The Third Department of Internal Medicine,School of Medicine, Osaka University,Osaka

Transcription

1 MECHANISM OF SERUM CHOLESTEROL-LOWERING ACTION OF AN ANABOLIC STEROID IN CONTRAST TO A GLUCORTICOID AKIRA KUMAGAI, MASAHIRO YAMAMOTO, SABURO YANO AND YUICHI YAMAMURA The Third Department of Internal Medicine,School of Medicine, Osaka University,Osaka It has been reported by several groups of workers that prolonged treatment with large doses of glucocorticoids elicited a hypercholesteremic response in man (Adlersberg et al., 1951; Skanse et al., 1959), rabbit (Stumpf and Wilens, 1954; Forbes et al., 1962), rat (Boyd, 1961), and dog (Wang et al., 1955), while anabolic steroids had a serum cholesterol-lowering action in man (Furman et al., 1958; Hellman et al., 1962), rat (Mosbach et al., 1963), dog (Abell et al., 1962) and cockerel (Campbell et al., 1960a and b). The mode of action of these hormones remains in doubt. The present studies were undertaken to investigate the machanism(s) by which these hormones affect the serum cholesterol level. 2-Hydroxymethylene-17ƒ -methyldihydrotestosterone (HMD) was chosen as an example of anabolic steroids and cortisone acetate as that of glucocorticoids. Effects of these hormones on different sites of the metabolic pathways of cholesterol were determined, using the radioisotopic methods. MATERIALS AND METHODS Treatment of animals Male rats of the Wistar strain, weighing from 170 to 200g, were fed ad libitum with commercial labolatory chow diet ("MF" of Oriental Yeast Co., Tokyo) before and during the experimental period. Animals were divided into 3 groups: Group I received daily intramuscular injections of 0.8mg of HMD and group II 2.0mg of cortisone acetate (Schering & Co., Berlin) per 100g of the body weight for 7 to 12 days. Group III, the control group, was injected with the same volume of saline during the same period. HMD was kindly supplied from Shionogi & Co., Osaka. Serum and liver cholesterol determination At the end of the 12th day of the hormonal treatment, rats were sacrificed after a 12 hr. fasting period. The serum and liver cholesterol were determined according to the method of Schoenheimer and Sperry (1934). Incorporation of acetate-1-14c into choresterol and other lipids Sodium acetate-1-14c (9.7mc/m mole) was purchased from Daiichi Pure Chemicals Co., Tokyo. Received for publication October 20, 1965.

2 ANABOLIC STEROID AND CHOLESTEROL At the end of the 7th day of the hormonal treatment, the hormone-treated and the control rats were fasted for 12 hrs. and received the intraperitoneal injection of 1ml of an isotonic saline solution containing 20ƒÊc of sodium acetate-1-14c. These animals were sacrificed from 15 to 240 mins. later after acetate-14-c injection. The livers, intestines, lungs, kidneys, testicles, brains, adrenals and adipose tissues were immediately removed and homogenized in a Waring blendor with 25 vols. of chloroform-methanol (2:1) mixture. The extract was purified according to the method of Folch et al. (1957). After the solvent was evaporated, the residue was dissolved in choloroform and chromatographed on the column of silicic acid (30g) following the modified method of Borgstrom (1952). Silicic acid was Mallinckrodt's analytical reagent, 100 mesh. Neutral lipid-fraction was eluted by 150ml of chloroform and phospholipid-fraction by 60ml of chloroform-methanol (1:1) mixture and 60ml of methanol. After the chloroform fraction was evaporated to dryness, the residue was dissolved in hexane and chromatographed on the column of Florisil (30g) according to the partial modi- form of 60 to 100 mesh, was first activated at 180 Ž for 12 hrs., then hydrated with 8.7% (w/w) of water. After then, several neutral lipid-fractions were separated, such as cholesterol esters, triglycerides and free cholesterol etc. These eluents were concentrated in vacuo and the aliquots were mounted on planchets. Radioactivities were determined with an end window type gas flow counter (Nuclear Chicago). The specific activity was expressed as counts per mg of each fraction after correction for self-absorption. In two preliminary experiments, phospholipids were separated by the usual method using acetone. The supernatant fraction was concentrated and hydrolysed with alcoholic KOH. The nonsaponifiable materials were extracted with petroleum ether. The extract was washed with water and evaporated to dryness. The residue was dissolved in acetone-ethanol (1:1) mixture and added with 1% (w/v) digitonin solution in 50% ethanol. The precipitate washed with acetone-ether (1:1) mixture and ether, was mounted on planchets for counting as the cholesterolfraction. Biliary excretion of cholesterol-414c and its metabolites At the end of 7 days' hormonal treatment, cannulation of the bile duct was performed in the rats with a polyethylene tube by an abdominal approach under hexobarbital anesthesia. After fasting for 12 hrs., 1ƒÊc of cholesterol-4-14c was injected intraperitoneally and bile was collected at intervals of 6 and 12 hrs.. Cholesterol-4-14C (33.8mc/m mole) purchased from Daiichi Pure Chemicals Co. was suspended in saline with the aid of Tween 80 (Siperstein et al., 1952). Aliquots of bile were spread on planchets and the radioactivities were determined (total 14C). The extraction of bile acids, nonsaponifiable materials and digitonin-precipitable 3 sterols from from bile was performed according to the method of Siperstein et al. (1952) and the radioactivities of these aliquots were determined. Fecal excretion of cholesterol-4 14C and its metabolites Rats were injected intramuscularly with HMD or cortisone in the same way. On the 4th day of hormonal treatment, the suspension of cholesterol-4-14c was injected intraperitoneally (2ƒÊc per 100g of the body weight) and feces were collected at intervals of 48 and 96 hrs. respectively after the administration of 14C. Ground feces were extracted for 6 hrs. with hot ethanol with the aid of a Soxhlet apparatus. Aliquots of the extracts were mounted on planchets and the radioactivities were determined (total 14C). Bile acids, nonsaponifiable materials and digitonin-precipitable materials were extracted as described above. Gastrointestinal absorption of cholesterol-4-14c and cholic acid-4-14c After 7 days' hormonal treatment, the hormone-treated and control rats were fasted for 24

3 KUMAGAI et al. Endocrinol.Japon. March 1966 hrs. and 1ƒÊc of cholesterol-4-14c or 0.01ƒÊc of cholic acid-4-14c was given into the stomach through a stainless steel gastric tube. Cholic acid-4-14c (3.2ƒÊc/m mole) was biosynthetically obtained according to the method of Bergstrom and Sjovall (1951). The radioactivity of cholesterol or bile acid-fraction in 1ml of serum and that of the whole liver were determined 1 or 2 hrs. after 14C ingestion. RESULTS Effects of HMD and cortisone on the serum cholesterol level The serum cholesterol levels of the HMD-treated rats (52.6 }2.4mg/dl-mean controls (69.8 }3.5mg/dl; No.,8) (0.001<P<0.01). On the contrary, higher serum cholesterol levels were observed in the cortisone-treated (88.2 }3.6mg/dl; No.,8), as compared with the controls (0.001<P<0.01). Effects of HMD and cortisone on the incorporation of acetate-1-14c into cholesterol and other lipids Preliminary experiments: The in vivo incorporation of acetate-1-14c into cholesterol and phospholipids in the various organs of rats 4 hrs. after acetate-14c injection was shown in Figure 1. The greatest incorporation into cholesterol was observed in the liver among the organs examined, as expressed as both in the whole organs (Fig.1) and per gram tissue. In the subsequent experiments, therefore, the incorporation into cholesterol was studied in the liver. Figures 2 and 3 show the time course studies of the in vivo incorporation of acetete-l-14c into cholesterol and phospholipids in the liver. The specific activities of cholesterol and phospholipids in the liver attained the peak in 30 mins. after the acetate-14c administration. Radioactivity In whole orssns Fig.1. Distribution of radioactivities of cholesterol and phospholipids in various organs-4 hrs. after intraperitoneal injection of acetate-1-14c

4 ANABOLIC STEROID AND CHOLESTEROL Time (minutes) Fig.2. Time course of the in vivo incorporation of acetate-1-14c into cholesterol of rat liver-after intraperitoneal injection of acetate-1-14c Time (minutes) Fig.3. Time course of the in vivo incorporation of acetate-1-14c into phospholipids of rat liver after intraperitoneal injection of acetate-1-14c Then, the specific activity of cholesterol declined gradually in the hormonetreated groups. It was shown in Table 1 that the specific activity of free cholesterol in the rat liver 15 mins. after the intraperitoneal injection of acetate-1-14c was much higher in both HMD and cortisone groups than the non-treated. Table 2 indicates that the specific activity of free cholesterol in the liver 4 hrs. after the acetate-14c administration was significantly lower in the HMD or cortisone-treated rats than the non-treated. These data might be interpreted to show that HMD or cortisone accelerated the turnover of cholesterol in the liver by both enhanced biosynthesis and increased degradation and excretion of cholesterol. Tables 1 and 2 also show that no difference was observed between the hormonetreated groups and the control group in the specific activity of cholesterol esters

5 KUMAGAI et al. Endocrinol. Japon. March 1966 Table1. Effects of HMD and cortisone administration on the incorporation of acetate-1-14c into cholesterol esters, free cholesterol, triglycerides and phospholipids of rat liver *Mean }standard error 15 mins. after acetate-14c injection, while the specific activity of cholesterol esters 4 hrs. after 14C injection was lower in the HMD or cortisone group than the control group. However, a much smaller percentage was incorporated into cholesterol. The incorporation of acetate-14c into triglycerides was slightly increased by cortisone, while almost no effects were observed on the biosynthesis of phospholipids by these hormones.

6 Vol.13, No.1 ANABOLIC STEROID AND CHOLESTEROL Table 2. Effects of HMD and cortisone administration on the incorporation of acetate-1-14c into cholesterol esters, free cholesterol, triglycerides and phospholids of rat liver Effects of HMD and cortisone on the biliary excretion of cholesterol-4-14c and its metabolites The quantity of bile from the bile fistula was greater in both the cortisone and the HMD group than the non-treated group as shown in Table 3. The radioactivities of the total 14C, bile acids, nonsaponifiable materials and 3ƒÀ-OH-sterols excreted into the bile after cholesterol-4-14c injection were significantly greater

7 KUMAGAI et al. Endocrinol. Japon. March 1966 Table 3. Effects of HMD and cortisone administration on the radioactivities of total intraperitoneal injection of cholesterol-4-14c in the bile duct-cannulated rats 0-6 hrs. after cholesterol-14c injection 6-12 hrs. after cholesterol-14c injection both in the HMD and the cortisone group than in the non-treated. The HMD or cortisone administration might increase the biliary excretion of cholesterol and its metabolites. About 2/3 of the total 14C excreted were derived from the bile acidfraction in each group and only a few percent from the nonsaponifiable materials.

8 Vol.13, No.1 ANABOLIC STEROID AND CHOLESTEROL Table 4. Effects of HMD and cortisone administration on the cumulative percentage of radioactivities of total 14C, bile acids, nonsaponifiable materials and 3ƒÀ-OHsterols excreted into feces after intraperitoneal injection of cholesterol-4-14c a) 0-48 hrs. after cholesterol-14c b) hrs. after cholesterol-14c injection c) 0-96 hrs. after cholesterol-14c injection

9 KUMAGAI et al. Endocrinol. Japon. March 1966 Although HMD had a hypocholesteremic action and cortisone had an opposite effect, no differences could be found between HMD and cortisone actions on the biosynthesis and degradation of cholesterol in the liver as well as the biliary excretion of cholesterol and its metabolites as described above. Effects of HMD and cortisone on the fecal excretion of cholesterol-4-14c and its metabolites It was shown in Table 4 that the cumulative percentages of the total 14C and bile acids-14c excreted in feces following the intraperitoneal injection of cholesterol- 4-14C, were higher in the HMD group, while slightly lower in the cortisone group, as compared with the non-treated group. On the contrary, the cumulative percentage of the nonsaponifiable materials-14c or 3ƒÀ-OH-sterols-14C was slightly increased by the cortisone administration, while almost no effect was observed by the HMD treatment. In our experimental conditions, about 2/3 of the total 14C in feces were excreted in the bile acid-fraction, while only a negligible percentage was excreted in the nonsaponifiable fraction. Thus, the distinct difference between HMD and cortisone action was observed in the fecal excretion of the total 14C and bile acids-14c after cholesterol-4-14c injection. Effects of HMD and cortisone on the gastrointestinal absorption of cholesterol-4-14c and cholic acid-4-14c Figures 4a and 4b show the effect of HMD and cortisone on the gastrointestinal absorption of cholesterol-4-14c. Cholesterol is absorbed or reabsorbed through the gastrointestinal tract and then transported as chylomicra via the thoracic duct into blood steam. Blood cholesterol is trapped into organs such as the liver and metabolized. The radioactivities of cholesterol of serum and the whole liver after cholesterol-4-14c ingestion could be regarded as the possible indicators of the gastrointestinal absorption of cholesterol. The radioactivity of cholesterol in serum 1 or 2 hrs. after the ingestion of cholesterol-4-14c was much higher in the cortisone group than in the non-treated HMD group, as shown in Figure 4a. Little difference was observed between the latter two groups. It was shown in Figure 4b that the radioactivity of cholesterol in the whole liver was also significantly higher in the cortisone group and slightly lower in the HMD group than in the control group. Next, the effects of HMD and cortisone on the gastrointestinal absorption of cholic acid-4-14-c were investigated. The majority of bile acids excreted into the intestine is reabsorbed and brought to the liver through the portal vein. Thus the radioactivity of bile acid in the whole liver following cholic acid-4-14c ingestion could be regarded as a possible indicator of the gastrointestinal(re)absorption of cholic acid. Figure 5 shows that the radioactivity of bile acides in the whole liver was significantly higher in the cortisone group than in the HMD or control group. Little difference was observed between the latter two groups. These data suggest that cortisone increased the gastrointestinal absorption of both cholesterol and bile acids, while HMD decreased it or had almost no effect on it.

10 Vol.13, No.1 ANABOLIC STEROID AND CHOLESTEROL Fig. 4. Effects of HMD and cortisone administration on the gastrointestinal absorption of cholesterol-4-14c given into the stomach through a gastric tube a) Radioactivity of cholesterol in 1ml of serum b) Radioactivity of cholesterol in the whole liver Fig. 5. Effects of HMD and cortisone administration on the gastrointestinal absorption of cholic acid-4-14c given into the stomach through a gastric tube

11 KUMAGAI et al. Endocrinol. Japon. March 1966 Effects of HMD and cortisone on distribution of cholesterol among various organs The incorporation of acetate-1-14c into cholesterol of various organs 4 hrs. after acetate-14c injection was shown in Figure 6. The radioactivity of cholesterol in the whole organs was decreased by the HMD or cortisone administration in the intestine and the lung as well as in the liver, but almost no effects were observed in the kidney and the testicle. Fig. 6. Effects of HMD and cortisone administration on distribution of radioactivity of cholesterol in various organs-4 hrs. after intraperitoneal injection of acetate-1-14c No evidence was obtained that the decreased incorporation of acetate-14c into cholesterol in the liver 4 hrs. after acetate-14c injection due to HMD or cortisone administration, was replaced by increased incorporation into cholesterol in the other organs. Effects of HMD and cortisone on release of cholesterol from liver into blooa The liver cholesterol content was increased with the HMD treatment (230 control group (162 }10mg/100g; No., 8)(0.001<P<0.01), but the decreased release of cholesterol from the liver into blood might not be the primary factor enough to explain the serum cholesterol-lowering action of HMD, because cortisone which has a hypercholesteremic effect also increased the liver cholesterol content (242

12 Vol.13, No.1 ANABOLIC STEROID AND CHOLESTEROL DISCUSSION Among the numerous factors that control cholesterol metabolism, the endocrine participation may be one of the most important factors. According to Altman et al.(1951) the cholesterol synthesis from acetate by the perfused rat liver was increased if cortisone was supplied in perfusing fluid. Rosenman et al.(1952 a) reported that cholesterol content in bile was increased by cortisone treatment. Boyd (1961) suggested that cortisone incresed cholesterol biosynthesis and shortened the biological half life from the data of the in vivo incorporation of acetate-1-14c into blood cholesterol. The in vivo incorporation of acetate-1-14c into cholesterol was much increased following the administration of testosterone propionate(raiford. and Wong, 1962). Furman et al.(1963) reported that the incorporation of acetate- tion. Many investigators found that thyroxine administration increased both biosynthesis and elimination of cholesterol(boyd, 1963). The most plausible explanation for the reduction of serum cholesterol by the use of thyroxine seems to be that elimination was increased to a greater degree than biosynthesis of cholesterol (Rosenman et al., 1952 b). The estrogenic action on biosynthesis and elimination of cholesterol also has been reported by many authors(boyd, 1963), but"over-all" effect on cholesterol metabolism of sex hormones was reported by Kritchevsky et al.(1961), that is, cholesterol was synthesized and metabolized in male rats at a much lower rate than in the castrated males or in the normal female rats. Forbes et al.(1962) reported that cortisol ingestion favored the absorption of cholesterol-14c from the gastrointestinal tract in accordance with our data. Mosbach and Bevans(1963) found that 17ƒ -methyl-testosterone ingestion inhibited the absorption of dietary dihydrocholesterol from the gastrointestinal tract, while the equivalent doses of testosterone were less effective than methyltestosterone. The data presented in this paper might show the possible mechanism of the serum cholesterol-lowering action of HMD and the hypercholesteremic response by the cortisone administration. Both HMD and cortisone accelerated biosynthesis as well as degradation of cholesterol in the liver. Both HMD and cortisone also increased the biliary excretion of cholesterol and its metabolites. But the distinct differences between HMD and cortisone were observed in the steps of the fecal excretion of chleterol and its metabolites and the gastrointestinal absorption of cholesterol or bile acids. It may be concluded from these facts that to determine the machanism by which a certain factor effects the serum cholesterol level, the effects of the factor on the whole steps of cholesterol metabolism should be checked using the same strain of animals. A series of experiments presented here might serve as an example of the screening methods of the serum cholesterol-lowering agents. SUMMARY Mechanism(s) of the hypocholesteremic action of an anabolic steroid, 2-hydroxymethylene-17ƒ -methyldihydrotesterone(hmd) were investigated in contrast to

13 KUMAGAI et al. Endocrinol. Japon. March 1966 those of the hypercholesteremic effect of a glucocorticoid, cortisone. HMD or cortisone increased the in vivo incorporation of acetate-1-14c into rat liver cholesterol 15 mins. after 14C injection (increased cholesterol biosynthesis) and decreased the incorporation 4 hrs. after 14C injection (increased cholesterol degradation and excretion by the liver). Almost the same results were observed in the intestine. HMD or cortisone also increased the biliary excretion of total 14C and bile acids- the contrary slightly decreased by cortisone. The gastrointestinal absorption of cholesterol-4-14c or cholic acid-4-14c was accelerated by cortisone, while almost no effect was observed by HMD. REFERENCES Abell, L. L., E. H. Mosbach and F. E. Kendall (1962). Circulation Research 10, 846. Adlersberg, D., L. E. Schaefer and S. R. Drachman (1951), J. Clin. Endocrinol. 11, 67. Altman, K. I., L. L. Millar and C. G. Bly (1951). Arch. Biochem. 31, 329. Bergstrom, S. and J. Sjovall (1951). Acta Chem. Scand. 5, BorgstrOm, B.(1952). Acta Physiol. Scand. 25, 101. Boyd, G. S.(1961). Federation Proc. 20, Suppl. 7, 152. Boyd, G. S. The control of lipid metabolism. Academic Press, London, p.79 (1963). Campbell, R. S. F., T. D. V. Lawrie, H. J. Lieberschuetz, S. G. MacAlpine, R. Pirrie, R. Rifkind and B. M. Rifkind (1960a). Ame. Heart J. 60, 799. Campbell, R. S. F., T. D. V. Lawrie, J. C. MacLaurin and R. Pirrie (1960b). Circulation Research 8, 78. Carroll, K. K.(1961). J. Lipid Res. 2, 135. Folch, J., M. Lees and G. H. S. Stanley (1957). J. Biol. Chem. 226, 497. Forbes, J. C., E. S. Higgins and O. M. Petterson (1962). Metabolism 11, Furman, R. H., R. P. Howard, L. N. Norcia. and E. C. Keaty (1958). Ame. J. Med. 24, 80. Furman, R. H., C. W. Robinson, Jr., R. H. Bradford, P. Alaupoic and L. N. Norcia (1963). Proc. Soc. Exptl. Biol. Med. 113, 789. Hellman, L., B. Zumoff. G. Kessler, R. S. Rosenfeld and T. F. Gallagher (1962). J. Clin. Invest. 41, Kritchevsky, D., E. Staple, J. L. Rabinowitz and M. W. Whitehouse (1961). Ame. J. Physiol. 200, 519. Mosbach. E. H., S. Zapp and L. L. Abell (1963). Proc. Soc. Exptl. Biol. Med. 114, 595. Mosbach, E. H. and M. Bevans (1963). Arch. Pathol. 75, 558. Raiford, R. L. and H. Y. C. Wong (1962). Circulation Research 11, 753. Rosenman, R. H., M. Friedman and S. O. Byers (1952a). Endocrinology 51, 142. Rosenman, R. H., S. O. Byers and M. Friedman (1952b). J. Clin. Endocrinol. 12, Schoenheimer, R. and W. M. Sperry (1934). J. Biol. Chem. 106, 745. Siperstein, M. D., M. E. Jayko, I. L. Chaikoff and W. G. Dauben (1952). Proc. Soc. Exptl. Biol. Med. 81, 720. Skanse, B., W. Studnitz and N. Skoog (1959). Acta Endocrinol. (Kbh.) 31, 442. Stumpf, H. H. and S. L. Wilens (1954). Proc. Soc. Exptl. Biol. Med. 86, 219. Wang, C. I., L. E. Schaefer and D. Adlersberg (1955). Endocrinology 56, 628.