Camp. Biochem. Physiol. Vol. 103A, No. 2, pp. 417420, 1992 0300-9629/92 $5.00 + 0.00 Printed in Great Britain 0 1992 Pergamon Press Ltd

DEVELOPMENT OF HEPATIC MICROSOMAL ACTIVITY OF 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE AND CHOLESTEROL 7cr-HYDROXYLASE

IN THE YOUNG CHICK

PEDRO KARREA,* ELENA VILLAREAL, IGNACIO MOYA, PABLO AGUIRRE and JESUS PALACIOS

Department of Biochemistry and Molecular and Cellular Biology, University College of La Rioja, University of Zaragoza, C/Obispo Bustamante no. 3, 26001 Logroiio, Spain. Tel.: 34-41-24-48-11;

Fax: 34-41-25-94-31

(Received 4 November 1991; accepted 25 March 1992)

Abstract-l. The 3-hydroxy-3-methylglutaryl-CoA reductase activity increased from 1 to 4 weeks of age, but decreased from 4 to 8 weeks of age.

2. Cholesterol 7a-hydroxylase activity increased from 1 to 4 weeks, decreased from 4 to 6 weeks, and increased again from 6 to 8 weeks of age.

3. Serum total and free cholesterol concentrations decreased from 1 to 6 weeks of age, but increased from 6 to 8 weeks of age.

INTRODUCTION

Young chicks present substantial variations in diges- tive function during the first weeks of age. Freeman (1976), Katongole and March (1980), and Polin and Hussein (1982) described that fat digestibility increase until 8 weeks of age, concluding that the absorptive mechanisms for lipid and protein are not fully devel- oped in the young chick. Gomez and Polin (1976) and Katongole and March (1980) reported that increases in the apparent absorption of fat were observed when bile acids (BA) were included in diet, giving evidence of this immature condition in digestive function. Krogdahl(1985) reported that changes in BA concen- tration and secretion were observed in the proximal jejunum during the first weeks of age in chicks, concluding that BA secretion seems to be the limiting factor in lipid digestion in young chicks. Finally, Iiiarrea et al. (1989) have observed changes in the concentration of biliary and serum BA during the first weeks after hatching.

Cholesterol metabolism and diet could play very important roles in concentration changes of biliary BA. The present study was performed to determine cholesterol metabolism implications, by feeding a fat-free animal diet. The experiment investigated the development of hepatic micro- somal activities of 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA) reductase (EC 1 .l .1.34) and cholesterol 7a-hydroxylase (EC 1.14.-.-), the respect- ive rate controlling enzyme activities for cholesterol (Linn, 1967) and bile acid (Danielsson et al., 1967) synthesis, and the evolution of serum lipids levels and glucose in chicks at 1,2,3,4,5,6,7 and 8 weeks after hatching.

*To whom correspondence should be addressed.

MATERIALS AND METHODS

Animals

Ninety-two l-day-old Warren chicks, Callus domesticus, obtained from Explotacion Agraria Ejemplar “Casas Blan- cas” (Rodezno, La Rioja, Spain), were used in this study. The chicks were kept in electrically heated, thermostatically controlled battery brooders, and were fed the diet described in Table 1. Samples were obtained from six chicks at 1, 2, 3. 4. 5. 6. 7 and 8 weeks of age. All birds were killed bv decapitation and blood samples were collected. The livers were removed, washed, chilled on ice, weighed and prepared for analyses. Serum samples were frozen at -30°C until assayed.

Chemicals and enzymes

pL-3-Hydroxy-3-methyl-[3-‘4C]glutaryl-CoA (47.4 mCi/ mmol), and [4-‘4C]cholesterol (59.4 mCi/mmol) were pur- chased from New England Nuclear, Boston, MA). The RS-mevalonic acid, glucose-6-phosphate, B-nicotinamide adenine dinucleotide phosphate, CoA, glucose-6-phosphate dehydrogenase, DL-HMG-CoA, cholesterol, 7-ketocholes- terol were obtained from Sigma Chemical Co. (St Louis, MO). Triton X-100, ethylenediaminetetraacetic acid di- sodium salt, adenosine triphosphate and dithiothreitol were obtained from Merck (Darmstadt, Germany). Leupeptin hemisulfate and trypsin inhibitor from soybeans was ob- tained from Fluka (Buchs, Switzerland). ‘la-Hydroxycholes- terol was prepared by reduction of 7-ketocholesterol with sodium borohydrate (Malinow et al., 1975). All the other chemicals were of the highest grade available commercially.

Enzyme assays

Microsomes from the livers were prepared and treated as described Burger er al. (1984). Assays for HMG-CoA reductase and cholesterol 7a-hydroxylase were carried out as described by Shapiro Ed al. (1974) and Hylemon et nl. (1985).

Free and total cholesterol, triglycerides, phospholipids, free fatty acids (FFA), and glucose concentrations in serum samples were estimated enzymatically as described by Allain et al. (1974), Richmond (1973), Fossati and Principe (1982),

417

418 PEDRO IRARREA et al.

Table I. Composition of diet

Amount Ingredients and analysis (g per kg of diet)

Corn 381.4 Soybean meal 43% protein 296.4 Barley 246.9 Alfalfa dehydrated 20 Linoleic acid 13 Delipided milk protein IO m-Methionine 1.5 Sodium chloride 2 Dicalcium phosphate 18.8 Calcium carbonate 6 Vitamin and mineral mix* 4 Calculated analysis of diet:

ME (kcal/kg) 2800 Protein (%) 19.0 Fat (%) 3.38 Methionine (%) 0.45 Calcium (%) I .oo Phosphorus (available, %) 0.45

*Supplies per kilogram: vitamin A, 10,0001.U.; vitamin D,, 2400 I.U.; vitamin E, 9.0 I.U.; menadione sodium bisulphite, 2.5mg; vitamin B,, 4.5mg; vitamin B,, 1.1 mg; vitamin B,,, 0.015 mg; niacin, 25 mg; pantothenic acid, 7.5 mg; folacin, 0.5 mg; choline chloride, 275 mg; Zn, 64 mg; Mn, 93 mg; Fe, 38 mg; Cu, 5 mg; Co, 0.5 mg; Se, 1 mg.

Takayama et al. (1977), Mizuno et al. (1980), and Barham and Trinder (1972), respectively. High density lipoprotein (HDL) was separated as described Lopez-Virella et al.

(1977) and its cholesterol level determined. The estimations of cholesterol in low density lipoprotein (LDL) were calcu- lated from the difference between total cholesterol and cholesterol in HDL plus triglycerides divided by 5 (Burger er al., 1984). Reagents were obtained from bioMCrieux (Marcy l’Etoile, France).

Analyses of variance were used to estimate the statistical differences (Dunn and Clark, 1974). Tukey’s method was used for mean separation. Estimates of correlation co- efficients among variables were based on mean values.

RESULTS

Hepatic HMG-CoA reductase activity progress- ively increased from 1 to 4 weeks and then decreased from 4 to 7 weeks of age (P < 0.05; Table 2). Hepatic cholesterol 7a-hydroxylase activity showed a pro- gressive increase from 1 to 4 weeks and a particular decrease at 6 weeks of age (P c 0.05; Table 2). Serum total cholesterol concentration decreased from 1 to 6 weeks (P < 0.05) and increased from 7 to 8 weeks of age (Table 2). Serum free cholesterol concentration decreased from 1 to 3 weeks, and increased from 7 to 8 weeks of age (P < 0.05; Table 2). The free choles-

terol: total cholesterol ratio decreased from 2 to 3 weeks (0.2W.14), and increased progressively from 3 to 6 weeks of age. An inverse correlation was ob- served between the development of hepatic HMG- CoA reductase activity and that of serum free cholesterol concentration (r = -0.74, P < 0.005).

The LDL cholesterol concentration increased from 2 to 5 weeks, and did not vary from 5 to 8 weeks of age (P < 0.05; Table 2). The HDL cholesterol con- centration decreased from 2 to 5 weeks, but increased at 7 and 8 weeks of age (P < 0.05; Table 2). The LDL cholesterol : total cholesterol ratio increased from 2 to 5 weeks (0.374.74), and decreased progressively from 6 to 8 weeks of age (0.714.49). The HDL cholesterol: total cholesterol ratio decreased from 2 to 5 weeks (0.5W.19), and increased progressively from 5 to 8 weeks of age (0.22-0.45). The LDL cholesterol : HDL cholesterol ratio increased from 2 to 5 weeks (0.65-4.00), and decreased progressively from 6 to 8 weeks of age (3.30-1.10).

Serum triglyceride and phospholipid concen- trations decreased progressively until 4 weeks of age (P < 0.05; Table 3). A correlation was noted between the change in serum total cholesterol and that of the serum triglyceride concentration (r = 0.80, P < 0.05) and phospholipid concentration (Y = 0.81, P < 0.05). Serum FFA concentration showed an irregular change with maximal values at 2,5 and 8 weeks of age (P < 0.05; Table 3). Serum glucose concentration also showed an irregular change with minimal values at 2, 5, 6 and 8 weeks of age (P < 0.05; Table 3).

DISCUSSION

Development of hepatic HMG-CoA reductase ac- tivity presented a transitory increase while serum total and free cholesterol concentrations decreased. Moreover, the proportion in LDL cholesterol changed inversely with variations of total serum cholesterol level from 2 to 8 weeks of age. This result suggests a constant LDL cholesterol biosynthesis: catabolism ratio and a transitory decrease of the HDL cholesterol biosynthesis : catabolism ratio. The intestinal biosynthesis, reabsorption and release of cholesterol could vary (Alejandre er al., 1981), and the endogenous and/or reabsorbed cholesterol and absorbed fat could exert changing effects on the feedback control of hepatic cholesterol synthesis and release in the young chick.

Table 2. Development of hepatic enzyme activities and levels of serum cholesterol

Serum (mg/lOO ml)

Age HMG-CoA* Cholesterol? Total Free Cholesterol Cholesterol LDL-chol: (weeks) reductasc ‘la-hydroxylase cholesterol cholesterol LDL HDL Total-chol

I 374 * 48a 4.7 f 0.4a I51 + l2a 32 k 2a 101 ?r l0a 38 * 3x 0.69 i_ 0. IOa 2 988 + 78b 6.6 + 0.7ab 120+3b 24 f 2b 44 + 5b 67*4b 0.37 + 0.05b 3 I266 +_ 67~ 10.0 k 0.6bd 112+2b I6 k Ic M) + 6bc 44 * 4a 0.54 + 0.06~ 4 1593f6od 15.6 + 0.8~ 100 + 3bcd 171 Ic 62+6bc 28 f 2cd 0.62 f O.OBac 5 I378 f 7Ocd 13.2 + l.Odc 87f4cd l5+_ Ic 64 i 5c 16 + 2d 0.74 f 0. IOa 6 835 + 3lbe 10.7 + 0.7bd 79 + 4d I6 + Ic 56i5bc 17&2d 0.71 * 0.1 la 7 654 + 50ef 14.4 + 0.8~ 106 + 6bc 21+2bc 60+7bc 39 f 5ac 0.57 + 0.06c 8 577 f 37af 16.7 f 0.9~ 112+ IOb 22 + 2b 55+5bc 50 + 6a 0.49 + 0.06bc

Mean f SEM of six chicks in duplicate. l Picomoles of mevalonic acid synthesized per minute per milligram of microsomal protein. TPicomoles of [“Cjcholesterol into 7a-[“C]hydroxycholesterol per minute per milligram of microsomal protein. (a-e) Concentration values followed by the same letter within a column are not significantly different (P < 0.05).

LDL-chol: HDL-chol

2.65 k 0.26ab 0.65 f 0.07b I .36 k 0.14bc 2.21 + 0.2Oacd 4.00 * 0.37e 3.30 f 0.3lde 1.53+0.14&c l.l0tO.I2bc

Cholesterol metabolism in the chick 419

Table 3. Evolution of serum lioids and nlucose

Serum (mg/lOO ml)

AS (weeks) Triglycerides

I 62.2 + 2.7a 2 45.7 k 2.9bc 3 43.7 + 2.4bc 4 51.9 & 3.5b 5 35.7 * 1.9c 6 32.9 k 1.8~ 7 35.8 * 1.3~ 8 37.0 + 1.4c

Choline phospholipids

188.6 + 6.2a 181.6 + 6.la 175.4 f 1.7a 150.4 f 6.3b 144.6 f 6.3b 132.6 i 3.lb 153.6 & 4.9b 135.0 + 3.8b

FFA’

21.0 + I.Oa 26.2 f 1.4b 13.6 _+ I.Ocd 11.8 k 0.5~ 16.3 + 0.7de 13.1 f l.Ocd 11.9f0.7C 18.7 + I.Oae

Glucose

267.2 + 5.0a 238.1 f 2.9b 259.3 + 2.9a 265.7 f 2.4a 235.5 + 3.0b 232.7 f 3.8be 243.7 f 3.lb 218.1 + 2.3~

Mean f SEM of six chicks in duplicate. *Free fatty acids. (a-e) Concentration values followed by the same letter within a column are not significantly

different (P < 0.05).

Values of hepatic HMG-CoA reductase activity were similar to those described by Alejandre et al. (1981), at 1 and 2 weeks of age in male White- Leghorn chicks, and by Burger et al. (1984), at 7 and 8 weeks of age, with higher serum cholesterol levels in female broiler chickens and female White-Leghorn chicks, respectively, using different diets. This value was greater than the one described by Qureshi et al. (1983), at 8 weeks of age, with higher serum choles- terol levels in male broiler chickens. Values of LDL and HDL cholesterol levels were smaller than those reported by Burger et al. (1984) at 7 weeks of age, but the LDL cholesterol : HDL cholesterol ratio was simi- lar. Values of LDL and HDL cholesterol levels were lower and similar, respectively, to the ones reported by Qureshi et al. (1983) at 8 weeks of age, and the LDL cholesterol: HDL cholesterol ratio was smaller. Total serum cholesterol level was similar to the results obtained by Diller et al. (1960) at 5 weeks of age in male White-Leghorn chicks but lower than those previously observed after feeding them a diet that contained animal fat (Ifiarrea et al., 1989).

Cholesterol 7a-hydroxylase activity varied from 1 to 6 weeks in a similar way to hepatic HMG-CoA reductase activity, but it did not decrease at 7 to 8 weeks, suggesting that no increases in the intestinal absorption of BA and/or changing effects on the feedback control of hepatic BA synthesis in the growing chick could occur at these last ages studied. Values of cholesterol 7a-hydroxylase activity were higher than those reported in the chick (Qureshi et al., 1983; Burger et al., 1984). Optimal hydroxylase specific activity using 0.3 mg/ml of microsomal pro- tein in the enzyme assay was found. Hepatic HMG- CoA reductase and cholesterol 7a-hydroxylase activities, and HDL cholesterol concentration evol- utions (Schwartz et al., 1978) could contribute to the changes of proximal jejunal and biliary BA concen- tration previously reported (Krogdahl, 1985; Iiiarrea et al., 1989).

Serum triglyceride and phospholipid concen- trations showed an early decrease and then plateaued, in agreement with the conclusions of an immature condition of digestive function in the young chicks. Development of serum triglycerides concentrations was inversely related to the gradual development of BA reabsorption and serum BA concentrations (Beil et al., 1982; Itiarrea et al., 1989), but directly related

a reflection of the development in BA and pancreatic lipase secretion (Katangole and March, 1980; Krog dahl, 1985; Ifiarrea et al., 1989). Values of serum triglyceride level were smaller than those reported by Burger et al. (1984) at 7 weeks of age in female broiler chickens, by Kubena et al. (1988, 1989) and Bailey et al. (1989) at 3 weeks of age in male broiler chickens, and by Huff et al. (1988) in male broiler chickens. Diet and strain could be principal causes of the differences found.

FFA concentrations generally decreased with age. Glucose concentrations also showed a general de- crease but with inverse changes to FFA concen- tration, following a caloric homeostasis behaviour (Fredrickson and Gordon, 1958) and suggesting a more effective metabolic combustible utilization. Val- ues of serum glucose level were smaller than those reported by Kubena et al. (1988) and Bailey et al. (1989) at 3 weeks of age in male broiler chickens.

These results show that changes in the hepatic cholesterol metabolism occur in the young chick fed a fat-free animal diet, and that changes of intestinal lipid absorption and of lipoprotein metabolism could be related.

SUMMARY

Hepatic microsomal activities of 3-hydroxy-3- methylglutaryl-CoA reductase and cholesterol 7cr- hydroxylase, serum concentrations of cholesterol, triglycerides, choline phospholipids, free fatty acids and glucose were determined in chicks at 1, 2, 3, 4, 5, 6, 7 and 8 weeks of age. The 3-hydroxy-3-methyl- glutaryl-CoA reductase activity increased from 1 to 4 weeks of age, but decreased from 4 to 8 weeks of age. Cholesterol 7a-hydroxylase activity increased from 1 to 4 weeks, decreased from 4 to 6 weeks, and increased again from 6 to 8 weeks of age. Serum total and free cholesterol concentrations decreased from 1 to 6 weeks of age, but increased from 6 to 8 weeks of age. The low density lipoprotein (LDL) choles- terol: total cholesterol ratio increased from 2 to 5 weeks (0.37-0.74), and decreased progressively from 6 to 8 weeks of age (0.714.49). The LDL-choles- terol : high density lipoprotein (HDL)-cholesterol ratio increased from 2 to 5 weeks (0.654.00), and decreased progressively from 6 to 8 weeks of age

to the concentration of fatty acid binding protein as (3.30-1.10).

420 PEDRO h&4 dtltEA et al.

Acknowledgements-This work was supported by grants from Instituto de Estudios Riojanos, Convenio Caja de Ahorros de Zaragoza Aragon y Rioja-University of Zaragoza, and Spanish Ministry of Education and Science. Appreciation is expressed to General de Piensos S.A. (GENSA), and Explotacion Ejemplar Casas Blancas.

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