Circannual variations in mevalonate utilization in frog (Rana esculenta)

Comp. Biochem. Physiol.Vol. 97B,No. 3, pp. 597~500, 1990 Printed in Great Britain

0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie

CIRCANNUAL VARIATIONS IN MEVALONATE UTILIZATION IN FROG (RANA ESCULENTA) G. BRUSCALUPI,F. CASTELLANOand A. TRENTALANCE Dipartimento di Biologia Cellulare e dello Sviluppo, Universita' di Roma I "La Sapienza", Piazza A. Moro 5, 00185 Roma, Italy (Received 9 May 1990) Abstract--1. The fate of mevalonate, the product of HMGCoA reductase, was studied in male and female

frogs (Rana esculenta) in order to explain the circannual variations of enzyme activity. 2. The incorporation of 2-14CMVA into unsaponifiable lipids, cholesterol and dolichol in liver, plasma and eggs was followed. 3. Labeled MVA shows a different utilization depending on season and sex. In spring and summer cholesterol synthesis is related to hepatic reserve storage in both sexes, while the peak of enzyme activity, present only in females in fall, seems committed to cholesterol export into the blood and uptake by the oocytes. 4. The presence of a MVA-derivedprotein identifiablewith vitellogeninand labeled on the lipid moiety, suggests that HMGCoA reductase activity in fall is committed to the lipidation of this protein essential for oocyte maturation.

INTRODUCTION Lipid metabolism in frogs presents circannual fluctuations reflecting environmental changes and physiological needs, namely gametogenesis. Variations of respiratory quotient in Rana pipiens suggest a differential utilization of energetic sources during the year with a preferential consumption of lipids in summer (Fromm and Johnson, 1955). Changes in weight, composition and enzymatic activities of liver throughout the year have been reported (Pasanen and Koskela, 1974; Schlageke and Blum, 1974; Chieffi et al., 1980; Rastogi et al., 1983; Scapin et al., 1984; Bruscalupi et al., 1989) probably related to fluctuations of blood hormones, in particular sex hormones. Actually both estrogens (D'Istria et al., 1974; Paolucci and Botte, 1988) and androgens (Specker and Moore, 1980) vary during the year with the higher levels between fall and winter. In the same period an increase of vitellogenin, the precursor of egg yolk proteins, is detectable in female plasma (Giorgi et al., 1982). Its synthesis in the liver, the uptake by oocytes and the enzymatic cleavage into yolk proteins lipovitellin and phosvitin seem to be estrogen-induced processes (Wallace and Dupont, 1968; Follet and Redshaw, 1974). We have previously reported circannual variations of hepatic cholesterol content with a peak in spring in both sexes (Bruscalupi et al., 1989). In this period 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase, the key enzyme of cholesterogenesis, is high in both sexes, while in fall it shows an extra peak only in females without corresponding cholesterol storage. This suggests that cholesterol produced by HMGCoA reductase is exported into the blood for some extra hepatic requirement, such as gametogenesis. In order to verify this hypothesis, we followed the fate of mevalonate (MVA), the direct product of the enzyme, supplied to frogs (Rana esculenta) of

both sexes "in vivo" as 2-14C-MVA. The presence of labeled unsaponifiable lipids, cholesterol and dolichols in liver, plasma and eggs was measured. Moreover, the presence of any radioactive plasma protein in both sexes was analyzed. MATERIALS AND METHODS

Animals Male and female frogs, Rana esculenta, (23.2 + 5.5g average body weight) kept in an outdoor terrarium were collected at random every season. Frogs were injected into the dorsal sac with 0.16/zCi 2-14C-MVA/g body wt and killed 15 hr later. Animals were killed after anaesthesia with 0.2% MS222 (tricaine methane sulphonate). Blood was collected by cardiac puncture with a heparinized syringe in the presence of 1 mM phenylmethylsulfonylfluoride and plasma prepared by centrifugation at 3000 rpm for 10 min. Liver and eggs were rapidly removed and weighed. Some male frogs were injected with 0.5 g of benzoate estradiol into the dorsal sac and killed 10 days later, when the levels of circulating vitellogenin are high (Smith et aL, 1978). Egg preparation Eggs freshly removed were homogenized in 5 vols of 0.15 M NaCI. After two washes with the same vols, they were resuspended in 3 vols of 0.5 M NaC1 and 500 #1 were used for cholesterol determination. Cholesterol extraction Cholesterol extraction was performed on 500 #1 of egg homogenate, chopped liver (500 mg) and 100 #1 of plasma according to Trentalance et al. (1984). Briefly,samples were precipitated with 10% TCA and TCA insoluble material was saponified in 30% alcoholic KOH. After the addition of aH cholesterol working as an internal standard, unsaponifiable lipids were extracted three times with petroleum benzene and separated by TLC with benzene:ethylacetate (5:l,v/v) as solvent system. Lipids were revealed by iodine vapors, spots scraped into scintillation vials and radioactivity measured in a Packard Tri-carb scintillation counter.

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Dolichol extraction

This was performed on 500 mg of chopped liver according to Marino et al. (1988). Samples were saponified in 60% KOH in the presence of 0.25% pyrogallol. After saponification, unsaponifiable lipids were extracted with diethyl ether/petroleum benzene (I:1, v/v). The extract, washed with methanol:H20 (1 : 1, v/v) was separated with chromatography on Sep-Pak C18 Cartridge with different proportions of propanol:methanol. Each fraction was chromatographed on TLC silica gel with 1% methanol in benzene as the solvent system. Lipids were revealed by iodine vapors, spots scraped into scintillation vials and radioactivity counted.

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Electrophoresis o f plasma proteins

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Spring Summer

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Dolichyl-P

This was performed on 100 #g protein under denaturing conditions according to Laemmli (1970) and under native conditions according to Davis (1964). Proteins were stained with Coomassie Brilliant Blue. Native gels were sliced into 0.5cm segments and solubilized with protosol:H20 (9: 1, v/v) at 60°C for 1 hr and radioactivity measured after the addition of the scintillation cocktail.

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Protein assay

Proteins were measured according to Lowry et al. (1951), using bovine serum albumin as the standard.

RESULTS AND DISCUSSION Seasonal and sex related changes of 14C-MVA conversion into hepatic unsaponifiable lipids and cholesterol were followed, and the results are reported in Table 1. Incorporation of 2-14C labeled MVA into liver unsaponifiable lipids showed a similar pattern in both sexes, with an increase in those periods (spring and summer) characterized by reserve storage. Incorporation into liver cholesterol was significantly higher in females, except in summer, and represented almost 40% of total unsaponifiable iipids in spring and fall. No difference in the synthesis of dolichol and dolichyl-phosphate was detectable in male and female frogs. As shown in Fig. 1, in both sexes labeled MVA incorporation had a huge increase in summer into dolichol and in summer and spring into dolichylphosphate. In both sexes 2-]4C-MVA incorporation into hepatic dolichyl-phosphate was always higher than into dolichol. Radioactivity incorporation into total unsaponifiable lipids and cholesterol of plasma was also measured. As shown in Table 2, a higher incorporation of 2-14C-MVA into unsaponifiable lipids in female plasma was detectable in winter and fall and almost totally ascribable to cholesterol. When the presence of labeled lipids in eggs was considered, the incorporation of MVA-derived radioactivity into un-

UL (dpm)

Winter

Spring S u m m e r Fall Season Male ~ Female Fig. 1. Incorporation of 2-14C-MVA into hepatic dolichol and dolichyl-phosphate of male and female frogs. 2-14C MVA was injected into the dorsal sac, animals were killed 15 hr later and dolichol and dolichyl-phosphate extracted from the liver as described in Materials and Methods. saponifiable lipids and cholesterol showed a similar pattern and a progressive increase in fall and winter (Fig. 2). The incorporation of 2-C-MVA into plasma proteins was studied in both sexes and in estrogenized males, measuring the radioactivity in solubilized gel fractions. After 5% S D S - P A G E separation, winter female and estrogenized male plasma showed the appearance of a peptide with a mol. wt of 180,000-200,000 comigrating with the vitellogenin standard. No radioactivity was measurable in any sample analyzed in denaturing conditions. Radioactivity distribution of proteins separated by native gel electrophoresis is reported in Fig. 3. No labeled components were detectable in male plasma; one labeled gel fraction was detectable in winter female and in estrogenized male plasma. Rf value of both these fractions was 0.24, corresponding to the relative mobility of authentic vitellogenin in the same electrophoretic system. Data reported here suggest that MVA is preferentially utilized for cholesterol than for dolichols synthesis and that hepatic cholesterol production has different roles depending on season and sex; in

Table 1. Incorporation of 2J4C-MVA into hepatic lipids Male Female C C/UL UL C (dpm) (%) (dpm) (dpm)

C/UL (%)

Season Winter 654,962_+107,19 64,438 _+4594 9.6 670,013:J:67,653 103,739_+5511 15.50 Spring 1,196,657_+36,487 100,063_ 37,967 8.2 1,192,727+ 249,287 452,128± 29,255 37.90 Summer 2,308,640+_363,452 109,903_+30,445 4.7 1,996,250± 280,557 "11,591__3503 0.05 Fall 444,675± 102,645 84,165+ 12,124 19.0 596,596+ 95,504 215,053± 13,745 36.00 Frogs were injectedwith 0.16#Ci/g body wt and killed 15 hr later and unsaponifiablelipids (UL) and cholesterol (C) extracted from 0.5 g liver as described in Materials and Methods. Data reported are the average of three determinations+_SD.

Mevalonate utilization in frog Table

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2. Incorporationof 2J4C-MVAinto plasma lipids

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C

C/UL

UL

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Season Winter 4062 1008 24.8 14,500 10,381 71.6 Spring 8250 3734 45.0 7718 1519 19.6 Summer 6125 2426 39.0 4437 768 17.0 Fall 8004 5885 74.0 21,300 20,945 98.3 Frogs were injected with 0.16/~Ci/g body wt and killed 15hr later and total unsaponifiablelipids(UL) and cholesterol(C) extractedfrom 100 #1 plasmaas described in Materialsand Methods. particular in females newly synthesized cholesterol seems mostly committed to lipid accumulation in spring and summer and to the export into the blood to allow the uptake by the growing oocytes in fall. Moreover in this period of the year the appearance in plasma of a labeled MVA-derived protein, comigrating with standard vitellogenin, is detectable. Its labeling is due to the lipid moiety since it is detectable only in non-denaturing conditions. A labeled protein with identical migration properties is also detectable in plasma of the estrogenized male. All these observations indicated that the hepatic H M G C o A reductase activity in fall is at least partially committed to the production of this lipoprotein. This conclusion is supported by the following considerations: vitellogenin is synthesized in the liver by an estrogen-mediated process that increases gene transcription (Brock and Shapiro, 1983); its molecule has a high cholesterol content (Smith et aL, 1978) that seems essential for its function; estrogens have a stimulatory effect on H M G C o A reductase activity

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(Philipp and Shapiro, 1981). Our results provide indications that the estrogen induction of vitellogcnin production is accomplished by increasing both the synthesis of the protein portion and the activity of H M G C o A reductase essential for the lipidation of the molecule. Differently from cholesterol, the production of dolichol from MVA in both sexes presents similar seasonal patterns. The MVA conversion into

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dolichyl-P is much higher than into dolichol. This result disagrees with the hepatic content of these compounds that we have previously reported (Marino et al., 1988), suggesting a modification of the turnover or interconversion process. The increased synthesis in summer of dolichyl-P, a key intermediate in the glycosylation process, is consistent with the high N-acetylglucosamine incorporation into glycoproteins (Leoni, pers. commun). Both these processes could be related to the intense metabolic activity and the high request of glycosylated molecules in this period. On the other hand, the enhanced synthesis of dolichol, a molecule suggested to be involved in maintaining the stability of the membrane bilayer, could be related to the modulation of the activity of membrane associated enzyme, particularly requested in poikilothermic animals. Acknowledgements--Vitellogenin standard was a kind gift of Professor A. Polzonetti-Magni (Universita' di Camerino, Italy). Part of the data presented in this paper is the outcome of the final thesis work of Dr. D. Marcozzi and Dr. P. Manzi. This work was partially supported by a 1988 grant from Ministero della Pubblica Istruzione of Italy. REFERENCES

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cycle of Rana esculenta L. Comp. Biochem. Physiol. 72B, 501-506. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lowry O. H., Rosebrough N. J., Karr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Marino M., Girelli A. M., Leoni S. and Trentalance A. (1988) Frog liver dolichols: separation and quantitative determination related to seasonality. Comp. Biochem. Physiol. 91B, 193-195. Paolucci M. and Botte V. (1988) Estradiol-binding molecules in the hepatocytes of the female water frog Rana esculenta and plasma estradiol and vitellogenin levels during the reproductive cycle. Gen. comp. Endocr. 70, 466-476. Pasanen S. and Koskela P. (1974) Seasonal and age variation in the metabolism of the common frog Rana temporaria L. in Northern Finland. Comp. Biochem. Physiol. 47A, 653~54. Philipp B. W. and Shapiro D. T. (1981) Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl CoA reductase and acetyl CoA carboxylase in Xenopus laevis. J. biol. Chem. 256, 2922-2927. Rastogi R. K., Izza-Vitiello, Di Meglio M., Di Matteo L., Franzese R., Di Costanzo M. G., Minucci S., Iela L. and Chietii G. (1983) Ovarian activity and reproduction in the frog Rana esculenta. J. Zool. 200, 233-247. Scapin S., Autuori F., Baldini P., Incerpi S., Luly P. and Sartori C. (1984) Tyrosine aminotransferase activity of frog (Rana esculenta) liver III. A circannual study. Comp. Biochem. Physiol. 79B, 369-374. Schlageke R. and Blum V. (1974) Seasonal variations in fat body metabolism of the green frog Rana esculenta (L.). Experienta 34, 456-457. Smith D. F., Penning T. M., Ansari A. Q., Munday K. A. and Akhtar M. (1978) Oestrogen-induced cholesterol and fatty acid biosynthesis in Xenopus laevis liver during vitellogenic response. Biochem. J. 174, 353-361. Specker J. L. and Moore F. L. (1980) Annual cycle of plasma androgens and testicular composition in the rough-skinned newt, Tachira granulosa. Gen. comp. Endocr. 42, 297-303. Trentalance A., Leoni S., Magiantini M. T., Spagnuolo S., Feingold K., Hughes-Fulford M., Siperstein M., Cooper A. and Erickson S. K. (1984) Regulation of 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol synthesis and esterification during the first cell cycle of liver regeneration. Biochim. biophys. Acta 794, 142-151. Wallace R. A. and Dupont J. N. (1968) The induced synthesis and transport of yolk proteins and their accumulation by the oocyte in Xenopus laevis. J. Cell. Physiol. 72(suppl), 73-89.