruvate by resting peritoneal macrophages and is oxidised in mitochondria. Glycolytic enzymes other than phosphoglucoisomerase and lactate dehydrogenase could not be substantially demonstrated. Glucose-6-phosphate dehydrogenase was detected. The presence of glucose- 6-phosphate dehydrogenase along with phosphoglucoisomerase indicates the operation of the hexose monophosphate shunt as a pathway supplementary to glycolysis. Resting rat peritoneal macrophages thus appear to utilize extracellular lactate as their main energy source instead of glucose, bypass glycolysis and have active hexose monophosphate shunt. Keywords. Carbohydrate metabolism; macrophage. Introduction Carbohydrate metabolism of macrophages has been a subject of conjecture. Studies conducted by various workers indicate the existence of the glycolytic pathway (Leake et al., 1964) and hexose monophosphate (Η Μ Ρ) shunt (Tsan, 1977; Schneider and Baggiolini, 1980) mostly in activated macrophages (Cohen et al., 1980; Damiani et αl., 1980) or during phagocytosis (Paradisi et al., 1979). Oxidative metabolism has also been shown to increase in these cells during phagocytosis (Oren et al., 1963; Ouchi et al., 1965). Knowledge about the bioenergetic enzyme make-up of resting macrophages is far from complete. Information about the difference between resting and activated peritoneal macrophages will be helpful in understanding the biochemical basis of macrophage activation. The present investigation has therefore been carried out to examine the enzymic constitution of resting peritoneal macrophages of rats in relation to their carbohydrate metabolism. Materials and methods Female albino rats ( g) from the Animal Colony, Industrial Toxicology Research Centre, Lucknow were used for the experiments. The animals were maintained under normal conditions of husbandry and on a pellet diet supplied by Hindustan Lever Ltd., Bombay. Abbreviations used: HMP, Hexose monophosphate; HBSS, Hanks balanced salt solution; G6PDH, glucose-6-phosphate dehydrogenase; P GM, phosphoglucomutase; PGI, phosphoglucoisomerase; FDPald, fructose diphosphate aldolase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; TCA, trichloroacetic acid. 415

2 416 Bansal Preparation of macrophages and cell free lavage Macrophages were lavaged from the peritoneal cavity into 5 ml of physiological saline containing heparin (5 I.U./ml). Cell free lavage was separated by centrifugation at 4 C for 5 min at 350 g. The cell pellet was suspended in Hanks balanced salt solution (HBSS) and the cell concentration adjusted to cells/ml. The cell preparation was enriched in macrophages by the following methods. Monolayer formation Into Leighton tubes containing flying cover slips, 0 5 ml of the cell suspension was inoculated. The tubes were incubated at 37 C for 2h. Monolayer formation was checked under a light microscope. The monolayers were washed thrice with HBSS and viability of the cells was determined. Osmotic shock treatment The cell suspension was centrifuged as before. The cell pellet was resuspended in 0 05 Μ NaCl for 1 min and then an equal volume of 0 25 Μ NaCl solution was added with gentle shaking. After centrifugation, the cell pellet was suspended in physiological saline and cell concentration adjusted to cells/ml. The cells obtained by osmotic shock treatment were checked by differential counts after staining with Giemsa stain (1 g Giemsa stain dissolved in 66 ml of distilled water, kept in a water bath at C for 90 min and mixed thoroughly with 66 ml of methanol. The stain was allowed to stand for 7 days and then filtered). The suspension of macrophages (2 ml) was centrifuged and cell pellet suspended in 0 4 ml of homologous serum. Smears were prepared using 0 02 ml of the suspension by carefully spreading over clean dry slides. The smears were air dried, fixed with methanol for 5 min and stained. For staining, stock Giemsa stain was diluted 1:2 with distilled water and flooded over the smears carefully. After 5 min the slides were washed with distilled water till the smears became pink. The smears were then air dried and finally mounted in glycerinei jelly. Viability of cells was checked by the dye uptake method of Hanks and Wallace (1958) using 1 % aqueous eosin. The ratio of dye to cell suspension was 1:2. The cells were examined under a light microscope after 1 min. Stained cells were considered as dead. The monolayers were similarly incubated with the dye for 1 min, washed 3 times with HBSS and examined for cell viability. Isolation of mitochondria The cell suspension obtained after osmotic shock treatment was centrifuged at 750 g for 10 min. The pellet was resuspended in distilled water and cell concentration adjusted to cells/ml. The suspension was kept in ice and homogenized in a Potter-Elvehjem-type homogenizer. Mitochondria were prepared from 2 5 ml of homogenate. The homogenate was centrifuged at 750 g for 10 min. The supernatant fraction obtained was centrifuged at 11,900 0 for 15 min in a K-24 refrigerated centrifuge at 4 o C. The supernatant was collected and retained

3 Carbohydrate metabolism in the rat peritoneal macrophages 417 for use. The pellet, containing mitochondria from cells was suspended in 1 ml of distilled water. The presence of mitochondria was ascertained by staining with Janus Green B. To 0 18 ml of suspension, 0 02 ml of 0 1 % Janus Green Β was added. After incubating for 20 min one drop of suspension was mounted on a clean dry slide and observed under a microscope. Enzyme assay Supernatant obtained after centrifugation at 11,900 g was assayed for glucose-6- phosphate dehydrogenase (G6PDH) (EC ) activity was assayed by the method of Kornberg and Horecker (1955). Phosphoghicornutase (PGM) (EC ), glucose- 6-phosphatase(EC 3 l 3 9), phosphoglucoisomerase (PGI) (EC ), fructose diphosphate aldolase (FDPald)(EC ) and lactate dehydrogenase (LDH) (EC ) were assayed in the cell homogenate in water using 0 1 ml enzyme by the methods of Najjar (1955), Swanson (1955), Lohmann (1933), Sibley and Lehninger (1949) and Kornberg (1955), respectively. For fructose diphosphatase the cells were homogenised in 0 05 Μ lactate buffer, ph 3 5 and the homogenate was centrifuged at 350 g for 20 min. The enzyme activity was assayed in the supernatant using 0 1 ml enzyme by the method of Pogell and McGilvery (1952). The enzymes assayed in the cell free lavage included PGI, FDPald and LDH by the methods of Lohmann (1933), Sibley and Lehninger (1949) and Kornberg (1955), respectively. For glycogen Phosphorylase (EC ) the macrophage homogenate was prepared in 0 1 Μ sodium fluoride and the enzyme assayed by the method of Sutherland (1955). Malate dehydrogenase (MDH) (EC ) and cytochrome c oxidase (EC ) activities were assayed in mitochondrial suspension by the method of Ochoa (1955) and Cooperstein and Lazaro (1951), respectively. Protein was estimated in macrophage homogenate, 11,900 g supernatant and cell free lavage by the method of Lowry et al. (1951) after precipitation with trichloroacetic acid (TCA). Lactate was estimated in macrophage homogenate and cell free lavage by the method of Barker and Summerson (1941). Results After osmotic shock treatment the differential count of the cell suspension showed that up to 96% of the cells were macrophages; viability was 93%. The viability of macrophages in monolayers was 95%. Therefore the osmotic shock method was used to obtain macrophages in suspension for the studies. In resting rat peritoneal macrophages, glycogen Phosphorylase, PGM, glucose-6- phosphatase, fructose diphosphatase and FDPald could not be detected. Only PGI, G6PDH, LDH, MDH and cytochrome c oxidase were found in measurable quantities. In the cell-free lavage PGI, FDPald and LDH were present (table 1). Negligible amounts of lactate were present in macrophages, while lavage contained significantly high amounts. Protein concentration of lavage was high (table 2). Discussion Stimulated peritoneal macrophages derive energy primarily from glycolysis (Harris and Barclay, 1955; Paradisi et al., 1979; Schneider and Baggiolini, 1980) as indicated

4 418 Bansal Table 1. Activities of various enzymes in rat resting peritoneal macrophages and cell-free lavage. Values are mean of results of 5 different experiments. a Average number of macrophages from each rat is b Volume of physiological saline used to collect the lavage from each rat is 5 0 ml. Table 2. Lactate and protein in rat resting peritoneal macrophages and cell-free lavage. Values are mean of results of 5 different experiments. by the measurements of glucose consumption and lactate production during active phagocytosis (Leake et αl., 1964). However the mode by which energy is obtained by resting macrophages is still a subject of concern and the energetics of these cells has not so far been explained precisely. Macrophage morphology and function have so far been studied by using macrophage cultures as suspensions or monolayers. The suspension is a mixed population of many cell types, while the monolayer represents a pure population of macrophages. Since surface morphology gets altered in monolayers (Leake et al., 1975) with possible changes in biochemical functions, it was preferred to study macrophages in suspension. Attempts have been made to develop a method for getting a pure population of macrophages in suspensions. The osmotic shock treatment method developed in our laboratory is the method of choice, since differential count of the cells obtained by this method show that upto 96% of the cells are macrophages. Cell viability (93%) is comparable to that of monolayers. The absence of glycogen in peritoneal macrophages has been reported by Hofmann and Decker (1979). In the present study glycogen Phosphorylase, PGM and glucose-6-phosphatase could not be detected in the resting cells. However, PGI and LDH were present. The LDH isoenzyme pattern of alveolar macrophages (Bansal and Kaw, 1981) showed all the 5 isoenzymes contrary to that of peritoneal macrophages in which LDH 1 and LDH 2 were missing; LDH 4 and LDH 5 were more prominent in peritoneal than in alveolar macrophages. Fructose diphosphatase and FDPald were not detected. The absence of enzymes of the glycolytic pathway other

5 Carbohydrate metabolism in the rat peritoneal macrophages 419 than PGI and LDH is interesting. The presence of the HMP shunt has been reported in cells stimulated during phagocytosis (Tsan, 1977; Schneider and Baggiolini, 1980), and PGI, in resting macrophages may have a role in this pathway alone. The presence of LDH alone does not extend the view of active glycolysis in resting rat peritoneal macrophages. The energy metabolism of cells depends very much on their environment. In the case of peritoneal macrophages, it appears to be affected by the composition of the lavage fluid since lactate is present in lavage fluid in high concentrations, while the resting cells have negligible amounts. Macrophages have been shown to utilize lactate as their energy substrate. Lactate is freely diffusible across the plasma membrane and is taken up from the medium till the concentration in the extracellular compartment falls below the blood concentration (Tierney, 1971). Perfused lung slices incubated with lactate and glucose showed lactate to have a sparing action on glucose; the concentration of glucose did not affect the oxidation of lactate (Wolfe et al., 1979). However when the lactate concentration is high in the medium, LDH acts more as lactate oxidase producing pyruvate and the lactate oxidase activity exceeds hexokinase activity by 30-fold suppressing the glycolytic pathway to a great extent. The resultant pyruvate is oxidised through the TCA cycle. The enzymes FDPald, PGI and LDH in lavage fluid probably produce lactate through the glycolytic pathway. Thus glycolysis operates extracellularly and the cells utilize the lactate as their energy substrate. The HMP shunt appears to operate in resting peritoneal macrophages. The liver, which is oxygen deficient, obtains energy both from HMP shunt and glycolysis. However, glycolysis is suppressed under the conditions that prevail in the liver (Lehninger, 1972). These conditions can be compared to the environment of peritoneal macrophages, and this taken together with the fact that lactate concentration affects glycolysis, would indicate that peritoneal macrophages obtain energy by the oxidation of glucose-6-phosphate via the HMP shunt. It is thus possible that resting peritoneal macrophages utilize extracellular lactate and convert it to pyruvate Figure 1. Diagramatic representation of carbohydrate metabolism in resting rat peritoneal macrophage.

6 420 Bansal which can be channellised to the TCA cycle while also maintaining the H M P shunt and keeping cellular glycolysis suppressed. The presence of M D H and cytochrome c oxidase in resting macrophage mitochondria indicates that pyruvate can be oxidised to CO 2. Thus it is apparent that resting rat peritoneal macrophages derive energy from the intracellular oxidation of exogenous lactate to generate ATP via the TCA cycle. Oxidative metabolism is supported by the presence of numerous mitochondria. The salient feature is the uptake of lactate and its utilization, as shown diagrammatically in figure 1. Acknowledgements The author is grateful to Dr. C. R. Krishna Murti, Dr. G. G. Sanwal, Dr. J. L. Kaw and Dr. P. N. Viswanathan for their constant encouragement and suggestions, and to Council of Scientific and Industrial Research, New Delhi for providing the financial assistance. References Bansal, S. K. and Kaw, J. L. (1981) Toxicol. Lett., 7, 279. Barker, S. B. and Summerson, W. H. (1941) J. Biol. Chem., 138, 535. Cohen, M. S., Ryan, J. L, Yohe, W. B. and Root, R. K. (1979) Curr. Chemother. Infect. Dis. Proc. Int. Ciongr. Chemother. 2,804. Cooperstein, S. J. and Lazaro, A. (1951) J. Biol. Chem., 189, 665. Damiani, G., Kiyotaki, C, Soeller, W., Sasada, M., Peisach, J. and Bloom, B. R. (1980) J. Exp. Med., 152, 808. Hanks, J. S. and Wallace, J. Η. (1958) Proc. Soc. Exp. Biol. Med., 98, 188. Harris, Η. and Barclay, W. R. (1955) Br. J. Exp. Pathol., 36, 592. Hofmann, F. and Decker, Κ. (1979) Hoppe-Seyler s Ζ. Physiol. Chem., 360, 905. Kornberg, A. (1955) Methods Enzymol, 1, 441. Kornberg, A. and Horecker, B. L. (1955) Methods Enzymol., 1, 323. Leake, Ε. S., Gonzales-Ojeda, D. and Myrvik, Q. N. (1964) Exp. Cell Res., 33, 553. Leake, Ε. S., Wright, Μ. J. and Myrvik, Q. N. (1975) J. Reticuloendothel. Soc, 17, 370. Lehninger, A. L. (1972) Biochemistry (New York: Worth Publ. Inc.). p. 337 Lohmann, K. (1933) Biochem. Z., 262, 137. Lowry, C. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265. Najjar, V. Α. (1955) Methods Enzymol, 1, 294. Ochoa, S. (1955) Methods Enzymol, 1, 735. Oren, R., Farnahm, Α. Ε., Saito, Κ., Milofsky, Ε. and Karnovsky, Μ. L. (1963) J. Cell Biol., 17, 487. Ouchi, Ε., Selvaraj, R. J. and Sbarra, A. J. (1965) Exp. Cell Res., 40, 456. Paradisi, F., D Onofrio, C., Pepe, G., Cifarelli, A. and Piceolo, D. (1979) Ric. Clin. Lab., 9, 47. Pogell, B, M. and McGilvery, R. W. (1952) J. Biol. Chem., 197, 293. Schneider, C. and Baggiolini, M. (1980) Proc. Natl. Acad. Sci. USA, 77, 414. Sibley, J. A. and Lehninger, Α. L. (1949) J. Biol. Chem., 177, 859. Sutherland, Ε. W. (1955) Methods Enzymol, 1, 215. Swanson, S. Μ. (1955) Methods Enzymol, 2, 541. Tierney, D. F. (1971) Arch. Int. Med., 137, 858. Tsan, Μ. F. (1977) Blood, 50, 935. Wolfe, R. R., Hochachka, P. W., Trelstad, R. L. and Bruke, J. F. (1979) Am. J. Physiol., 236, E276.