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Effect of 1,3-Butylene Glycol on Growth and In Vivo and In Vitro Lipogenesis by Turkey Poults
METABOLISM AND NUTRITION Effect of 1,3-Butylene Glycol on Growth and In Vivo and In Vitro Lipogenesis by Turkey Poults R. W. ROSEBROUGH and N. C. STEELE Nonruminant Animal Nutrition Laboratory, US Department of Agriculture, Beltsville, Maryland 20705 (Received for publication September 12, 1980)
1981 Poultry Science 60:1448-1453
INTRODUCTION Lipid metabolism has been extensively studied in t h e chick (Leveille et al., 1 9 6 8 ; O'Hea and Leveille, 1 9 6 8 , 1 9 6 9 ) . Avian lipid metabolism, like t h a t in t h e rat, is increased by mealfeeding (Muiruri et al., 1975) and decreased b y increasing t h e protein level in t h e diet (Leveille and Yeh, 1 9 7 2 ) ; unlike t h a t in t h e rat, it is decreased b y insulin (Hazlewood, 1 9 7 1 ) . T h e liver is also t h e major point of de novo fatty acid synthesis in chicks; adipose tissue serves mainly for storage of fatty acids (Goodridge, 1 9 6 8 ) . T h e controlling steps in avian lipogenesis also p r o b a b l y are different from those of t h e m a m m a l . T h e avian liver does n o t contain a high capacity glucose p h o s p h o r y l a t i o n system as does t h e m a m m a l i a n liver; therefore, t h e initial step in glycolysis m a y be limiting in birds (Leveille et al, 1 9 7 5 ) . T h e fact t h a t fatty acid C o A derivatives inhibit acetyl CoA carboxylase in chick liver suggests t h a t fatty acid activation may partly inhibit lipogenesis in birds (Goodridge et al, 1 9 7 4 ) . Also, unlike t h e m a m m a l , t h e newly h a t c h e d chick m u s t a d a p t t o a high c a r b o h y d r a t e diet in t h e " n e o n a t a l " period, which is unlike t h e diet c o n s u m e d in ovo. A l t h o u g h m u c h w o r k has been d o n e o n t h e regulation of c a r b o h y d r a t e and lipid m e tabolism in t h e chick during t h e p o s t h a t c h period, few studies have elucidated m e t a b o l i c p a t h w a y s in t h e developing t u r k e y p o u l t . T h e t u r k e y differs from t h e chicken in t h a t t h e
growing period is m u c h longer; therefore, observations m a d e with 21-day-old chicks may n o t be applicable t o t h e t u r k e y poult. Also, t h e t u r k e y poult has a m u c h higher protein requirem e n t (30 vs. 23%) than does t h e chick, and this r e q u i r e m e n t m a y c o m p o u n d i n t e r p r e t a t i o n of lipogenic rates derived from chick data. T h e purposes of t h e experiments were 1) to examine b o t h in vitro and in vivo lipogenesis in t h e t u r k e y p o u l t and 2) t o c o m p a r e t h e growth and lipogenic rates of t u r k e y poults fed 0, 1 2 . 5 , and 2 5 % of their dietary energy as 1,3-butylene glycol (BG). Butylene glycol has been extensively used as a t o o l to investigate t h e regulation of lipogenesis in rats. T h e hepatic conversion of BG of B - h y d r o x y b u t y r a t e induces ketosis b y decreasing t h e availability of free c o e n z y m e A for oxidative metabolism. The subsequent increase in acylated c o e n z y m e A decreases t h e activity of ATP-citrate lyase, an e n z y m e which reflects lipogenesis. Thus, a dietary t r e a t m e n t t h a t decreases c o e n z y m e A availability ( R o m s o s et al., 1975a) illustrates t h e possible control of de novo lipogenesis in t h e poult. EXPERIMENTAL PROCEDURE Animals. A series of 21 day growth trials were c o n d u c t e d with Large White t u r k e y poults. Day-old poults were r a n d o m l y assigned t o pens (12 per pen) in electrically heated b a t t e r y b r o o d e r s . T h e poults were fed o n e of
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ABSTRACT A series of feeding trials lasting 21 days was conducted with Large White turkey poults to determine the effects of 0, 12.5, and 25% energy as 1,3-butylene glycol (BG) on growth and on both in vivo and in vitro lipogenesis. The substitution of 12.5 and 25% of the energy as BG depressed growth and feed efficiency of 21-day-old poults (P<.01). The relative liver size was increased by BG (P<.01) while liver lipid per 100 g of body weight was decreased (P<.01) by BG. In vivo lipogenesis, determined by the incorporation of tritiated water into liver fatty acids, was decreased (P<05) by BG. The evolvement of C 0 2 from both (1- 14 C) acetate and from (U-' 4 C) glucose was decreased by BG. The results of this study indicate that while lipogenesis can be decreased by BG, growth is also decreased. Therefore, the regulation of growth parallels the regulation of lipid synthesis in the turkey poult. (Key words: lipogenesis, growth, regulation of metabolism, turkey poult, butylene glycol)
EFFECT OF 1,3-BUTYLENE GLYCOL ON GROWTH AND LIPOGENESIS three diets calculated t o contain 0, 1 2 . 5 , or 2 5 % of t h e metabolizable energy as BG (Table 1). A n h y d r o u s dextrose was purchased from Corn Products Inc., Englewood Cliffs, NJ and was calculated to provide 4.0 kcal ME/g. Butylene glycol was provided by N o r m a n Baker of Celanese Chemical Co., New York, NY. Miller and Dymsza's ( 1 9 6 7 ) value of 6.0 kcal ME/g was used for BG. Each diet was replicated eight times; each pen was considered as a dietary replication. Feed and water were provided ad libitum t h r o u g h o u t t h e 21-day
New England Nuclear, Boston, MA. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other suitable products.
trials. Final weights of poults were d e t e r m i n e d after a 4 hr fasting period. In Vivo Lipogenesis. At t h e e n d of t h e trial period, t w o birds were r a n d o m l y selected from each pen and injected intraperitoneally with tritiated water in .9% saline. Doses were adjusted so t h a t each bird received 100 /iCi/100 g of b o d y weight. After 30 m i n , t h e poults were sacrificed and t h e livers excised. Liver lipid was extracted with 20 volumes of 2 : 1 chloroformm e t h a n o l at 6 0 C for 2 4 hr (Folch et ah, 1 9 5 7 ) . The lipid extract was saponified with 10% potassium h y d r o x i d e in m e t h a n o l . After acidification with c o n c e n t r a t e d hydrochloric acid, t h e fatty acid fraction was removed with t w o successive h e x a n e washes, evaporated t o dryness, and dispersed in Aquasol-2. 1 Radioactivity was determined by liquid scintillation spectroscopy and corrected for quench by t h e channels ratio m e t h o d . Activity was t h e n
TABLE 1. Composition of the experimental dietsS Dietary energy at 1,3-butylene glycol % 12.5
Ingredient
Soybean meal (49% protein) Ground yellow corn Meat and bone meal (50% protein) Alfalfa meal (17% protein) Dried whey Isolated soy protein (C—1) Soybean oil Iodized salt Selenium premix a D, L-methionine Dicalcium phosphate (anhydrous) Vitamin mix 0 Trace mineral mix c Glucose^ 1,3-butylene glycol e Cellulose^
490 121 50 25 25 19 40 3 1 1 22 5 1 200
(g/kg) 490 191 50 25 25 19 40 3 1 1 22 5 1 100 67 33
25
490 191 50 25 25 19 40 3 1 1 22 5 1 134 66
Provides .1 mg of Se/kg of diet. Provides per kilogram of diet: vitamin A, 12000 IU; vitamin D 3 , 3000 ICU; vitamin E, 38 IU; vitamin K, 10 mg; thiamine, 10 mg; riboflavin, 10 mg; pyridoxine, 10 mg; pantothenic acid, 20 mg; niacin, 100 mg; biotin, 2mg;choline 2000 mg; folic acid, 2 mg; vitamin B 1 2 , 100 Mg; ethoxyquin, 150 mg. c
Supplies milligrams per kilogram of diet: Mn, 100; Zn, 100; Fe, 100; Cu, 10; Co, 1; I, 3.
Anhydrous dextrose (Cerelose-2000), Corn Products, Englewood Cliffs, NJ. Calculated to contain 4 kcal ME/g. Graciously provided by Norman Baker, Celanese Chemical Company, New York, NY. Calculated to contain 6 kcal ME/g. f Solka-floc, Brown Company, Brown, NH. "Diets were calculated to contain 300 g of protein, 18.8 g of lysine, 5.9 g of methionine, 4.6 g of cysteine, and 3 200 kcal ME/kg of diet.
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content and liver size. The overall null hypothesis of inequality of means was tested with an analysis of variance while differences between pairs of means was tested with Duncan's new multiple range test (Kirk, 1968). RESULTS AND DISCUSSION The results of this study show that BG decreases growth rate and feed efficiency (P<.01) of 21-day-old turkey poults (Table 2). These findings with turkey poults are different from those of Davenport and Griffith (1969), which showed that 5% dietary BG, providing 11% of the caloric intake, did not depress chick growth rates or those of Romsos et al. (1975b), which showed that 27% of the dietary energy as BG was required to decrease chick growth rates. The lower level of BG used in the present study (6.7%) provided 12.5% of the poults energy intake and decreased growth while the similar level did not affect chick growth. Perhaps there is some species difference between chicks and poults concerning tolerance to energy substitutions at an early age. Dietary BG is a known method of inducing ketosis (Tobin et al., 1972); therefore, the turkey poult may also be intolerant of nutritional ketosis. Because feed intakes were similar for all diet treatments and the diets were calculated to be isocaloric, the results of this study are due to differences in the utilization of similar metabolizable energy intakes from different energy sources and not to an inhibition of feed intake. The results of this study can also be contrasted with those of
TABLE 2. The effect of dietary 1,3-butylene glycol (BG) on growth, liver lipid, and in vivo lipogenesis by turkey poults (mean ± SE; n = 8per each dietary treatment group) Dietary energy as BG, % 0 Weight at 21 days, g Total feed intake, g Feed/gain Relative liver size, g of liver/100 g of body weight Liver lipid, mg/100 g of body weight In vivo lipogenesis1
1
12.5 525.3 ± 6.3a 730.1 ± 36.5 1.56 ± .06 a 2.20 ±
.12a
181.72+ .59 a 3185 ± 99a
25
436.6 ± 7.9 b 740.7 ±12.1 1.74 ± . 0 3 b 2.90 ±
.05b
122.10 ± .79 b 2720 ± 79 b
Analysis of variance
435.8 ± 11.0 b 775.7 ± 19.6 2.06 + .04C
P<.01 NS P<.01
.llb
P-C01
119.80 + .63b 2163 ± 103 c
P<.01 P<.01
2.88 ±
' ' Means within a row followed by a common superscript are not statistically different (P>.01). Disintegrations per minute of tritiated water incorporated/100 g of body weight into liver fatty acids.
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expressed as disintegrations per minute per 100 g of body weight recovered as fatty acids. Total lipid content per liver was also determined gravimetrically. In Vitro Lipogenesis. Two poults from each pen were randomly selected, sacrificed, and livers excised. Livers were sliced (150 to 200 mg) with a Stadie-Riggs hand microtome and incubated in 3 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM sodium acetate and 5 mM glucose. The buffer was also routinely supplemented with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 2% bovine serum albumin. One microcurie of either D-(U-14C) glucose or (1- 14 C) acetate was added to determine lipogenic rates from either glucose or acetate. The slices were incubated for 2 hr at 39 C in 25 ml Erlenmeyer flasks. After the 2 hr period, .1 ml of 30% KOH was injected onto filter paper in the center wells of the incubation flasks to trap C 0 2 , and .3 ml of 4N H 2 S 0 4 was injected into the medium to stop the reaction and liberate CO2. The slices were incubated for an additional 1 hr to ensure adequate trapping of CO2, and the filter papers were then placed in Aquasol-2. The liver slices were extracted in 10 ml of 2:1 chloroform-methanol for 24 hr at 60 C. The lipid extract was saponified with 5% potassium hydroxide in methanol and acidified with concentrated hydrochloric acid. Fatty acids were extracted with two successive hexane washes. The evolution of CO2 and the de novo lipogenesis from the radioactive substrates were expressed on a relative liver size to avoid complications caused by differences in lipid
EFFECT OF 1,3-BUTYLENE GLYCOL ON GROWTH AND LIPOGENESIS
1965). Although independent of the source of acetyl groups, the tritiated water method nonetheless is related to acetate incorporation into fatty acids (Lowenstein, 1970). A comparison between in vivo lipogenesis and liver lipid content indicates that at the 25% BG energy level the decrease in lipogenesis was not accompanied by a further decrease in liver lipid. It is important to note that liver lipid content may be a result of long-term metabolism and may not be totally related to short-term rates of lipogenesis such as tritiated water incorporation into fatty acids. Butylene glycol decreases the in vitro oxidation of both glucose and acetate as well as the use of both of these substrates for lipogenesis (P<.05; Table 3). The decrease in lipogenesis from glucose noted in poults fed 25% BG agrees with the chick data of Romsos et al. (1975b); however, when absolute values are compared with those of the chick, the turkey poult appears to have a lesser ability to convert glucose to fatty acids. Therefore, the poult, even though of similar weight to the chick at 21 days of age, may be metabolically less mature than the chick. The decrease in glucose and acetate oxidation accompanying the decrease in lipogenesis suggests a dual control on metabolism elicited by feeding BG: first, a decrease in the metabolism of glucose through glycolysis and, second, a decrease in the addition of two carbon units during lipogenesis by a decrease in coenzyme A availability. Work by Mehlman and Veech (1972) demonstrated that dietary BG increased
TABLE 3. The effect of dietary 1,3-butylene glycol (BG) on in vitro lipogenesis by turkey poults (mean ± SE; n = 8 per each dietary treatment group)
Substrate (U- 14 C) glucose Analysis of variance (1- 14 C) acetate Analysis of variance
Dietary energy asBG, %
C 0 2 , Mmoles/100g of body weight
0 12.5 25.0
1.58+ 1.02 ± .83 + P<.01 20.94 ± 18.61 ± 14.3 5 ± P<.05
0 12.5 25.0
.17 a .09 b .08 c 1.84 a 1.55 a 1.77 b
Lipid synthesis, Mmoles/ 100 g of body weigh td 2.18 ± 1.85 ± 1.28 + P<.05 8.04 ± 3.90 ± 3.66 ± P<.05
.18 a .18a .26b 1.45 a .80b .76b
a ' 'cMeans within a column for each isotopically labeled substrate followed by a common superscript are not statistically different (P>.05). Lipid synthesis expressed as Mmoles of (1-14C) sodium acetate or (U-14C) glucose incorporated into fatty acids per 2 hr incubation time.
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Mehlman et al. (1966) and Stoewsand et al. (1965), which showed that differences in weight gain by rats consuming BG could be directly attributable to differences in feed intake rather than to differences in the utilization of dietary energy derived from BG. An analysis of the effects of BG on liver weight and lipid content reveals that, in contrast to results with the chick, BG increases (P<.01; Table 2) the relative liver size of the turkey poult. Butylene glycol also decreases (P<.01) liver lipid content, although, as with liver weight, the substitution of 25% of the energy as BG had no more effect than did substitution of 12.5%. An examination of in vivo lipogenesis indicates that BG decreases (P<.01; Table 2) the incorporation of tritiated water into liver fatty acids. The relatively short period of labeling we used insured that the label did not translocate and that fatty acids synthesized during this time remained in the liver (Leveille, 1969). The use of tritiated water as an indicator of in vivo lipogenesis is superior to the use of specific isotopically labeled lipid precursors such as ( 14 C) glucose or ( 14 C) acetate. In particular, the use of labeled acetate requires the assumption that the rate of incorporation of acetate into acetyl coenzyme A is similar for all treatments and that acetate specific activities in vivo are also similar, regardless of treatments (Romsos and Leveille, 1974). In contrast, the use of tritiated water measures the total lipogenic rate because tritium is introduced into fatty acids only during lipogenesis (Fain and Skow,
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In conclusion, we have s h o w n t h a t dietary BG decreases in vivo and in vitro lipogenesis b y t h e t u r k e y poult. Moreover, t h e decrease in liver fat n o t e d in t h e present s t u d y was related t o t h e feeding of BG, although t h e role of t h e hepatic redox state is not clear cut. T h e decrease in lipogenesis caused by dietary BG could n o t be a t t r i b u t e d t o changes in feed intake as feed intakes were similar for all dietary t r e a t m e n t s . Because BG was s u b s t i t u t e d isocalorically for glucose, energy intakes were also similar for all dietary t r e a t m e n t s . T h e results of this s t u d y also indicate t h a t dietary energy c a n n o t be partitioned into t h a t required for lean tissue growth o r lipid synthesis, because an energy source t h a t is an inhibitor of lipogenesis also decreases t h e a p p a r e n t growth rates.
REFERENCES Davenport, R. F„ and M. Griffith, 1969. Effect of varying levels of dietary 1,3-butanediol on growing chicks. Poultry Sci. 4 8 : 1 3 6 5 - 1371. Fain, J. N., and R. O. Skow, 1965. Fatty acid synthesis in vivo in maternal and fetal tissues in the rat. Amer. J. Physiol. 210: 1 9 - 2 5 . Folch, J., M. Lees, and G. H. Sloane-Stanley, 1957. A simple method for the isolation and purification
of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Goodridge, A. G., 1968. Metabolism of glucoseU- 14 C in vitro in adipose tissue from embryonic and growing chicks. Amer. J. Physiol. 214: 897-901. Goodridge, A. G., A. Garay, and P. Silpananta, 1974. Regulation of lipogenesis and the total activities of lipogenic enzymes in the primary culture of hepatocytes from prenatal and early postnatal chicks. J. Biol. Chem. 249:1469-1475. Hazlewood, R. L., 1971. Endocrine control of avian carbohydrate metabolism. Poultry Sci. 50:9—18. Kirk, R. E., 1968. Experimental design procedures for the behavioral sciences. Wadsworth Publ. Co., Belmont, CA. Leveille, G. A., 1969. In vitro hepatic lipogenesis in the hen and chick. Comp. Biochem. Physiol. 28:431-43 5. Leveille, G. A., E. K. O'Hea, and K. Chakabarty, 1968. In vivo lipogenesis in the domestic chicken. Proc. Soc. Exp. Biol. Med. 128: 3 9 8 - 4 0 1 . Leveille, G. A., D. R. Romsos, Y. Y. Yeh, and E. K. O'Hea, 1975. Lipid biosynthesis in the chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanisms. Poultry Sci. 54:1075-1093. LeveUle, G. A., and Y. Y. Yeh, 1972. Influence of intermittent fasting on protein-free feeding on lipid metabolism in young cockerels. J. Nutr. 102:733-740. Lowenstein, J., 1970. Effect of (-)-hydroxycitrate on fatty acid synthesis by rat liver in vivo. J. Biol. Chem. 246:629-632. Mehlman, M. A., D. G. Therriault, W. Porter, G. S. Stoewsand, and H. A. Dymsza, 1966. Distribution of lipids in rats fed 1,3-butanediol. J. Nutr. 88:215-218. Mehlman, M. A., and R. L. Veech, 1972. Redox and phosphorylation states and metabolite concentrations in frozen clamped livers of rats fed diets containing 1,3-butanediol and DL-carnitine. J. Nutr. 102:45-51. Miller, S. A., and H. A. Dymsza, 1967. Utilization by the rat of 1,3-butanediol as a synthetic source of dietary energy. J. Nutr. 79—87. Muiruri, K. L., D. R. Romsos, and G. A. Leveille. 1975. Influence of meal frequency on in vivo hepatic fatty acid synthesis, lipogenic enzyme and glucose tolerance in the chicken. J. Nutr. 105:963-971. O'Hea, E. K., and G. A. Leveille, 1968. Lipogenesis in isolated adipose tissue of the domestic chick (Gallus domesticus). Comp. Biochem. Biophys. 26:111-120. O'Hea, E. K., and G. A. Leveille, 1969. Lipid biosynthesis and transport in the domestic chick (Gallus domesticus). Comp. Biochem. Physiol. 30: 149-159. Romsos, D. R., P. S. Belo, and G. A. Leveille, 1974. Effect of 1,3-butanediol on hepatic fatty acid synthesis and metabolite levels in the rat. J. Nutr. 104:1438-1445. Romsos, D. R., P. S. Belo, and G. A. Leveille, 1975a. Butanediol and lipid metabolism. Fed. Proc. 34:2186-2190. Romsos, D. R., P. S. Belo, E. R. Miller, and G. A.
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acetyl c o e n z y m e A and decreased c o e n z y m e A availability. Acetate use for b o t h oxidation a n d lipogenesis can be limited by t h e availability of c o e n z y m e A (Yeh and Leveille, 1 9 7 1 ) . T h e integration of BG into lipid and carboh y d r a t e metabolism involves t h e f o r m a t i o n of B - h y d r o x y b u t y a t e and a c e t o a c e t a t e by t h e liver and a change in t h e cytoplasmic r e d o x state ( R o m s o s et ah, 1 9 7 5 a ) . T h e increase in t h e cytoplasmic r e d o x s t a t e decreases glycolysis and glucose utilization for lipogenesis in rats. A n increase in hepatic r e d o x also was accompanied b y an increase in acyl c o e n z y m e A's, a decrease in fatty acid o x i d a t i o n , and a decrease in citrate cleavage activity (Romso's et ah, 1 9 7 4 ) . Although R o m s o s et ah ( 1 9 7 5 b ) did n o t show a change in hepatic r e d o x in chicks following t h e feeding of BG, we have f o u n d t h a t BG increases t h e r e d o x state and decreases t h e liver fat of t h e t u r k e y hen (Rosebrough et ah, 1 9 8 0 ) . F u r t h e r m o r e , R o m s o s et ah ( 1 9 7 5 b ) reported t h a t while BG did n o t affect glucose or acetate conversion t o fatty acids by chick liver, BG did increase t h e r e d o x of circulating metabolites in chicks. It is possible t h a t t h e relationship b e t w e e n circulating and hepatic redox metabolites is n o t as defined in t h e bird as in t h e rat.
EFFECT OF 1,3-BUTYLENE GLYCOL ON GROWTH AND LIPOGENESIS Leveille, 1975b. Influence of dietary 1,3-butanediol on weight gain, blood and liver metabolites, and lipogenesis in the pig and chick J. Nutr. 105:161-170. Romsos, D. R., and G. A. Leveille, 1974. Effect of dietary fructose on in vitro and in vivo fatty acid synthesis in the rat Biochim. Biophys. Acta 360:1-11. Rosebrough, R. W., E. Geis, P. James, H. Ota, and J. Whitehead, 1980. Effects of dietary energy substitutions on reproductive performance, feed efficiency, and lipogenic enzyme activity in Large White turkey hens. Poultry Sci. 59:
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1485-1492. Stoewsand, G. A., H. A. Dymsza, M. A. Mehlman, and D. G. Therriault, 1965. Influence of 1,3-butanediol on tissue lipids of cold-exposed rats. J. Nutr. 87:464-^68. Tobin, R. B., M. A. Mehlman, and M. Parker, 1972. Effect of 1,3-butanediol and propionic acid on blood ketones, lipids and metal ions in rats. J. Nutr. 102:1001-1008. Yeh, Y. Y., and G. A. Leveille, 1971. Studies on the relationship between lipogenesis and the level of coenzyme A derivatives, lactate and pyruvate in chick liver. J. Nutr. 101: 911-918.
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1981 Poultry Science 60:1448-1453
INTRODUCTION Lipid metabolism has been extensively studied in t h e chick (Leveille et al., 1 9 6 8 ; O'Hea and Leveille, 1 9 6 8 , 1 9 6 9 ) . Avian lipid metabolism, like t h a t in t h e rat, is increased by mealfeeding (Muiruri et al., 1975) and decreased b y increasing t h e protein level in t h e diet (Leveille and Yeh, 1 9 7 2 ) ; unlike t h a t in t h e rat, it is decreased b y insulin (Hazlewood, 1 9 7 1 ) . T h e liver is also t h e major point of de novo fatty acid synthesis in chicks; adipose tissue serves mainly for storage of fatty acids (Goodridge, 1 9 6 8 ) . T h e controlling steps in avian lipogenesis also p r o b a b l y are different from those of t h e m a m m a l . T h e avian liver does n o t contain a high capacity glucose p h o s p h o r y l a t i o n system as does t h e m a m m a l i a n liver; therefore, t h e initial step in glycolysis m a y be limiting in birds (Leveille et al, 1 9 7 5 ) . T h e fact t h a t fatty acid C o A derivatives inhibit acetyl CoA carboxylase in chick liver suggests t h a t fatty acid activation may partly inhibit lipogenesis in birds (Goodridge et al, 1 9 7 4 ) . Also, unlike t h e m a m m a l , t h e newly h a t c h e d chick m u s t a d a p t t o a high c a r b o h y d r a t e diet in t h e " n e o n a t a l " period, which is unlike t h e diet c o n s u m e d in ovo. A l t h o u g h m u c h w o r k has been d o n e o n t h e regulation of c a r b o h y d r a t e and lipid m e tabolism in t h e chick during t h e p o s t h a t c h period, few studies have elucidated m e t a b o l i c p a t h w a y s in t h e developing t u r k e y p o u l t . T h e t u r k e y differs from t h e chicken in t h a t t h e
growing period is m u c h longer; therefore, observations m a d e with 21-day-old chicks may n o t be applicable t o t h e t u r k e y poult. Also, t h e t u r k e y poult has a m u c h higher protein requirem e n t (30 vs. 23%) than does t h e chick, and this r e q u i r e m e n t m a y c o m p o u n d i n t e r p r e t a t i o n of lipogenic rates derived from chick data. T h e purposes of t h e experiments were 1) to examine b o t h in vitro and in vivo lipogenesis in t h e t u r k e y p o u l t and 2) t o c o m p a r e t h e growth and lipogenic rates of t u r k e y poults fed 0, 1 2 . 5 , and 2 5 % of their dietary energy as 1,3-butylene glycol (BG). Butylene glycol has been extensively used as a t o o l to investigate t h e regulation of lipogenesis in rats. T h e hepatic conversion of BG of B - h y d r o x y b u t y r a t e induces ketosis b y decreasing t h e availability of free c o e n z y m e A for oxidative metabolism. The subsequent increase in acylated c o e n z y m e A decreases t h e activity of ATP-citrate lyase, an e n z y m e which reflects lipogenesis. Thus, a dietary t r e a t m e n t t h a t decreases c o e n z y m e A availability ( R o m s o s et al., 1975a) illustrates t h e possible control of de novo lipogenesis in t h e poult. EXPERIMENTAL PROCEDURE Animals. A series of 21 day growth trials were c o n d u c t e d with Large White t u r k e y poults. Day-old poults were r a n d o m l y assigned t o pens (12 per pen) in electrically heated b a t t e r y b r o o d e r s . T h e poults were fed o n e of
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ABSTRACT A series of feeding trials lasting 21 days was conducted with Large White turkey poults to determine the effects of 0, 12.5, and 25% energy as 1,3-butylene glycol (BG) on growth and on both in vivo and in vitro lipogenesis. The substitution of 12.5 and 25% of the energy as BG depressed growth and feed efficiency of 21-day-old poults (P<.01). The relative liver size was increased by BG (P<.01) while liver lipid per 100 g of body weight was decreased (P<.01) by BG. In vivo lipogenesis, determined by the incorporation of tritiated water into liver fatty acids, was decreased (P<05) by BG. The evolvement of C 0 2 from both (1- 14 C) acetate and from (U-' 4 C) glucose was decreased by BG. The results of this study indicate that while lipogenesis can be decreased by BG, growth is also decreased. Therefore, the regulation of growth parallels the regulation of lipid synthesis in the turkey poult. (Key words: lipogenesis, growth, regulation of metabolism, turkey poult, butylene glycol)
EFFECT OF 1,3-BUTYLENE GLYCOL ON GROWTH AND LIPOGENESIS three diets calculated t o contain 0, 1 2 . 5 , or 2 5 % of t h e metabolizable energy as BG (Table 1). A n h y d r o u s dextrose was purchased from Corn Products Inc., Englewood Cliffs, NJ and was calculated to provide 4.0 kcal ME/g. Butylene glycol was provided by N o r m a n Baker of Celanese Chemical Co., New York, NY. Miller and Dymsza's ( 1 9 6 7 ) value of 6.0 kcal ME/g was used for BG. Each diet was replicated eight times; each pen was considered as a dietary replication. Feed and water were provided ad libitum t h r o u g h o u t t h e 21-day
New England Nuclear, Boston, MA. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other suitable products.
trials. Final weights of poults were d e t e r m i n e d after a 4 hr fasting period. In Vivo Lipogenesis. At t h e e n d of t h e trial period, t w o birds were r a n d o m l y selected from each pen and injected intraperitoneally with tritiated water in .9% saline. Doses were adjusted so t h a t each bird received 100 /iCi/100 g of b o d y weight. After 30 m i n , t h e poults were sacrificed and t h e livers excised. Liver lipid was extracted with 20 volumes of 2 : 1 chloroformm e t h a n o l at 6 0 C for 2 4 hr (Folch et ah, 1 9 5 7 ) . The lipid extract was saponified with 10% potassium h y d r o x i d e in m e t h a n o l . After acidification with c o n c e n t r a t e d hydrochloric acid, t h e fatty acid fraction was removed with t w o successive h e x a n e washes, evaporated t o dryness, and dispersed in Aquasol-2. 1 Radioactivity was determined by liquid scintillation spectroscopy and corrected for quench by t h e channels ratio m e t h o d . Activity was t h e n
TABLE 1. Composition of the experimental dietsS Dietary energy at 1,3-butylene glycol % 12.5
Ingredient
Soybean meal (49% protein) Ground yellow corn Meat and bone meal (50% protein) Alfalfa meal (17% protein) Dried whey Isolated soy protein (C—1) Soybean oil Iodized salt Selenium premix a D, L-methionine Dicalcium phosphate (anhydrous) Vitamin mix 0 Trace mineral mix c Glucose^ 1,3-butylene glycol e Cellulose^
490 121 50 25 25 19 40 3 1 1 22 5 1 200
(g/kg) 490 191 50 25 25 19 40 3 1 1 22 5 1 100 67 33
25
490 191 50 25 25 19 40 3 1 1 22 5 1 134 66
Provides .1 mg of Se/kg of diet. Provides per kilogram of diet: vitamin A, 12000 IU; vitamin D 3 , 3000 ICU; vitamin E, 38 IU; vitamin K, 10 mg; thiamine, 10 mg; riboflavin, 10 mg; pyridoxine, 10 mg; pantothenic acid, 20 mg; niacin, 100 mg; biotin, 2mg;choline 2000 mg; folic acid, 2 mg; vitamin B 1 2 , 100 Mg; ethoxyquin, 150 mg. c
Supplies milligrams per kilogram of diet: Mn, 100; Zn, 100; Fe, 100; Cu, 10; Co, 1; I, 3.
Anhydrous dextrose (Cerelose-2000), Corn Products, Englewood Cliffs, NJ. Calculated to contain 4 kcal ME/g. Graciously provided by Norman Baker, Celanese Chemical Company, New York, NY. Calculated to contain 6 kcal ME/g. f Solka-floc, Brown Company, Brown, NH. "Diets were calculated to contain 300 g of protein, 18.8 g of lysine, 5.9 g of methionine, 4.6 g of cysteine, and 3 200 kcal ME/kg of diet.
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content and liver size. The overall null hypothesis of inequality of means was tested with an analysis of variance while differences between pairs of means was tested with Duncan's new multiple range test (Kirk, 1968). RESULTS AND DISCUSSION The results of this study show that BG decreases growth rate and feed efficiency (P<.01) of 21-day-old turkey poults (Table 2). These findings with turkey poults are different from those of Davenport and Griffith (1969), which showed that 5% dietary BG, providing 11% of the caloric intake, did not depress chick growth rates or those of Romsos et al. (1975b), which showed that 27% of the dietary energy as BG was required to decrease chick growth rates. The lower level of BG used in the present study (6.7%) provided 12.5% of the poults energy intake and decreased growth while the similar level did not affect chick growth. Perhaps there is some species difference between chicks and poults concerning tolerance to energy substitutions at an early age. Dietary BG is a known method of inducing ketosis (Tobin et al., 1972); therefore, the turkey poult may also be intolerant of nutritional ketosis. Because feed intakes were similar for all diet treatments and the diets were calculated to be isocaloric, the results of this study are due to differences in the utilization of similar metabolizable energy intakes from different energy sources and not to an inhibition of feed intake. The results of this study can also be contrasted with those of
TABLE 2. The effect of dietary 1,3-butylene glycol (BG) on growth, liver lipid, and in vivo lipogenesis by turkey poults (mean ± SE; n = 8per each dietary treatment group) Dietary energy as BG, % 0 Weight at 21 days, g Total feed intake, g Feed/gain Relative liver size, g of liver/100 g of body weight Liver lipid, mg/100 g of body weight In vivo lipogenesis1
1
12.5 525.3 ± 6.3a 730.1 ± 36.5 1.56 ± .06 a 2.20 ±
.12a
181.72+ .59 a 3185 ± 99a
25
436.6 ± 7.9 b 740.7 ±12.1 1.74 ± . 0 3 b 2.90 ±
.05b
122.10 ± .79 b 2720 ± 79 b
Analysis of variance
435.8 ± 11.0 b 775.7 ± 19.6 2.06 + .04C
P<.01 NS P<.01
.llb
P-C01
119.80 + .63b 2163 ± 103 c
P<.01 P<.01
2.88 ±
' ' Means within a row followed by a common superscript are not statistically different (P>.01). Disintegrations per minute of tritiated water incorporated/100 g of body weight into liver fatty acids.
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expressed as disintegrations per minute per 100 g of body weight recovered as fatty acids. Total lipid content per liver was also determined gravimetrically. In Vitro Lipogenesis. Two poults from each pen were randomly selected, sacrificed, and livers excised. Livers were sliced (150 to 200 mg) with a Stadie-Riggs hand microtome and incubated in 3 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM sodium acetate and 5 mM glucose. The buffer was also routinely supplemented with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 2% bovine serum albumin. One microcurie of either D-(U-14C) glucose or (1- 14 C) acetate was added to determine lipogenic rates from either glucose or acetate. The slices were incubated for 2 hr at 39 C in 25 ml Erlenmeyer flasks. After the 2 hr period, .1 ml of 30% KOH was injected onto filter paper in the center wells of the incubation flasks to trap C 0 2 , and .3 ml of 4N H 2 S 0 4 was injected into the medium to stop the reaction and liberate CO2. The slices were incubated for an additional 1 hr to ensure adequate trapping of CO2, and the filter papers were then placed in Aquasol-2. The liver slices were extracted in 10 ml of 2:1 chloroform-methanol for 24 hr at 60 C. The lipid extract was saponified with 5% potassium hydroxide in methanol and acidified with concentrated hydrochloric acid. Fatty acids were extracted with two successive hexane washes. The evolution of CO2 and the de novo lipogenesis from the radioactive substrates were expressed on a relative liver size to avoid complications caused by differences in lipid
EFFECT OF 1,3-BUTYLENE GLYCOL ON GROWTH AND LIPOGENESIS
1965). Although independent of the source of acetyl groups, the tritiated water method nonetheless is related to acetate incorporation into fatty acids (Lowenstein, 1970). A comparison between in vivo lipogenesis and liver lipid content indicates that at the 25% BG energy level the decrease in lipogenesis was not accompanied by a further decrease in liver lipid. It is important to note that liver lipid content may be a result of long-term metabolism and may not be totally related to short-term rates of lipogenesis such as tritiated water incorporation into fatty acids. Butylene glycol decreases the in vitro oxidation of both glucose and acetate as well as the use of both of these substrates for lipogenesis (P<.05; Table 3). The decrease in lipogenesis from glucose noted in poults fed 25% BG agrees with the chick data of Romsos et al. (1975b); however, when absolute values are compared with those of the chick, the turkey poult appears to have a lesser ability to convert glucose to fatty acids. Therefore, the poult, even though of similar weight to the chick at 21 days of age, may be metabolically less mature than the chick. The decrease in glucose and acetate oxidation accompanying the decrease in lipogenesis suggests a dual control on metabolism elicited by feeding BG: first, a decrease in the metabolism of glucose through glycolysis and, second, a decrease in the addition of two carbon units during lipogenesis by a decrease in coenzyme A availability. Work by Mehlman and Veech (1972) demonstrated that dietary BG increased
TABLE 3. The effect of dietary 1,3-butylene glycol (BG) on in vitro lipogenesis by turkey poults (mean ± SE; n = 8 per each dietary treatment group)
Substrate (U- 14 C) glucose Analysis of variance (1- 14 C) acetate Analysis of variance
Dietary energy asBG, %
C 0 2 , Mmoles/100g of body weight
0 12.5 25.0
1.58+ 1.02 ± .83 + P<.01 20.94 ± 18.61 ± 14.3 5 ± P<.05
0 12.5 25.0
.17 a .09 b .08 c 1.84 a 1.55 a 1.77 b
Lipid synthesis, Mmoles/ 100 g of body weigh td 2.18 ± 1.85 ± 1.28 + P<.05 8.04 ± 3.90 ± 3.66 ± P<.05
.18 a .18a .26b 1.45 a .80b .76b
a ' 'cMeans within a column for each isotopically labeled substrate followed by a common superscript are not statistically different (P>.05). Lipid synthesis expressed as Mmoles of (1-14C) sodium acetate or (U-14C) glucose incorporated into fatty acids per 2 hr incubation time.
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Mehlman et al. (1966) and Stoewsand et al. (1965), which showed that differences in weight gain by rats consuming BG could be directly attributable to differences in feed intake rather than to differences in the utilization of dietary energy derived from BG. An analysis of the effects of BG on liver weight and lipid content reveals that, in contrast to results with the chick, BG increases (P<.01; Table 2) the relative liver size of the turkey poult. Butylene glycol also decreases (P<.01) liver lipid content, although, as with liver weight, the substitution of 25% of the energy as BG had no more effect than did substitution of 12.5%. An examination of in vivo lipogenesis indicates that BG decreases (P<.01; Table 2) the incorporation of tritiated water into liver fatty acids. The relatively short period of labeling we used insured that the label did not translocate and that fatty acids synthesized during this time remained in the liver (Leveille, 1969). The use of tritiated water as an indicator of in vivo lipogenesis is superior to the use of specific isotopically labeled lipid precursors such as ( 14 C) glucose or ( 14 C) acetate. In particular, the use of labeled acetate requires the assumption that the rate of incorporation of acetate into acetyl coenzyme A is similar for all treatments and that acetate specific activities in vivo are also similar, regardless of treatments (Romsos and Leveille, 1974). In contrast, the use of tritiated water measures the total lipogenic rate because tritium is introduced into fatty acids only during lipogenesis (Fain and Skow,
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In conclusion, we have s h o w n t h a t dietary BG decreases in vivo and in vitro lipogenesis b y t h e t u r k e y poult. Moreover, t h e decrease in liver fat n o t e d in t h e present s t u d y was related t o t h e feeding of BG, although t h e role of t h e hepatic redox state is not clear cut. T h e decrease in lipogenesis caused by dietary BG could n o t be a t t r i b u t e d t o changes in feed intake as feed intakes were similar for all dietary t r e a t m e n t s . Because BG was s u b s t i t u t e d isocalorically for glucose, energy intakes were also similar for all dietary t r e a t m e n t s . T h e results of this s t u d y also indicate t h a t dietary energy c a n n o t be partitioned into t h a t required for lean tissue growth o r lipid synthesis, because an energy source t h a t is an inhibitor of lipogenesis also decreases t h e a p p a r e n t growth rates.
REFERENCES Davenport, R. F„ and M. Griffith, 1969. Effect of varying levels of dietary 1,3-butanediol on growing chicks. Poultry Sci. 4 8 : 1 3 6 5 - 1371. Fain, J. N., and R. O. Skow, 1965. Fatty acid synthesis in vivo in maternal and fetal tissues in the rat. Amer. J. Physiol. 210: 1 9 - 2 5 . Folch, J., M. Lees, and G. H. Sloane-Stanley, 1957. A simple method for the isolation and purification
of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Goodridge, A. G., 1968. Metabolism of glucoseU- 14 C in vitro in adipose tissue from embryonic and growing chicks. Amer. J. Physiol. 214: 897-901. Goodridge, A. G., A. Garay, and P. Silpananta, 1974. Regulation of lipogenesis and the total activities of lipogenic enzymes in the primary culture of hepatocytes from prenatal and early postnatal chicks. J. Biol. Chem. 249:1469-1475. Hazlewood, R. L., 1971. Endocrine control of avian carbohydrate metabolism. Poultry Sci. 50:9—18. Kirk, R. E., 1968. Experimental design procedures for the behavioral sciences. Wadsworth Publ. Co., Belmont, CA. Leveille, G. A., 1969. In vitro hepatic lipogenesis in the hen and chick. Comp. Biochem. Physiol. 28:431-43 5. Leveille, G. A., E. K. O'Hea, and K. Chakabarty, 1968. In vivo lipogenesis in the domestic chicken. Proc. Soc. Exp. Biol. Med. 128: 3 9 8 - 4 0 1 . Leveille, G. A., D. R. Romsos, Y. Y. Yeh, and E. K. O'Hea, 1975. Lipid biosynthesis in the chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanisms. Poultry Sci. 54:1075-1093. LeveUle, G. A., and Y. Y. Yeh, 1972. Influence of intermittent fasting on protein-free feeding on lipid metabolism in young cockerels. J. Nutr. 102:733-740. Lowenstein, J., 1970. Effect of (-)-hydroxycitrate on fatty acid synthesis by rat liver in vivo. J. Biol. Chem. 246:629-632. Mehlman, M. A., D. G. Therriault, W. Porter, G. S. Stoewsand, and H. A. Dymsza, 1966. Distribution of lipids in rats fed 1,3-butanediol. J. Nutr. 88:215-218. Mehlman, M. A., and R. L. Veech, 1972. Redox and phosphorylation states and metabolite concentrations in frozen clamped livers of rats fed diets containing 1,3-butanediol and DL-carnitine. J. Nutr. 102:45-51. Miller, S. A., and H. A. Dymsza, 1967. Utilization by the rat of 1,3-butanediol as a synthetic source of dietary energy. J. Nutr. 79—87. Muiruri, K. L., D. R. Romsos, and G. A. Leveille. 1975. Influence of meal frequency on in vivo hepatic fatty acid synthesis, lipogenic enzyme and glucose tolerance in the chicken. J. Nutr. 105:963-971. O'Hea, E. K., and G. A. Leveille, 1968. Lipogenesis in isolated adipose tissue of the domestic chick (Gallus domesticus). Comp. Biochem. Biophys. 26:111-120. O'Hea, E. K., and G. A. Leveille, 1969. Lipid biosynthesis and transport in the domestic chick (Gallus domesticus). Comp. Biochem. Physiol. 30: 149-159. Romsos, D. R., P. S. Belo, and G. A. Leveille, 1974. Effect of 1,3-butanediol on hepatic fatty acid synthesis and metabolite levels in the rat. J. Nutr. 104:1438-1445. Romsos, D. R., P. S. Belo, and G. A. Leveille, 1975a. Butanediol and lipid metabolism. Fed. Proc. 34:2186-2190. Romsos, D. R., P. S. Belo, E. R. Miller, and G. A.
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acetyl c o e n z y m e A and decreased c o e n z y m e A availability. Acetate use for b o t h oxidation a n d lipogenesis can be limited by t h e availability of c o e n z y m e A (Yeh and Leveille, 1 9 7 1 ) . T h e integration of BG into lipid and carboh y d r a t e metabolism involves t h e f o r m a t i o n of B - h y d r o x y b u t y a t e and a c e t o a c e t a t e by t h e liver and a change in t h e cytoplasmic r e d o x state ( R o m s o s et ah, 1 9 7 5 a ) . T h e increase in t h e cytoplasmic r e d o x s t a t e decreases glycolysis and glucose utilization for lipogenesis in rats. A n increase in hepatic r e d o x also was accompanied b y an increase in acyl c o e n z y m e A's, a decrease in fatty acid o x i d a t i o n , and a decrease in citrate cleavage activity (Romso's et ah, 1 9 7 4 ) . Although R o m s o s et ah ( 1 9 7 5 b ) did n o t show a change in hepatic r e d o x in chicks following t h e feeding of BG, we have f o u n d t h a t BG increases t h e r e d o x state and decreases t h e liver fat of t h e t u r k e y hen (Rosebrough et ah, 1 9 8 0 ) . F u r t h e r m o r e , R o m s o s et ah ( 1 9 7 5 b ) reported t h a t while BG did n o t affect glucose or acetate conversion t o fatty acids by chick liver, BG did increase t h e r e d o x of circulating metabolites in chicks. It is possible t h a t t h e relationship b e t w e e n circulating and hepatic redox metabolites is n o t as defined in t h e bird as in t h e rat.
EFFECT OF 1,3-BUTYLENE GLYCOL ON GROWTH AND LIPOGENESIS Leveille, 1975b. Influence of dietary 1,3-butanediol on weight gain, blood and liver metabolites, and lipogenesis in the pig and chick J. Nutr. 105:161-170. Romsos, D. R., and G. A. Leveille, 1974. Effect of dietary fructose on in vitro and in vivo fatty acid synthesis in the rat Biochim. Biophys. Acta 360:1-11. Rosebrough, R. W., E. Geis, P. James, H. Ota, and J. Whitehead, 1980. Effects of dietary energy substitutions on reproductive performance, feed efficiency, and lipogenic enzyme activity in Large White turkey hens. Poultry Sci. 59:
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1485-1492. Stoewsand, G. A., H. A. Dymsza, M. A. Mehlman, and D. G. Therriault, 1965. Influence of 1,3-butanediol on tissue lipids of cold-exposed rats. J. Nutr. 87:464-^68. Tobin, R. B., M. A. Mehlman, and M. Parker, 1972. Effect of 1,3-butanediol and propionic acid on blood ketones, lipids and metal ions in rats. J. Nutr. 102:1001-1008. Yeh, Y. Y., and G. A. Leveille, 1971. Studies on the relationship between lipogenesis and the level of coenzyme A derivatives, lactate and pyruvate in chick liver. J. Nutr. 101: 911-918.
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