BRIEF REPORTS / M P P B m BREFS
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Templeton, J. F., Sashi Kumar, V. P., Bose, D., Smyth, D. D., Kim, R. S., and LaBella, F. %. 1988. Digitalis-like pregnanes. Cardiac m d r e d effects of a glycoside of 140-hydroxyprogesterone. Can. J. Physiol. Phamacol. 66: 1420- 1424. Templeton, J. F., %ashiKumar, V. P., Bose, D., and LaBella, F. %. 1989. Cardiac glycoside-like structure and function of 50,140pregnanes. J. Med. Chem. 33: I977 - 1981.
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Weiland, J., Schwabe, K., Hubler, D., Schonfeld, W., and Repke, K. R. H. 1987. Glycosidation of chlormadinal acetate alters its actions on Na'1K'-transporting ATPase and cardiac contractility: a contribution to the endogenous digitalis problem. J. Enzyme Inhib. 2: 31 -36.
Differential effects of blood insulin levels on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats' Institute ste Fisiokogia Experimental, Consejo Nacional de Investigaciones Cient@cas y Ticnicas, Facultad de Ciertcias Bioquimicas y Farmac&uticas, Universidad Nacional de Rosario, Suigacha 570, 2000 Rosario, Argentina
Received May 27, 1991 CARNOVALE, C. E., CATANIA, V. A., MONTI,J. A., and CARRILLO, ha. C. 1992. Differential effects of blood insulin levels on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats. Can. J. Physiol. Pharmacol. 70: 727-731. Microsomal glutathione S-transferase, UDP-glucuronyl transferase, and aniline hydroxylase activities were determined in liver, renal cortex, and small intestine of control, streptozotocin-diabetic, alloxan-diabetic. and untreated insulin-injected male Wistar rats. Renal microsomal glutathione S-transferase activity showed a direct linear relationship with insulin blood levels, in agreement with our previous report on cytosolic glutathione S-transferase. This result suggests a possible regulatory mechanism of insulin that needs to be further examined. The hepatic microsomal UDP-glucuronyl transferase was only decreased in streptozotocin-diabetic rats and was not restored by insulin treatment. Intestinal UDP-glucuronyl transferase exhibited an opposite response in streptozotocin-treated animals that was not normalized by the administration of insulin. Hepatic aniline hydroxylase showed the same behaviour as intestinal UDP-glucuronyl transferase. These results suggest that streptozotocin and (or) its metabolites have a direct effect on hepatic and intestinal UDP-glucuronyl transferase activity and on hepatic aniline hydroxylase activity. On the other hand, insulin regulation of enzyme activity varies from one organ to another. Key words: insulin, streptozotocin, alloxan, glutathione S-transferase, UDP-glucursnyl transferase, aniline hydroxylase.
V. A., MONTI,J. A., et CARRILLO, ha. C. 1992. Differential effects of blood insulin levels CARNOVALE, C. E., CATANHA, on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats. Can. J. Physisl. Pharmacol. 70 : 727-731. On a dCterminC les activitks de glutathion S-transfkrase, UDP-glucuronyl transfkrase et aniline hydroxylase dans la foie, le cortex renal et l'intestin grCle de rats Wistar non traitks sous perfusion insulinique, rendus diabktiques par alloxane, rendus diabCtiques par streptozotocine et tCmoins. L'activitk de glutathion S-transferax microsomiale rCnale a montrk une relation linCaire directe avec les taux sanguins insuliniques, ce qui est en accord avec notre rapport antkrieur sur la glutathion S-transfkrase cytosolique. Ce rCsultat suggkre un mkcanisme rkgulateur possible de l'insuline, qui devra Ctre dkmontrk. L'UDP-glucuronyl transfkrase microsomiale hkpatique a Ctk diminuCe seulement chez les rats rendus diabktiques par streptozotocine et n'a pas CtC rktablie par un traitement i?i l'insuline. L'UDP-glucuronyl transfkrase intestinale a prCsentC, chez ces mCmes animux, une rCponse inverse qui n'a pas CtC normalisCe par l'administration d'insuline. L'aniline hydroxylase hkpatique s'est conagsrtie c o m e 1'UDP-glucuronyl transfdrase intestinale. Ces rksultats suggkrent que la streptozotocine et (OU)ses mCtabolites ont un effet direct sur I'activitC de 1'UDP-glucuronyl transfkrase intestinale et hkpatique, ainsi que sur I'activitC d'aniline hydroxylase hkpatique. Par ailleurs, la rkgulation insulinique des deux organes difEre. Mots clefs : insuline, streptozotocine, alloxane, glutathion S-transfkrase, UDP-glucuronyl transfkrase, aniline hydroxylase. [Traduit par la r&action]l Introduction The effects of experimentally induced diabetes on the microsomal metabolism of xenobiotics have been studied in several hboratories (A-Turk et al 1981; Warren et al 1983; Rouer et al. 1985; Watkim and 1987)' In general, it has 1~ preliminary abstract of this paper has been published in C o m u n . Biol. 1990, 9(2): 177. 2Author for correspondence and reprint requests. Printed in Cam& / Imprim6 au Cma&
been observed that alloxan or streptszotocin (SZ) induced diabetes decreased androgen-sensitive hepatic drug metabolism in male rats, whereas in female rats the same sex-dependent activities are increased (Reinke et al. 1978, 1979). Qn the other hand, sex-independent drug-metabolizing activities are not impaired or may even be (Al-Turk et al. 1980, 1981).-~thas been reported that diabetesis a physispathological inducer of hepatic cytockrome P45Q with high activity towards aniline (fast and Cook 1982; Yamazoe et al. 1989).
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Group 11 was studied 4 h after the injection of insulin as reported by Carnovale et d. (1990). Group 111 was studied '7 days aker the SZ injection. A group of rats were injected with insulin (0.2 IU/kg b . ~, .0.1 mL) after 7 days of SZ-induced diabetes and studied 4 R aker the insulin injection. Group IV was studied 48 h aker the alloxan injection. A group of animals were injected with insulin (0.2 IU/kg b . ~ . 0.1 , mL) after 48 R of alloxan-induced diabetes and studied 4 h after insulin administration. Assessment of the induced diabetes Plasma was used to measure glucose enzymatically (GOB-PAP test Wienner Lab.) (Kunst et al. 1983) and insulin by Radioimmunoassay (RIA) (Insulin Kit, Biodata, Milano, Italy) (Yalow and Berson 1960).
FIG.I. Blood glucose and insulin levels throughout the treatments. C, control rats (n = 4); UT + I, untreated rats studied 4 h after the injection of a single dose of insulin (0.2 IU/kg b.w., i.p.) (n = 4); SZ, SZ-treated rats (50 mg/kg b.w., i.v.) studied at 7 days after SZ administration (n = 6); SZ + I, SZ-diabetic rats studied 4 h after insulin administration (n = 5); AL, alloxan-treated rats (40 mg/kg b.w., i.v.) studied at 48 R after the drug administration (n = 6); AL + I, alloxan-diabetic rats studied 4 h after insulin administration (n = 5). Error bars represent the SEM. * Significantly different from control values.
Liver and intestinal microsomal 7-ethoxycoumarin metabolism were found to be increased in rats with SZ-induced diabetes (Al-Turk et al. 1980). Besides, we have reported a direct relationship between blood insulin levels and cytosolic glutathione S-transferase (GST) activity (phase HI detoxication system) in liver, renal cortex, and intestine from male rats (Carnovale et d. 1990). The present study was aimed at elucidating if the insulin regulatory mechanism reported on cytosolic GST is also I detoxifying enzymes such as exerted on microsomal phase H GST (GST,) and UDP-glucuronyl transferase (UDP-GT), as well as the cytochrome P-450 related enzyme aniline hydroxylase (AH), from liver, renal cortex, and small intestine of male Wistar rats.
Methods Animals and treatment Male Wistar rats (280 -350 g) were used throughout. The animals were allowed free access to food and water prior to the experiments. They were randomly divided into four groups: group 1 (control), injected i.v. through femoral vein with 0.5 mL of the SZ and alloxan vechicle; group II (untreated insulin), untreated rats injected s.c. with 0.1 mL of insulin (8.2 IU/kg b.w.); group I11 (SZ-treated), injected i.v. through femoral vein with SZ (58 mg/kg b. w. in 0.5 mL of freshly prepared 0.01 M sodium citrate, pH 4.5); group IV (dloxan treated), injected i.v. through femoral vein with alloxan (40 mg/kg b. w. in 8.5 mL of freshly prepared 0.81 WE sodium citrate, pH 4.5). Group I was studied at different periods of time, in accordance with the SZ or alloxan treatment.
+
Tissue preparations Animals were bled by cardiac puncture under pentobarbital anaesthesia between 14:W and 15:W to avoid possible effects of diurnal variations. Plasma samples were used to determine glucose and insulin levels. Liver was perfused with cold saline and the largest lobe was excised. Kidneys were removed, washed in cold saline, and cortical tissues were separated. The small intestine was rinsed with cold saline, opened lengthwise, and additional mucus was removed by blotting with moist tissue paper. Mucosa was freed from the underlying muscular layer by scraping with a glass slide on an icecold surface. Each tissue weight (liver, renal cortex, and intestinal mucosa) was constant from one experiment to another. Cytosolic and microsomal fractions were obtained as previously described (Peters 1988). Analytical assays GST activities in cytosolic and r m i c r o s o ~fractions were determined in the presence of l -cMoro-2,4-dinitrokmene (CBNB; 0.5 mM) as described (Habig et al. 1974). UBP-GT activiv was determined in the presence of p-nitrophenol (PNP) according to the method described by Lucier et al. (I977), with modifications (Cahnia et al. 1990). AH activity was measured by following the formation of p-aminophenol from aniline, according to Winston and Narayan (1988). Cytosolic and microsomal proteins from liver, renal cortex, and intestine were measured by the methold of Lowry et al. (1951) with bovine semm albumin as standard. Enzyme activities were expressed as nanomoles of product formed per minute per milligram of cytosolic or microsoml protein. Chemicals All the chemicals were of the highest quality commercially available. SZ, alloxan, bovine albumin, reduced glutathione, PNP, UBPGA (ammonium salt), UDP-N-acetyl glucosamine (sodium salt), and aniline were purchased from Sigma Chemical Co. (St. Louis, Mo). CBNB were purchased from Tokyo Kasei Kogyo Co. Ltd. (Japan). Statistical analysis Data were analyzed by analysis of variance with Bunnett's test for multiple comparisons against one control. A p value lower than 0.05 was judged to be significant. Regression line analysis was done by the l e s t squares method.
Results and discussion Blood ghcsse and insulin levels As expected, glucose levels were increased markedly coinciding with the decrease sf insulin levels in SZ- or alloxantreated rats compared with controls. The untreated rats showed, 4 h after the insulin injection, a 40 % increase in blood insulin levels relative to the basal value. The insulin treatment in SZ- or dloxan-diabetic rats caused a decrease in blood glucose levels that tended to normal values (see Fig. 1).
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KIDNEY
SMALL INTESTINE
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*
FIG. 2. Effect of different levels of blood insulin on GST,, UBP-GT, and AH from liver, kidney (renal cortex), and small intestine. C, control rats (n = 4); UT + I, untreated rats studied 4 h after the in~ectionof a single dose of insulin (0.2 IUIkg b.w., i.p.1 (n = 4); SZ, SZ-treated rats (50 mglkg b.w., i.v.) studied at 7 days after SZ administration (n = 6); SZ f I, SZ-diabetic rats studied 4 h after insulin administration (n = 5); AL, alloxan-treated rats (40 mglkg b.w., i.v.1 studied at 48 h after the drug administration (n = 6); AL + I, alloxandiabetic rats studied 4 h after insulin administration (n = 5). Enzyme activities are expressed as nan~molesper minute per milligram of protein. Error bars represent the SEM. * Significantly different from control values.
Enzyme activities Significant variations in cytosolic and microsomal tissue proteins were not observed throughout the treatments and, therefore enzyme activities were expressed per milligram of cytosolic or microsomal proteins. Glutathione S-transferuse activity In concordance with Our previous repon et al. 19901, a direct linear relationship (r > 0.56, p < 0.05, in all the cases) was obtained between cytosolic GST activity of the different tissues and the insulin plasma levels throughout the treatments (data not shown). Regarding GST, activity, the same linear relationship (r = 0.65, p < 0.05, n = 30) was seen in the renal cortex. On the other hand, there were no significant variations in hepatic and intestinal GST, with the different levels of blood insulin (see Fig. 2). These results suggest that the effect of insulin on GST, varies from one organ to another. The regulation by insulin observed in GST, from the three organs, but not in GST, from liver and intestine, indicates preferential induction of cytosolic enzymes in those enzymatic systems that are located in both loci, as was previously observed (Thomas et al. 1989; Catania and Carrillo 1990). Aniya et al. (1989) have also reported a difference in the
regulating mechanism of hepatic GST, and GST, in male Sprague-Dawley rats, but, in contrast with our results, they observed the regulatory action of insulin on GST,. This lack of agreement could be due to the different strain of rats used. In addition, Aniya et al. suggested that SZ causes an impairment of the gene for GST. However, we can exclude an effect of SZ, since we obtain the same behaviour with alloxan, suggesting that it is the lack of insulin that decreases the GST activity. UDP-ghcuroazyl transferuse activity Liver UBP-GT activity towards PNP diminished only in the liver of rats treated with SZ and the effect was not reversed by insulin administration. On the other hand, it did not change in alloxan-treated rats, indicating that the alteration was due to an effect of SZ and not the result of the deficiency of insulin caused by the diabetic state (see Fig. 2). Conversely, in previous work (Carnovale et al. 1987) we did not observe modifications in hepatic bilirubin UBP-GT in SZ-diabetic rats, suggesting differences in the SZ action upon different isozymes . On the other hand, intestinal PNP UDP-GT increased with SZ administration, and the effect was, again, due to the diabetogenic agent and not to the blood insulin Bevels, since
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CAN. J. PHYSHOL. PHARMACQL. VOL. 70, 1992
insulin treatment did not return the enzyme activity toward the normal value. Alloxan-diabetic rats did not show variations in PNP UDP-GT activity. Morrison and Hawksworth (1982) have suggested that the variations in glucuronidation sf PNP in SZ-treated male rats may be due to an alteration in the membrane environment of the enzyme rather than to a transferase modification by effect of SZ. It has been postulated (Mottino et al. 1991) that the lipidic environment of the hepatic UBP-GT is different from that of the intestinal UDP-GT. Thus, the alteration of the different microenvironment by SZ could be the cause of the opposite response of the UBP-GT activity in both tissues. Aniline hydroxylase activity Hepatic microsomal AH activity was increased in SZtreated rats, and the treatment with insulin did not abolish the effect of SZ. Alloxan treatment did not cause variations on AH activity indicating a SZ modification of the enzyme activity (see Fig. 2). Previous reports have shown an increase of AH activity in hepatic microsomes from alloxan- and SZ-diabetic Sprague Bawley rats (Past and Cook 1982; Rouer et al. 1985). Both laboratories have concluded that the alterations in aniline metabolism are the result of a deficiency of insulin and not an effect of the diabetogenic agent. In contrast, we have demonstrated a SZ-related effect, thus discounting a possible alteration caused by hypoinsulinemia. This difference between our observations and those made in other laboratories could be due to the different treatment schedules used in the investigations. We used acutely diabetic animals, while the cited authors used chronic diabetic rats. It is known that there are different effects of acute and chronic diabetes mellitus on hepatic dmg metabolism in the rat (Skett and Joels f 985; Watkins et al. 1988). The difference in effect might d s o be due to the strains sf rats used. On the other hand, we found that the increased rend AH activity in SZ- and allsxan-diabetic rats is diminished by the insulin treatment, without reaching basal values in the case of SZ-treated animals. These data could be in agreement with the hepatic data from Past and Cook (1982). However, we can not exclude a direct effect sf SZ on the enzyme, which dramatically increases its activity in SZ-diabetic rats. Insulin treatment decreased the higher activity without returning it to the control value. This behaviour suggests that the lack of insulin increased the activity and the restitution of the normal levels of the hormone decreased it slightly, unmasking the effect of SZ on the enzyme. In summary. our results suggest: (i) insulin regulates renal GSTm as well as cytosolic GST (Carnovale et al. 1998); (ii) SZ and (or) its metabolites has a direct effect on hepatic and intestinal PNP UDP-GT activity and on hepatic AH activity. Because SZ could impair the microsomal membrane environment, the results with PNP UDP-GT may support the hypothesis of the different composition of this environment in both these tissues. In addition, since SZ is probably converted to metabolites via the liver microsomal cytochrome B-450 enzyme system (Tjglve 1984), hepatic microsomd AH might have been induced by these metabolites.
This work was supported by a research grant from Consejo Nacional de Investigaeiones Cienta'ficas y TCcnicas (CONHCET), Argentina. The authors express their gratitude to Dr. JosC M . Pellegrino for his technical assistance.
Al-Turk, W . A., Stohs, S. J., and Roehe, E. B. 1980. Altered metabolism of 7-ethoxyeoumarin by hepatic, pulrnomry and intestinal microsomes from streptozotocin-diabetics rats. Drug Metab. Dispos. 8: 44-45. Al-Turk, W. A,, Stohs, S. J., and Rocke, E. B. 1981. Altered activities of hepatic and extrahepatic microsornal mixed function oxidase enzymes in diabetic and adrenalectomized diabetic rats. Pharmacology, 23: 337 - 345. Aniya, Y . , Ojiri, Y., Sunagawa, R., et ah, 1989. Glutathione Stransferases and chloroform toxicity in streptozotmin-induced diabetic rats. Jpn. J. Phamacol. 50: 263-269. Carnovale, C. E. Carrillo, M. C., and Rodriguez Garay, E. A. 1987. Reversible damage of hepatic excretion of bilirubin induced by streptszotocin in the rat. Comun. Biol. 5: 405 -413. Carnovale, C. E., Monti, J. A., Catania, V. A., and Carrillo, M. C. 1990. Possible role of blmd insulin levels on glutathione Stransferase activities from different tissues of male rats. Can. J. Physiol. Bhamacol. 68: 170- 173. Catania, V. A., and Carrills, M. C. 1990. Intestinal phase EE detoxification systems: effect of low-protein diet in weanling rats. Toxicol. Lett. 54: 263 -270. Catania, V. A., Luquita, M. G . , Carrills, M. C., and Mottino, A. D. 1990. Sex differences in spironolactone induction of rat intestinal and hepatic p-nitrophenol LTDP-glucuronyl transferase. Can. J. Physisl. Pharrnacol. 68: 1385- 1387. Habig, W. H., Pabst, M. J., and Jakoby, W. B. 1974. Glutathione Stransferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249: 7130-7139. Kunst, A., Draeger, B., and Ziegenhorn, J. 1983. Colorimetric methods with glucose oxidase and peroxidase. Edited by H. U. Bergmeyer. In Methods of enzymatic analysis. Vol. VI. 3rd ed. Verlar Chemie, Weiheim. gg. 178- 185. Lowry, 8.W., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Lucier, G. W., Sonawane, B. R., and MeDaniel, 01. S. 1977. Glucuronidation and deglucuronidation reactions in hepatic and extrahepatic tissues during perinatal development. Bmg Metab. Bispos. 5: 279 -287. Morrison, M. H., and Wawksworth, G . M. 1982. The effect of activators of glucuronyltransferase in streptozotocin-induced diabetic rat. Biochem. Bharmcol. 31: 1944- 1946. Mottino, A. D., Guibert, E. E., and Rodriguez Garay, E. A. 1991. Effect of spironolactone and phenobarbital on bilirubin glucuronidation in hepatic and extrahepatic microsomes. Biochem. Bharmacsl. 41: 1075- 1076. Past, M. R., and Cook, D. E. 1982. Effect of diabetes on rat liver cytochrome P-450. Evidence for a unique diabetes-dependent rat liver cytochrome P-450. Biochem. Pharmacol. 31: 3329 - 3334. Peters, W. H. M. 1988. Purification and partial characterization sf human intestinal glutathione S-transferases, Biochem. Pharmacol. 37: 2288 -2291. Reinke, L. A., Stohs, S. J., and Rosenberg, H. 1978. Altered activity of hepatic mixed-function monosxygenase enzymes in streptozotocin-induced diabetic rats. Xenobiotica, 8: 6 11 -6 19. Reinke, L. A., Stohs, S. J., and Rosenhrg, H. 1979. Increased aryl hydroxylase activity in hepatic microsomes from streptozotocindiabetic female rats. Xenobiotica, 8: 769 -778. Rouer, E., Beaune, Ph., and Leroux, J. P. 1985. Imrnunoquantitation of some cytochrome P-450 isszymes in liver microsomes from streptozstocin-diabetic rats. Experientia, 42: 1162- 1 163. Skett, B., and Joels, E. A. 1985. Different effects of acute and chronic diabetes mellitus on hepatic dmg metabolism in the rat. Biochem. Pharmacol. 34: 287 -289. Thomas, H.,Schladt, E., Knehr, M., and Oesch, IF. 1989. Effect of diabetes and starvation on the activity sf rat liver epoxide hydrolases, glutathione Stransferases and geroxisomal B-oxidation. Biochem. Pharmacol. 38: 4291 -4297. Tjalve, H. 1984. Streptozotocin: distribution, metabolism and mechanisms of action. Ugsala J. Med. Sci. 39: 145 - 157.
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Warren, B. L., Pak, R., Finlayson, M., eb (PI. 1983. Differential effects of diabetes on microsomal metabolism of various substrates. Comparison of streptozotocin and spontaneously diabetic Wistar rats. Biochem. Phamacol. 32: 327-335. Watkins, J. B., and Mangels, E. A. 1987. Hepatic biotransformation in lean and obese Wistar Kyoto rats: comparison to that in streptozotocin-treated Sprague -Dawley rats. Comp. Biochem. Physiol. $8: 159- 164. Watkins, J. B., Sanders, W. A., and Beck, L. V. 1988. The effect of long-term streptozotocin-induced diabetes on the hepatotoxicity of bromobenzene and tetrachloride and hepatic biotransformation in rats. Toxicol. Appl. Pharmacol. 93: 329-338.
Winston, G. W., and Narayan, S. 1988. Alteration of liver rnicrossmal monocsxigenases and substrate competition with aniline hydroxylase from rats chronically fed low-fat and high-fatcontaining alcohol diets. J. Biochem. Toxicol. 3: 19 1 - 2 12. Yalow, R. S.. and Berson, S. A. 1960. Inmunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39: 1157 - 1161. Yamazoe, Y., Murayama, N., Shimada, M., et a&. 1989. Cytochrome P-450 in livers of diabetic rats: regulation by growth hormone and insulin. Arch. Biochem. Biophys. 268: 567 -575.
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Templeton, J. F., Sashi Kumar, V. P., Bose, D., Smyth, D. D., Kim, R. S., and LaBella, F. %. 1988. Digitalis-like pregnanes. Cardiac m d r e d effects of a glycoside of 140-hydroxyprogesterone. Can. J. Physiol. Phamacol. 66: 1420- 1424. Templeton, J. F., %ashiKumar, V. P., Bose, D., and LaBella, F. %. 1989. Cardiac glycoside-like structure and function of 50,140pregnanes. J. Med. Chem. 33: I977 - 1981.
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Weiland, J., Schwabe, K., Hubler, D., Schonfeld, W., and Repke, K. R. H. 1987. Glycosidation of chlormadinal acetate alters its actions on Na'1K'-transporting ATPase and cardiac contractility: a contribution to the endogenous digitalis problem. J. Enzyme Inhib. 2: 31 -36.
Differential effects of blood insulin levels on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats' Institute ste Fisiokogia Experimental, Consejo Nacional de Investigaciones Cient@cas y Ticnicas, Facultad de Ciertcias Bioquimicas y Farmac&uticas, Universidad Nacional de Rosario, Suigacha 570, 2000 Rosario, Argentina
Received May 27, 1991 CARNOVALE, C. E., CATANIA, V. A., MONTI,J. A., and CARRILLO, ha. C. 1992. Differential effects of blood insulin levels on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats. Can. J. Physiol. Pharmacol. 70: 727-731. Microsomal glutathione S-transferase, UDP-glucuronyl transferase, and aniline hydroxylase activities were determined in liver, renal cortex, and small intestine of control, streptozotocin-diabetic, alloxan-diabetic. and untreated insulin-injected male Wistar rats. Renal microsomal glutathione S-transferase activity showed a direct linear relationship with insulin blood levels, in agreement with our previous report on cytosolic glutathione S-transferase. This result suggests a possible regulatory mechanism of insulin that needs to be further examined. The hepatic microsomal UDP-glucuronyl transferase was only decreased in streptozotocin-diabetic rats and was not restored by insulin treatment. Intestinal UDP-glucuronyl transferase exhibited an opposite response in streptozotocin-treated animals that was not normalized by the administration of insulin. Hepatic aniline hydroxylase showed the same behaviour as intestinal UDP-glucuronyl transferase. These results suggest that streptozotocin and (or) its metabolites have a direct effect on hepatic and intestinal UDP-glucuronyl transferase activity and on hepatic aniline hydroxylase activity. On the other hand, insulin regulation of enzyme activity varies from one organ to another. Key words: insulin, streptozotocin, alloxan, glutathione S-transferase, UDP-glucursnyl transferase, aniline hydroxylase.
V. A., MONTI,J. A., et CARRILLO, ha. C. 1992. Differential effects of blood insulin levels CARNOVALE, C. E., CATANHA, on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats. Can. J. Physisl. Pharmacol. 70 : 727-731. On a dCterminC les activitks de glutathion S-transfkrase, UDP-glucuronyl transfkrase et aniline hydroxylase dans la foie, le cortex renal et l'intestin grCle de rats Wistar non traitks sous perfusion insulinique, rendus diabktiques par alloxane, rendus diabCtiques par streptozotocine et tCmoins. L'activitk de glutathion S-transferax microsomiale rCnale a montrk une relation linCaire directe avec les taux sanguins insuliniques, ce qui est en accord avec notre rapport antkrieur sur la glutathion S-transfkrase cytosolique. Ce rCsultat suggkre un mkcanisme rkgulateur possible de l'insuline, qui devra Ctre dkmontrk. L'UDP-glucuronyl transfkrase microsomiale hkpatique a Ctk diminuCe seulement chez les rats rendus diabktiques par streptozotocine et n'a pas CtC rktablie par un traitement i?i l'insuline. L'UDP-glucuronyl transfkrase intestinale a prCsentC, chez ces mCmes animux, une rCponse inverse qui n'a pas CtC normalisCe par l'administration d'insuline. L'aniline hydroxylase hkpatique s'est conagsrtie c o m e 1'UDP-glucuronyl transfdrase intestinale. Ces rksultats suggkrent que la streptozotocine et (OU)ses mCtabolites ont un effet direct sur I'activitC de 1'UDP-glucuronyl transfkrase intestinale et hkpatique, ainsi que sur I'activitC d'aniline hydroxylase hkpatique. Par ailleurs, la rkgulation insulinique des deux organes difEre. Mots clefs : insuline, streptozotocine, alloxane, glutathion S-transfkrase, UDP-glucuronyl transfkrase, aniline hydroxylase. [Traduit par la r&action]l Introduction The effects of experimentally induced diabetes on the microsomal metabolism of xenobiotics have been studied in several hboratories (A-Turk et al 1981; Warren et al 1983; Rouer et al. 1985; Watkim and 1987)' In general, it has 1~ preliminary abstract of this paper has been published in C o m u n . Biol. 1990, 9(2): 177. 2Author for correspondence and reprint requests. Printed in Cam& / Imprim6 au Cma&
been observed that alloxan or streptszotocin (SZ) induced diabetes decreased androgen-sensitive hepatic drug metabolism in male rats, whereas in female rats the same sex-dependent activities are increased (Reinke et al. 1978, 1979). Qn the other hand, sex-independent drug-metabolizing activities are not impaired or may even be (Al-Turk et al. 1980, 1981).-~thas been reported that diabetesis a physispathological inducer of hepatic cytockrome P45Q with high activity towards aniline (fast and Cook 1982; Yamazoe et al. 1989).
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Group 11 was studied 4 h after the injection of insulin as reported by Carnovale et d. (1990). Group 111 was studied '7 days aker the SZ injection. A group of rats were injected with insulin (0.2 IU/kg b . ~, .0.1 mL) after 7 days of SZ-induced diabetes and studied 4 R aker the insulin injection. Group IV was studied 48 h aker the alloxan injection. A group of animals were injected with insulin (0.2 IU/kg b . ~ . 0.1 , mL) after 48 R of alloxan-induced diabetes and studied 4 h after insulin administration. Assessment of the induced diabetes Plasma was used to measure glucose enzymatically (GOB-PAP test Wienner Lab.) (Kunst et al. 1983) and insulin by Radioimmunoassay (RIA) (Insulin Kit, Biodata, Milano, Italy) (Yalow and Berson 1960).
FIG.I. Blood glucose and insulin levels throughout the treatments. C, control rats (n = 4); UT + I, untreated rats studied 4 h after the injection of a single dose of insulin (0.2 IU/kg b.w., i.p.) (n = 4); SZ, SZ-treated rats (50 mg/kg b.w., i.v.) studied at 7 days after SZ administration (n = 6); SZ + I, SZ-diabetic rats studied 4 h after insulin administration (n = 5); AL, alloxan-treated rats (40 mg/kg b.w., i.v.) studied at 48 R after the drug administration (n = 6); AL + I, alloxan-diabetic rats studied 4 h after insulin administration (n = 5). Error bars represent the SEM. * Significantly different from control values.
Liver and intestinal microsomal 7-ethoxycoumarin metabolism were found to be increased in rats with SZ-induced diabetes (Al-Turk et al. 1980). Besides, we have reported a direct relationship between blood insulin levels and cytosolic glutathione S-transferase (GST) activity (phase HI detoxication system) in liver, renal cortex, and intestine from male rats (Carnovale et d. 1990). The present study was aimed at elucidating if the insulin regulatory mechanism reported on cytosolic GST is also I detoxifying enzymes such as exerted on microsomal phase H GST (GST,) and UDP-glucuronyl transferase (UDP-GT), as well as the cytochrome P-450 related enzyme aniline hydroxylase (AH), from liver, renal cortex, and small intestine of male Wistar rats.
Methods Animals and treatment Male Wistar rats (280 -350 g) were used throughout. The animals were allowed free access to food and water prior to the experiments. They were randomly divided into four groups: group 1 (control), injected i.v. through femoral vein with 0.5 mL of the SZ and alloxan vechicle; group II (untreated insulin), untreated rats injected s.c. with 0.1 mL of insulin (8.2 IU/kg b.w.); group I11 (SZ-treated), injected i.v. through femoral vein with SZ (58 mg/kg b. w. in 0.5 mL of freshly prepared 0.01 M sodium citrate, pH 4.5); group IV (dloxan treated), injected i.v. through femoral vein with alloxan (40 mg/kg b. w. in 8.5 mL of freshly prepared 0.81 WE sodium citrate, pH 4.5). Group I was studied at different periods of time, in accordance with the SZ or alloxan treatment.
+
Tissue preparations Animals were bled by cardiac puncture under pentobarbital anaesthesia between 14:W and 15:W to avoid possible effects of diurnal variations. Plasma samples were used to determine glucose and insulin levels. Liver was perfused with cold saline and the largest lobe was excised. Kidneys were removed, washed in cold saline, and cortical tissues were separated. The small intestine was rinsed with cold saline, opened lengthwise, and additional mucus was removed by blotting with moist tissue paper. Mucosa was freed from the underlying muscular layer by scraping with a glass slide on an icecold surface. Each tissue weight (liver, renal cortex, and intestinal mucosa) was constant from one experiment to another. Cytosolic and microsomal fractions were obtained as previously described (Peters 1988). Analytical assays GST activities in cytosolic and r m i c r o s o ~fractions were determined in the presence of l -cMoro-2,4-dinitrokmene (CBNB; 0.5 mM) as described (Habig et al. 1974). UBP-GT activiv was determined in the presence of p-nitrophenol (PNP) according to the method described by Lucier et al. (I977), with modifications (Cahnia et al. 1990). AH activity was measured by following the formation of p-aminophenol from aniline, according to Winston and Narayan (1988). Cytosolic and microsomal proteins from liver, renal cortex, and intestine were measured by the methold of Lowry et al. (1951) with bovine semm albumin as standard. Enzyme activities were expressed as nanomoles of product formed per minute per milligram of cytosolic or microsoml protein. Chemicals All the chemicals were of the highest quality commercially available. SZ, alloxan, bovine albumin, reduced glutathione, PNP, UBPGA (ammonium salt), UDP-N-acetyl glucosamine (sodium salt), and aniline were purchased from Sigma Chemical Co. (St. Louis, Mo). CBNB were purchased from Tokyo Kasei Kogyo Co. Ltd. (Japan). Statistical analysis Data were analyzed by analysis of variance with Bunnett's test for multiple comparisons against one control. A p value lower than 0.05 was judged to be significant. Regression line analysis was done by the l e s t squares method.
Results and discussion Blood ghcsse and insulin levels As expected, glucose levels were increased markedly coinciding with the decrease sf insulin levels in SZ- or alloxantreated rats compared with controls. The untreated rats showed, 4 h after the insulin injection, a 40 % increase in blood insulin levels relative to the basal value. The insulin treatment in SZ- or dloxan-diabetic rats caused a decrease in blood glucose levels that tended to normal values (see Fig. 1).
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KIDNEY
SMALL INTESTINE
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*
FIG. 2. Effect of different levels of blood insulin on GST,, UBP-GT, and AH from liver, kidney (renal cortex), and small intestine. C, control rats (n = 4); UT + I, untreated rats studied 4 h after the in~ectionof a single dose of insulin (0.2 IUIkg b.w., i.p.1 (n = 4); SZ, SZ-treated rats (50 mglkg b.w., i.v.) studied at 7 days after SZ administration (n = 6); SZ f I, SZ-diabetic rats studied 4 h after insulin administration (n = 5); AL, alloxan-treated rats (40 mglkg b.w., i.v.1 studied at 48 h after the drug administration (n = 6); AL + I, alloxandiabetic rats studied 4 h after insulin administration (n = 5). Enzyme activities are expressed as nan~molesper minute per milligram of protein. Error bars represent the SEM. * Significantly different from control values.
Enzyme activities Significant variations in cytosolic and microsomal tissue proteins were not observed throughout the treatments and, therefore enzyme activities were expressed per milligram of cytosolic or microsomal proteins. Glutathione S-transferuse activity In concordance with Our previous repon et al. 19901, a direct linear relationship (r > 0.56, p < 0.05, in all the cases) was obtained between cytosolic GST activity of the different tissues and the insulin plasma levels throughout the treatments (data not shown). Regarding GST, activity, the same linear relationship (r = 0.65, p < 0.05, n = 30) was seen in the renal cortex. On the other hand, there were no significant variations in hepatic and intestinal GST, with the different levels of blood insulin (see Fig. 2). These results suggest that the effect of insulin on GST, varies from one organ to another. The regulation by insulin observed in GST, from the three organs, but not in GST, from liver and intestine, indicates preferential induction of cytosolic enzymes in those enzymatic systems that are located in both loci, as was previously observed (Thomas et al. 1989; Catania and Carrillo 1990). Aniya et al. (1989) have also reported a difference in the
regulating mechanism of hepatic GST, and GST, in male Sprague-Dawley rats, but, in contrast with our results, they observed the regulatory action of insulin on GST,. This lack of agreement could be due to the different strain of rats used. In addition, Aniya et al. suggested that SZ causes an impairment of the gene for GST. However, we can exclude an effect of SZ, since we obtain the same behaviour with alloxan, suggesting that it is the lack of insulin that decreases the GST activity. UDP-ghcuroazyl transferuse activity Liver UBP-GT activity towards PNP diminished only in the liver of rats treated with SZ and the effect was not reversed by insulin administration. On the other hand, it did not change in alloxan-treated rats, indicating that the alteration was due to an effect of SZ and not the result of the deficiency of insulin caused by the diabetic state (see Fig. 2). Conversely, in previous work (Carnovale et al. 1987) we did not observe modifications in hepatic bilirubin UBP-GT in SZ-diabetic rats, suggesting differences in the SZ action upon different isozymes . On the other hand, intestinal PNP UDP-GT increased with SZ administration, and the effect was, again, due to the diabetogenic agent and not to the blood insulin Bevels, since
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CAN. J. PHYSHOL. PHARMACQL. VOL. 70, 1992
insulin treatment did not return the enzyme activity toward the normal value. Alloxan-diabetic rats did not show variations in PNP UDP-GT activity. Morrison and Hawksworth (1982) have suggested that the variations in glucuronidation sf PNP in SZ-treated male rats may be due to an alteration in the membrane environment of the enzyme rather than to a transferase modification by effect of SZ. It has been postulated (Mottino et al. 1991) that the lipidic environment of the hepatic UBP-GT is different from that of the intestinal UDP-GT. Thus, the alteration of the different microenvironment by SZ could be the cause of the opposite response of the UBP-GT activity in both tissues. Aniline hydroxylase activity Hepatic microsomal AH activity was increased in SZtreated rats, and the treatment with insulin did not abolish the effect of SZ. Alloxan treatment did not cause variations on AH activity indicating a SZ modification of the enzyme activity (see Fig. 2). Previous reports have shown an increase of AH activity in hepatic microsomes from alloxan- and SZ-diabetic Sprague Bawley rats (Past and Cook 1982; Rouer et al. 1985). Both laboratories have concluded that the alterations in aniline metabolism are the result of a deficiency of insulin and not an effect of the diabetogenic agent. In contrast, we have demonstrated a SZ-related effect, thus discounting a possible alteration caused by hypoinsulinemia. This difference between our observations and those made in other laboratories could be due to the different treatment schedules used in the investigations. We used acutely diabetic animals, while the cited authors used chronic diabetic rats. It is known that there are different effects of acute and chronic diabetes mellitus on hepatic dmg metabolism in the rat (Skett and Joels f 985; Watkins et al. 1988). The difference in effect might d s o be due to the strains sf rats used. On the other hand, we found that the increased rend AH activity in SZ- and allsxan-diabetic rats is diminished by the insulin treatment, without reaching basal values in the case of SZ-treated animals. These data could be in agreement with the hepatic data from Past and Cook (1982). However, we can not exclude a direct effect sf SZ on the enzyme, which dramatically increases its activity in SZ-diabetic rats. Insulin treatment decreased the higher activity without returning it to the control value. This behaviour suggests that the lack of insulin increased the activity and the restitution of the normal levels of the hormone decreased it slightly, unmasking the effect of SZ on the enzyme. In summary. our results suggest: (i) insulin regulates renal GSTm as well as cytosolic GST (Carnovale et al. 1998); (ii) SZ and (or) its metabolites has a direct effect on hepatic and intestinal PNP UDP-GT activity and on hepatic AH activity. Because SZ could impair the microsomal membrane environment, the results with PNP UDP-GT may support the hypothesis of the different composition of this environment in both these tissues. In addition, since SZ is probably converted to metabolites via the liver microsomal cytochrome B-450 enzyme system (Tjglve 1984), hepatic microsomd AH might have been induced by these metabolites.
This work was supported by a research grant from Consejo Nacional de Investigaeiones Cienta'ficas y TCcnicas (CONHCET), Argentina. The authors express their gratitude to Dr. JosC M . Pellegrino for his technical assistance.
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