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Effect of Increasing Dietary Energy on Hepatic Lipogenesis in Growing Chicks
Effect of Increasing D i e t a r y E n e r g y o n H e p a t i c L i p o g e n e s i s in G r o w i n g C h i c k s . II. Increasing E n e r g y b y F a t or P r o t e i n S u p p l e m e n t a t i o n . KEHCHI TANAKA, SHIGERU OHTANI, and KAKICHI SHIGENO Department of Poultry and Animal Sciences, Gifu University, Gifu, Gifu, Japan 501—11 (Received for publication May 17, 1982)
INTRODUCTION
MATERIALS AND METHODS
Isocaloric substitution of carbohydrate with either fat or protein decreases in vitro hepatic lipogenesis by chicks (Yeh and Leveille, 1969; Tanaka et al., 1979b,c) and turkey hens (Rosebrough et al, 1981). However, interpretation of the effect of quantity of dietary carbohydrate on hepatic lipogenesis may be confounded because the decrease in lipogenesis could be attributed either to a decrease in carbohydrate or to an increase in fat or protein. Therefore, we have previously reported that increases in the energy consumed by chicks in the form of carbohydrate resulted in increases in the rate of hepatic fatty acid synthesis and the activities of nicotinamide adenine dinucleotide phosphatemalate dehydrogenase (NADP-MDH) and citrate cleavage enzyme to support fatty acid biosynthesis (Tanaka et al., 1983). Hence, in the present experiment, we studied the effects of changing the dietary metabolizable energy through supplementation of fat or protein on in vitro lipogenesis as measured by the incorporation of 14C-labeled actate into fatty acid by liver slices of growing chicks. In addition, the activities of NADP-malate dehydrogenase, citrate cleavage enzyme, and pentose phosphate pathway dehydrogenases were also studied.
Day-old meat type chicks obtained from a local hatchery were used in this experiment. They were raised on wire floors. Feed and water were made available at all times. At 28 days of age, all chicks were weighed individually and divided into four groups. Thereafter, they were randomly distributed to different treatments with 5 chicks assigned to each treatment. Chicks were housed in portable wire battery cages from which water was freely accessible. The levels of dietary energy were increased through fat (Experiment 1) or protein supplementation (Experiment 2). The chicks were force-fed the experimental diet (Table 1) for 7 days. Room temperature was maintained at 25 C and lights were on 24 hr a day. The force-feeding procedures have been described previously (Tanaka et al., 1981). At the termination of the 7-day experimental period, all chicks were weighed and then sacrificed by decapitation. Blood samples were collected from which serum was later extracted. Both the liver and abdominal fat were immediately taken and weighed. The liver samples were temporarily placed in an ice-cold saline solution (.9% NaCl) and were later used to determine the rate of hepatic lipogenesis.
452
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ABSTRACT The effect of increasing the energy content of the diet through supplementation of various levels of fat or protein on hepatic lipogenesis and activities of associated enzymes of liver was examined in force-fed growing chicks. Hepatic lipogenesis was significantly decreased as the dietary metabolizable energy level was increased through supplementation of fat or protein. The activity of nicotinamide adenine dinucleotide phosphate-malate dehydrogenase (NADPMDH) (EC 1.1.1.40) in liver was higher (P<.01) in chicks fed diets containing the lowest energy level. The activity of citrate cleavage enzyme (EC 4.1.3.8) in liver was significantly depressed as the dietary metabolizable energy level increased through supplementation of fat, whereas increasing the dietary metabolizable energy level through protein supplementation resulted in a significant increase in citrate cleavage enzyme activity in liver. Nonesterified fatty acid concentration in serum was significantly increased as the dietary metabolizable energy level was increased through supplementation of fat. (Key words: force-fed growing chicks, hepatic lipogenesis, NADP-malate dehydrogenase, citrate cleavage enzyme, nonesterified fatty acid) 1983 Poultry Science 62:452-458
FAT OR PROTEIN AND HEPATIC LIPOGENESIS
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Body weight gain, feed consumption, metabolizable energy (ME) intake, and liver and abdominal fat weights of chicks fed diets containing graded levels of ME are shown in Table 2. Body weight gains were significantly increased as dietary ME was increased through supplementation of fat or protein. Abdominal fat weight, expressed as percentage of body weight, significantly increased as dietary ME was increased through supplementation of fat or protein. Liver weight, expressed as percentage of body weight, was not affected by treatments. Table 3 shown the effect of increasing dietary ME by fat or protein supplementation on CO2 production, fatty acid and glycerideglycerol synthesis in liver slices, and on the activities of various enzymes in the liver of growing chicks. The oxidation of 14 C-labeled acetate in liver slices tended to be decreased or significantly decreased as the dietary ME was increased through supplementation of fat (Experiment 1), whereas increasing the dietary ME through protein supplementation had no influence on the oxidation of 14 C-labeled acetate in liver slices (Experiment 2). The incorporation of 14 C-labeled acetate into fatty acids was significantly depressed as dietary ME was increased through supplementation of fat or protein. Increasing dietary ME through fat supplementation had no influence on the activity of NADP-MDH (EC 1.1.1.40) in liver (Experiment 1). In Experiment 2, the activity of NADP-MDH in liver was higher (P<.01) in chicks fed the diet containing the lowest ME. The activity of citrate cleavage enzyme (CCE, EC 4.1.3.8) in liver was significantly depressed as the dietary ME was increased through supplementation of fat (Experiment 1), whereas increasing dietary ME through protein supplementation resulted in a significant increase
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 7, 2015
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Experimental procedures used to determine enzyme activities in the liver, as well as the determinations of the concentrations of nonesterifed fatty acids (NEFA) of serum and various lipid fractions of liver, have been described previously (Tanaka et al., 1979b). All data were statistically analyzed by means of analyses of variance (Snedecor and Cochran, 1968), and differences between the treatments were determined by Duncan's multiple range test (Duncan, 1955). RESULTS
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62.3
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4.0 +
514.9
269.3
.4
724.4
692.9
3.6 ±
406.1
331.2
.4&b
.4
198.7 ± 10.3 C
1.4 ±
3.1 ±
124.5 ± 2 6 . l b
.3
.9±
a
345.5 549.2
324.3 243.7
1.0
846.8
804.5
3.4+
469.4
162.4+ 15.4 C
414.4
83.3 ± 16.2b
SD for five chicks.
Probability of a significant treatment effect; ns, not significant.
' ' ' Values (mean ± SD for 5 chicks) in the same row with different superscript letters are significantly different from
Experiment 1 Body weight gain, g/chick/7 days 46.7 + 10.2 2 * Feed consumption, g/chick/7 days 378.0 Carbohydrate ME intake, kcal/chick/7 days 783.8 Protein ME intake, kcal/chick/7 days 312.7 Fat ME intake, kcal/chick/7 days 11.0 Liver weight, g/100 g body .2 weight 3.0 + Abdominal fat weight, g/100 g body weight .6 ± .2a Experiment 2 Body weight gain, g/chick/7 days - 1 6 . 2 ± 10.0 a Feed consumption, g/chick/7 days 258.0 Carbohydrate ME intake, kcal/chick/7 days 644.5 Protein ME intake, 55.2 kcal/chick/7 days Fat ME intake, kcal/chick/7 55.4 days Liver weight, g/100 g body weight 3.4+ .1 Abdominal fat weight, 1.0 ± .2*b g/100 g body weight
Experimental diets
TABLE 2. Influence of changing dietary fat or protein and energy levels on body weight gains feed consump abdominal fat weights of growing chicks
oaded from http://ps.oxfordjournals.org/ at UCSF Library on April 7, 2015
863.6 1532.2 91.3 54.0 4.5 11.4 105.6
556.2 1489.8 81.6 85.7 2.3 10.8 60.4 + 161.0 ± 226.5b + 43.3 + 11.4a .5 ± 1.4 + ± 22.3ab
± 86.2b ± 218.2b ± 24.4 ± 12.8 .5 ± + .9 ± 14.8b
693.8 ± 815.2 ± 86.6 ± 47.8 ± 4.5 ± 12.1 ± 127.0 ±
467.8 ± 936.3 ± 69.0+ 83.2 ± 2.1 ± 11.4 ± 39.8 ± 180.3 116.4 a 33.1 11.0 a .4 2.1 17.lb
51.3ab 158.1* 27.8 16.4 .9 .4 7.4 a
Probability of a significant treatment; ns, not significant.
4
Activities expressed as nanomoles substrate converted to product per minute per milligram protein at 25 C.
NADP-MDH, Nicotinamide adenine dinucleotide phosphatemalate dehydrogenase; G6PDH, glucose-6-phosphate d hydrogenase; CCE, citrate cleavage enzyme.
3
Results expressed as nanomoles substrate converted to product indicated per 100 mg liver per 3 hr incubation pe 14 C-l-acetate.
2
1
± 60.3 ± 303.lc + 31.3 ± 20.1b ± .4 ± 1.2 ± 15.4a
t 83.2b ± 252.6C ± 43.3 ± 18.1 ± .4 ± 1.3 ± 10.6C
' ' Values in the same row with different supercript letters are significantly different from one another (P<.05). Valu
860.2 2393.5 114.3 111.4 4.1 11.2 88.0
Experiment 2 14 C 0 2 production2 F a t t y acid s y n t h e s i s 2 Glyceride-glycerol synthesis 2 NADP-MDH 4 G6PDH4 6PGDH4 CCE4
a
604.4 1953.6 92.3 105.8 2.1 12.0 89.2
Experiment 1 14 C 0 2 production2 F a t t y acid synthesis 2 Glyceride-glycerol synthesis 2 NADP-MDH 3 ' 4 G6PDH3'4 6PGDH3'4 CCE4
Experimental diet
TABLE 3. Influence of changing dietary fat or protein and energy levels on 14CO} production and hepatic several enzymes in liver of growing chicks
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1
1.7 .3b 1.3
270.5 + 36.1
14.4 ± 3.3 b 4.4 ± . 5 b 30.8 ± 2.0
144.8 ± 18.8 a
7.6 ± 3.8 ± 34.7 ±
275.6 ± 34.0
OuEq/liter serum)
6.5 ± 1.4a 3.2 ± . 2 a 29.0 ± 5.3
(mg/g liver)
192.9 ± 18.9 b
(juEq/liter serum)-
5.7 ± 2.0 3.1 ± . 3 a 34.0 ± 1.4
(mg/g liver) 1.6 .3a .9
293.2 ±42.1
7.5 + 1.8 a 3.8 ± . 5 a b 33.8+ 3.5
217.4+ 24.1 C
5.3 ± 3.0+ 34.2 +
Probability of a significant treatment effect; ns, not significant.
' ' Values in the same row with different superscript letters are significantly different one another (P<.05). Values are
Serum NEFA
Triglyceride Total cholesterol Phospholipids
Experiment 2 Liver
NEFA
Serum
Triglyceride Total cholesterol Phospholipids
Experiment 1 Liver
Experimental diet
TABLE 4. Influence of changing dietary fat or protein and energy levels on the contents of various lipids in liver and no in serum of growing chicks
oaded from http://ps.oxfordjournals.org/ at UCSF Library on April 7, 2015
FAT OR PROTEIN AND HEPATIC LIPOGENESIS
DISCUSSION Increasing the level of dietary fat or protein has been reported to depress fatty acid synthesis in growing chicks (Yeh and Leveille, 1969, 1970; Tanaka et al, 1979b,c). In these studies, however, changes in dietary fat or protein were at the expense of dietary carbohydrate, because dietary energy was held constant. Thus, the changes observed might have resulted from the increase in dietary fat or protein or from the decrease in dietary carbohydrate. In the present experiments, effects of dietary fat or protein on hapatic lipogenesis were investigated in growing chicks given purified diets with various levels of ME by the supplementation of fat or protein. Increasing ME intake with increasing fat or protein consumption caused a decrease in the rate of hepatic fatty acid synthesis in growing chicks. Yeh and Leveille (1970, 1971) have demonstrated that when chicks were fed a high-fat diet or fasted short-term, the rate of hepatic fatty acid synthesis was depressed and plasma NEFA concentration was increased as were hepatic long-chain acyl CoA levels. The observations that plasma NEFA and hepatic long-chain acyl CoA concentrations were inversely correlated with the rate of hepatic fatty acid synthesis suggested that these metabolites might act as inhibitors of fatty acid synthesis (Yeh and Leveille, 1970, 1971; Yeh et al, 1970). In other words, an increased NEFA in liver, as reflected by the circulating NEFA concentration, would result in an
increased rate of fatty acid activation (to CoA derivatives) and in decreased free CoA. Free CoA participates in the citrate cleavage recation to form acetyl CoA. Fatty acid activation would thereby compete with the citrate cleavage enzyme for free CoA and would reduce rate of cytoplasmic acetyl CoA generation in the chick liver (Yeh and Leveille, 1971). Also, Yeh and Leveille (1971) have reported decreased free CoA with an increase in long-chain acyl CoA in the chick liver. Long-chain acyl CoA derivatives would inhibit citrate transport from the mitochondria to the cytoplasm (Halperin et al, 1972; Goodridge, 1973). Citrate is not only a substrate for fatty acid synthesis but is also an activator of acetyl CoA carboxylase. As such, long-chain acyl CoA derivatives would inhibit the activation of acetyl CoA carboxylase, which appears to play a key role in the regulation of fatty acid synthesis in animal tissues (Tubbs and Garland, 1964). In Experiment 1, increasing ME intake with increasing fat consumption could induce increases in long-chain acyl CoA derivatives and a decrease in free CoA in chick liver. Subsequently, an inhibition of acetyl CoA carboxylase activity and a reduced rate of cytoplasmic acetyl CoA generation appear to cause a depression in the rate of hepatic fatty acid synthesis in growing chicks. In Experiment 2, the increase in dietary ME through supplementation of protein also resulted in a decreased rate of hepatic fatty acid synthesis. However, unlike Experiment 1, NEFA concentration in serum was not affected by-increasing protein consumption in chicks. Therefore, it seems likely that factors other than changes in longchain acyl CoA were involved in the inhibition of fatty acid synthesis resulting from increased dietary ME brought about by the supplementation of protein. Yeh and Leveille (1971) have proposed that the availability of reducing equivalents to support fatty acid synthesis might be a factor in limiting fatty acid synthesis in chicks fed high-protein diets. O'Hea and Leveille (1969) have suggested that in view of the activity of NADP-MDH in chick liver, transhydrogenation of NADPH in the cell cytoplasm is probably the major source of reducing equivalent for the support of fatty acid biosynthesis in the chick. There are numerous reports demonstrating that NADPMDH is involved in supplying NADPH to support fatty acid synthesis, and changes in this activity are highly correlated with changes in
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 7, 2015
in CCE in the liver. Increasing dietary ME through fat or protein supplementation had no influence on activities of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) in the liver. The concentrations of various lipid fractions in liver and serum NEFA of chicks fed diets containing graded levels of ME are shown in Table 4. The concentration of serum NEFA was significantly increased as dietary ME was increased through supplementation of fat (Experiment 1). Compared to the other treatments, chicks fed the diet containing the lowest ME in Experiments 1 and 2 demonstrated a significantly increased total cholesterol. Triglyceride content in liver was significantly decreased as dietary ME was increased through supplementation of protein (Experiment 2).
457
458
TANAKA ET AL.
REFERENCES Duncan, D. B., 1955. Multiple range and multiple F test. Biometrics 11:1—42. Frenkel, R., M. J. Stark, and J. Stafford, 1972. Increased "malic enzyme" activity during adaptation to a low protein diet. Biochim. Biophys. Res. Commun. 49:1684-1689. Gibson, D. M., R. T. Lyons, D. F. Scott, and Y. Muto, 1972. Synthesis and degradation of the lipogenic enzymes of rat liver. Adv. Enzyme Regul. 10: 187-204. Goodridge, A. G., 1973. Regulation of fatty acid synthesis in isolated hepatocytes: evidence for a physiological role for long chain fatty acyl Coenzyme A. J. Biol. Chem. 248:4318-4326. Halperin, M. L., B. H. Robinson and I. B. Fritz, 1972. Effects of palmityl CoA on citrate and malate transport by rat liver mitochondria. Proc. Natl. Acad. Sci. 69:1003-1007. Leveille, G. A., and R. W. Hanson, 1966. Adaptive changes in enzyme activity and metabolic pathways in adipose tissue from meal-fed rats. J. Lipid Res. 7 : 4 6 - 5 5 . Leveille, G. A., and Y. -Y. Yeh, 1972. Influence of intermittent fasting or protein-free feeding on lipid metabolism in young cockerels. J. Nutr. 102:733-740. O'Hea, E. K., and G. A. Leveille, 1969. Influence of fasting and refeeding on lipogenesis and enzymatic activity of pig adipose tissue. J. Nutr.
99:345-352. Rosebrough, R. W., N. C. Steele, and L. T. Frobish, 1981. Effects of dietary fat on reproductive performance and in vitro lipogenesis by the turkey hen. Poultry Sci. 60:1931-1938. Romsos, D. R., and G. A. Leveille, 1974. Effect of diet on activity of enzymes involved in fatty acid and cholesterol synthesis. Adv. Lipid Res. 12: 97-146. Snedecor, G. W., and W. G. Cochran, 1968. Statistical methods. 6th ed. The Iowa State Coll. Press, Ames, IA. Tanaka, K., N. Akazaki, C. M. Collado, S. Ohtani, and K. Shigeno, 1979a. Effects of dietary essential fatty acids on the accumulation of lipids in the liver of growing chicks. Jap. J. Zootech. Sci. 50:563-573. Tanaka, N. Akazaki, C. M. Collado, S. Ohtani, and K. Shigeno, 1981. Effect of dietary essential fatty acid deficiency on hepatic lipogenesis in the growing chick. Jap. Poultry Sci. 18:120-125. Tanaka, K., K. Kitahara, and K. Shigeno, 1979b. Effect of dietary protein level on lipid metabolism in growing chicks. Jap. J. Zootech. Sci. 50:44-54. Tanaka, K., K. Kitahara, and K. Shigeno, 1979c. Effect of dietary fat level on lipid metabolism in growing chicks. Jap. J. Zootech. Sci. 50:100— 107. Tanaka, K., N. Takagi, S. Ohtani, and K. Shigeno, 1983. Effect of increasing dietary energy on hepatic lipogenesis in growing chicks. I. Increasing energy by carbohydrate supplementation. Poultry Sci. 6 2 : 4 4 5 - 4 5 1 . Tubbs, P. K., and P. B. Garland, 1964. Variations in tissue contents of Coenzyme A thioesters and possible metabolic implications. Biochem. J. 93:550-556. Yeh, Y. -Y., and G. A. Leveille, 1969. Effect of dietary protein on hepatic lipogenesis in growing chicks. J. Nutr. 98:356-366. Yeh, Y. -Y., and G. A. Leveille, 1970. Hepatic fatty acid synthsis and plasma free fatty acid level in chicks subjected to short periods of food restriction and refeeding. J. Nutr. 100:1389—1398. 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. Yeh, Y. -Y., G. A. Leveille, and J. H. Wiley, 1970. Influence of dietary lipid on lipogenesis and on the activity of malic and citrate cleavage enzyme in liver of the growing chick. J. Nutr. 100: 917-924.
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t h e rate of fatty acid synthesis (Tanaka et al, 1979a,b,c, 1 9 8 1 ; Leveille and Hanson, 1 9 6 6 ; Leveille and Yeh, 1 9 7 2 ; O'Hea and Leveille, 1 9 6 9 ; Frenkel et al, 1 9 7 2 ; Gibson et al, 1 9 7 2 ; R o m s o s and Leveille, 1 9 7 4 ) . F u r t h e r o m r e , if glucose p r o d u c t i o n is e n h a n c e d w h e n dietary protein is increased, as suggested by Yeh and Leveille ( 1 9 6 9 ) , this would utilize cytoplasmic N A D H and further reduce t h a t available for t r a n s h y d r o g e n a t i o n . In E x p e r i m e n t 2, t h e activity of NADP-MDH was significantly increased as t h e dietary ME was increased by t h e s u p p l e m e n t a t i o n of p r o t e i n . We would therefore suggest t h a t t h e availability of cytoplasmic reducing equivalents is p r o b a b l y the major factor limiting fatty acid synthesis in chicks fed high dietary protein.
INTRODUCTION
MATERIALS AND METHODS
Isocaloric substitution of carbohydrate with either fat or protein decreases in vitro hepatic lipogenesis by chicks (Yeh and Leveille, 1969; Tanaka et al., 1979b,c) and turkey hens (Rosebrough et al, 1981). However, interpretation of the effect of quantity of dietary carbohydrate on hepatic lipogenesis may be confounded because the decrease in lipogenesis could be attributed either to a decrease in carbohydrate or to an increase in fat or protein. Therefore, we have previously reported that increases in the energy consumed by chicks in the form of carbohydrate resulted in increases in the rate of hepatic fatty acid synthesis and the activities of nicotinamide adenine dinucleotide phosphatemalate dehydrogenase (NADP-MDH) and citrate cleavage enzyme to support fatty acid biosynthesis (Tanaka et al., 1983). Hence, in the present experiment, we studied the effects of changing the dietary metabolizable energy through supplementation of fat or protein on in vitro lipogenesis as measured by the incorporation of 14C-labeled actate into fatty acid by liver slices of growing chicks. In addition, the activities of NADP-malate dehydrogenase, citrate cleavage enzyme, and pentose phosphate pathway dehydrogenases were also studied.
Day-old meat type chicks obtained from a local hatchery were used in this experiment. They were raised on wire floors. Feed and water were made available at all times. At 28 days of age, all chicks were weighed individually and divided into four groups. Thereafter, they were randomly distributed to different treatments with 5 chicks assigned to each treatment. Chicks were housed in portable wire battery cages from which water was freely accessible. The levels of dietary energy were increased through fat (Experiment 1) or protein supplementation (Experiment 2). The chicks were force-fed the experimental diet (Table 1) for 7 days. Room temperature was maintained at 25 C and lights were on 24 hr a day. The force-feeding procedures have been described previously (Tanaka et al., 1981). At the termination of the 7-day experimental period, all chicks were weighed and then sacrificed by decapitation. Blood samples were collected from which serum was later extracted. Both the liver and abdominal fat were immediately taken and weighed. The liver samples were temporarily placed in an ice-cold saline solution (.9% NaCl) and were later used to determine the rate of hepatic lipogenesis.
452
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 7, 2015
ABSTRACT The effect of increasing the energy content of the diet through supplementation of various levels of fat or protein on hepatic lipogenesis and activities of associated enzymes of liver was examined in force-fed growing chicks. Hepatic lipogenesis was significantly decreased as the dietary metabolizable energy level was increased through supplementation of fat or protein. The activity of nicotinamide adenine dinucleotide phosphate-malate dehydrogenase (NADPMDH) (EC 1.1.1.40) in liver was higher (P<.01) in chicks fed diets containing the lowest energy level. The activity of citrate cleavage enzyme (EC 4.1.3.8) in liver was significantly depressed as the dietary metabolizable energy level increased through supplementation of fat, whereas increasing the dietary metabolizable energy level through protein supplementation resulted in a significant increase in citrate cleavage enzyme activity in liver. Nonesterified fatty acid concentration in serum was significantly increased as the dietary metabolizable energy level was increased through supplementation of fat. (Key words: force-fed growing chicks, hepatic lipogenesis, NADP-malate dehydrogenase, citrate cleavage enzyme, nonesterified fatty acid) 1983 Poultry Science 62:452-458
FAT OR PROTEIN AND HEPATIC LIPOGENESIS
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Body weight gain, feed consumption, metabolizable energy (ME) intake, and liver and abdominal fat weights of chicks fed diets containing graded levels of ME are shown in Table 2. Body weight gains were significantly increased as dietary ME was increased through supplementation of fat or protein. Abdominal fat weight, expressed as percentage of body weight, significantly increased as dietary ME was increased through supplementation of fat or protein. Liver weight, expressed as percentage of body weight, was not affected by treatments. Table 3 shown the effect of increasing dietary ME by fat or protein supplementation on CO2 production, fatty acid and glycerideglycerol synthesis in liver slices, and on the activities of various enzymes in the liver of growing chicks. The oxidation of 14 C-labeled acetate in liver slices tended to be decreased or significantly decreased as the dietary ME was increased through supplementation of fat (Experiment 1), whereas increasing the dietary ME through protein supplementation had no influence on the oxidation of 14 C-labeled acetate in liver slices (Experiment 2). The incorporation of 14 C-labeled acetate into fatty acids was significantly depressed as dietary ME was increased through supplementation of fat or protein. Increasing dietary ME through fat supplementation had no influence on the activity of NADP-MDH (EC 1.1.1.40) in liver (Experiment 1). In Experiment 2, the activity of NADP-MDH in liver was higher (P<.01) in chicks fed the diet containing the lowest ME. The activity of citrate cleavage enzyme (CCE, EC 4.1.3.8) in liver was significantly depressed as the dietary ME was increased through supplementation of fat (Experiment 1), whereas increasing dietary ME through protein supplementation resulted in a significant increase
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 7, 2015
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Experimental procedures used to determine enzyme activities in the liver, as well as the determinations of the concentrations of nonesterifed fatty acids (NEFA) of serum and various lipid fractions of liver, have been described previously (Tanaka et al., 1979b). All data were statistically analyzed by means of analyses of variance (Snedecor and Cochran, 1968), and differences between the treatments were determined by Duncan's multiple range test (Duncan, 1955). RESULTS
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692.9
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406.1
331.2
.4&b
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1.4 ±
3.1 ±
124.5 ± 2 6 . l b
.3
.9±
a
345.5 549.2
324.3 243.7
1.0
846.8
804.5
3.4+
469.4
162.4+ 15.4 C
414.4
83.3 ± 16.2b
SD for five chicks.
Probability of a significant treatment effect; ns, not significant.
' ' ' Values (mean ± SD for 5 chicks) in the same row with different superscript letters are significantly different from
Experiment 1 Body weight gain, g/chick/7 days 46.7 + 10.2 2 * Feed consumption, g/chick/7 days 378.0 Carbohydrate ME intake, kcal/chick/7 days 783.8 Protein ME intake, kcal/chick/7 days 312.7 Fat ME intake, kcal/chick/7 days 11.0 Liver weight, g/100 g body .2 weight 3.0 + Abdominal fat weight, g/100 g body weight .6 ± .2a Experiment 2 Body weight gain, g/chick/7 days - 1 6 . 2 ± 10.0 a Feed consumption, g/chick/7 days 258.0 Carbohydrate ME intake, kcal/chick/7 days 644.5 Protein ME intake, 55.2 kcal/chick/7 days Fat ME intake, kcal/chick/7 55.4 days Liver weight, g/100 g body weight 3.4+ .1 Abdominal fat weight, 1.0 ± .2*b g/100 g body weight
Experimental diets
TABLE 2. Influence of changing dietary fat or protein and energy levels on body weight gains feed consump abdominal fat weights of growing chicks
oaded from http://ps.oxfordjournals.org/ at UCSF Library on April 7, 2015
863.6 1532.2 91.3 54.0 4.5 11.4 105.6
556.2 1489.8 81.6 85.7 2.3 10.8 60.4 + 161.0 ± 226.5b + 43.3 + 11.4a .5 ± 1.4 + ± 22.3ab
± 86.2b ± 218.2b ± 24.4 ± 12.8 .5 ± + .9 ± 14.8b
693.8 ± 815.2 ± 86.6 ± 47.8 ± 4.5 ± 12.1 ± 127.0 ±
467.8 ± 936.3 ± 69.0+ 83.2 ± 2.1 ± 11.4 ± 39.8 ± 180.3 116.4 a 33.1 11.0 a .4 2.1 17.lb
51.3ab 158.1* 27.8 16.4 .9 .4 7.4 a
Probability of a significant treatment; ns, not significant.
4
Activities expressed as nanomoles substrate converted to product per minute per milligram protein at 25 C.
NADP-MDH, Nicotinamide adenine dinucleotide phosphatemalate dehydrogenase; G6PDH, glucose-6-phosphate d hydrogenase; CCE, citrate cleavage enzyme.
3
Results expressed as nanomoles substrate converted to product indicated per 100 mg liver per 3 hr incubation pe 14 C-l-acetate.
2
1
± 60.3 ± 303.lc + 31.3 ± 20.1b ± .4 ± 1.2 ± 15.4a
t 83.2b ± 252.6C ± 43.3 ± 18.1 ± .4 ± 1.3 ± 10.6C
' ' Values in the same row with different supercript letters are significantly different from one another (P<.05). Valu
860.2 2393.5 114.3 111.4 4.1 11.2 88.0
Experiment 2 14 C 0 2 production2 F a t t y acid s y n t h e s i s 2 Glyceride-glycerol synthesis 2 NADP-MDH 4 G6PDH4 6PGDH4 CCE4
a
604.4 1953.6 92.3 105.8 2.1 12.0 89.2
Experiment 1 14 C 0 2 production2 F a t t y acid synthesis 2 Glyceride-glycerol synthesis 2 NADP-MDH 3 ' 4 G6PDH3'4 6PGDH3'4 CCE4
Experimental diet
TABLE 3. Influence of changing dietary fat or protein and energy levels on 14CO} production and hepatic several enzymes in liver of growing chicks
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1
1.7 .3b 1.3
270.5 + 36.1
14.4 ± 3.3 b 4.4 ± . 5 b 30.8 ± 2.0
144.8 ± 18.8 a
7.6 ± 3.8 ± 34.7 ±
275.6 ± 34.0
OuEq/liter serum)
6.5 ± 1.4a 3.2 ± . 2 a 29.0 ± 5.3
(mg/g liver)
192.9 ± 18.9 b
(juEq/liter serum)-
5.7 ± 2.0 3.1 ± . 3 a 34.0 ± 1.4
(mg/g liver) 1.6 .3a .9
293.2 ±42.1
7.5 + 1.8 a 3.8 ± . 5 a b 33.8+ 3.5
217.4+ 24.1 C
5.3 ± 3.0+ 34.2 +
Probability of a significant treatment effect; ns, not significant.
' ' Values in the same row with different superscript letters are significantly different one another (P<.05). Values are
Serum NEFA
Triglyceride Total cholesterol Phospholipids
Experiment 2 Liver
NEFA
Serum
Triglyceride Total cholesterol Phospholipids
Experiment 1 Liver
Experimental diet
TABLE 4. Influence of changing dietary fat or protein and energy levels on the contents of various lipids in liver and no in serum of growing chicks
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FAT OR PROTEIN AND HEPATIC LIPOGENESIS
DISCUSSION Increasing the level of dietary fat or protein has been reported to depress fatty acid synthesis in growing chicks (Yeh and Leveille, 1969, 1970; Tanaka et al, 1979b,c). In these studies, however, changes in dietary fat or protein were at the expense of dietary carbohydrate, because dietary energy was held constant. Thus, the changes observed might have resulted from the increase in dietary fat or protein or from the decrease in dietary carbohydrate. In the present experiments, effects of dietary fat or protein on hapatic lipogenesis were investigated in growing chicks given purified diets with various levels of ME by the supplementation of fat or protein. Increasing ME intake with increasing fat or protein consumption caused a decrease in the rate of hepatic fatty acid synthesis in growing chicks. Yeh and Leveille (1970, 1971) have demonstrated that when chicks were fed a high-fat diet or fasted short-term, the rate of hepatic fatty acid synthesis was depressed and plasma NEFA concentration was increased as were hepatic long-chain acyl CoA levels. The observations that plasma NEFA and hepatic long-chain acyl CoA concentrations were inversely correlated with the rate of hepatic fatty acid synthesis suggested that these metabolites might act as inhibitors of fatty acid synthesis (Yeh and Leveille, 1970, 1971; Yeh et al, 1970). In other words, an increased NEFA in liver, as reflected by the circulating NEFA concentration, would result in an
increased rate of fatty acid activation (to CoA derivatives) and in decreased free CoA. Free CoA participates in the citrate cleavage recation to form acetyl CoA. Fatty acid activation would thereby compete with the citrate cleavage enzyme for free CoA and would reduce rate of cytoplasmic acetyl CoA generation in the chick liver (Yeh and Leveille, 1971). Also, Yeh and Leveille (1971) have reported decreased free CoA with an increase in long-chain acyl CoA in the chick liver. Long-chain acyl CoA derivatives would inhibit citrate transport from the mitochondria to the cytoplasm (Halperin et al, 1972; Goodridge, 1973). Citrate is not only a substrate for fatty acid synthesis but is also an activator of acetyl CoA carboxylase. As such, long-chain acyl CoA derivatives would inhibit the activation of acetyl CoA carboxylase, which appears to play a key role in the regulation of fatty acid synthesis in animal tissues (Tubbs and Garland, 1964). In Experiment 1, increasing ME intake with increasing fat consumption could induce increases in long-chain acyl CoA derivatives and a decrease in free CoA in chick liver. Subsequently, an inhibition of acetyl CoA carboxylase activity and a reduced rate of cytoplasmic acetyl CoA generation appear to cause a depression in the rate of hepatic fatty acid synthesis in growing chicks. In Experiment 2, the increase in dietary ME through supplementation of protein also resulted in a decreased rate of hepatic fatty acid synthesis. However, unlike Experiment 1, NEFA concentration in serum was not affected by-increasing protein consumption in chicks. Therefore, it seems likely that factors other than changes in longchain acyl CoA were involved in the inhibition of fatty acid synthesis resulting from increased dietary ME brought about by the supplementation of protein. Yeh and Leveille (1971) have proposed that the availability of reducing equivalents to support fatty acid synthesis might be a factor in limiting fatty acid synthesis in chicks fed high-protein diets. O'Hea and Leveille (1969) have suggested that in view of the activity of NADP-MDH in chick liver, transhydrogenation of NADPH in the cell cytoplasm is probably the major source of reducing equivalent for the support of fatty acid biosynthesis in the chick. There are numerous reports demonstrating that NADPMDH is involved in supplying NADPH to support fatty acid synthesis, and changes in this activity are highly correlated with changes in
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in CCE in the liver. Increasing dietary ME through fat or protein supplementation had no influence on activities of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) in the liver. The concentrations of various lipid fractions in liver and serum NEFA of chicks fed diets containing graded levels of ME are shown in Table 4. The concentration of serum NEFA was significantly increased as dietary ME was increased through supplementation of fat (Experiment 1). Compared to the other treatments, chicks fed the diet containing the lowest ME in Experiments 1 and 2 demonstrated a significantly increased total cholesterol. Triglyceride content in liver was significantly decreased as dietary ME was increased through supplementation of protein (Experiment 2).
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REFERENCES Duncan, D. B., 1955. Multiple range and multiple F test. Biometrics 11:1—42. Frenkel, R., M. J. Stark, and J. Stafford, 1972. Increased "malic enzyme" activity during adaptation to a low protein diet. Biochim. Biophys. Res. Commun. 49:1684-1689. Gibson, D. M., R. T. Lyons, D. F. Scott, and Y. Muto, 1972. Synthesis and degradation of the lipogenic enzymes of rat liver. Adv. Enzyme Regul. 10: 187-204. Goodridge, A. G., 1973. Regulation of fatty acid synthesis in isolated hepatocytes: evidence for a physiological role for long chain fatty acyl Coenzyme A. J. Biol. Chem. 248:4318-4326. Halperin, M. L., B. H. Robinson and I. B. Fritz, 1972. Effects of palmityl CoA on citrate and malate transport by rat liver mitochondria. Proc. Natl. Acad. Sci. 69:1003-1007. Leveille, G. A., and R. W. Hanson, 1966. Adaptive changes in enzyme activity and metabolic pathways in adipose tissue from meal-fed rats. J. Lipid Res. 7 : 4 6 - 5 5 . Leveille, G. A., and Y. -Y. Yeh, 1972. Influence of intermittent fasting or protein-free feeding on lipid metabolism in young cockerels. J. Nutr. 102:733-740. O'Hea, E. K., and G. A. Leveille, 1969. Influence of fasting and refeeding on lipogenesis and enzymatic activity of pig adipose tissue. J. Nutr.
99:345-352. Rosebrough, R. W., N. C. Steele, and L. T. Frobish, 1981. Effects of dietary fat on reproductive performance and in vitro lipogenesis by the turkey hen. Poultry Sci. 60:1931-1938. Romsos, D. R., and G. A. Leveille, 1974. Effect of diet on activity of enzymes involved in fatty acid and cholesterol synthesis. Adv. Lipid Res. 12: 97-146. Snedecor, G. W., and W. G. Cochran, 1968. Statistical methods. 6th ed. The Iowa State Coll. Press, Ames, IA. Tanaka, K., N. Akazaki, C. M. Collado, S. Ohtani, and K. Shigeno, 1979a. Effects of dietary essential fatty acids on the accumulation of lipids in the liver of growing chicks. Jap. J. Zootech. Sci. 50:563-573. Tanaka, N. Akazaki, C. M. Collado, S. Ohtani, and K. Shigeno, 1981. Effect of dietary essential fatty acid deficiency on hepatic lipogenesis in the growing chick. Jap. Poultry Sci. 18:120-125. Tanaka, K., K. Kitahara, and K. Shigeno, 1979b. Effect of dietary protein level on lipid metabolism in growing chicks. Jap. J. Zootech. Sci. 50:44-54. Tanaka, K., K. Kitahara, and K. Shigeno, 1979c. Effect of dietary fat level on lipid metabolism in growing chicks. Jap. J. Zootech. Sci. 50:100— 107. Tanaka, K., N. Takagi, S. Ohtani, and K. Shigeno, 1983. Effect of increasing dietary energy on hepatic lipogenesis in growing chicks. I. Increasing energy by carbohydrate supplementation. Poultry Sci. 6 2 : 4 4 5 - 4 5 1 . Tubbs, P. K., and P. B. Garland, 1964. Variations in tissue contents of Coenzyme A thioesters and possible metabolic implications. Biochem. J. 93:550-556. Yeh, Y. -Y., and G. A. Leveille, 1969. Effect of dietary protein on hepatic lipogenesis in growing chicks. J. Nutr. 98:356-366. Yeh, Y. -Y., and G. A. Leveille, 1970. Hepatic fatty acid synthsis and plasma free fatty acid level in chicks subjected to short periods of food restriction and refeeding. J. Nutr. 100:1389—1398. 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. Yeh, Y. -Y., G. A. Leveille, and J. H. Wiley, 1970. Influence of dietary lipid on lipogenesis and on the activity of malic and citrate cleavage enzyme in liver of the growing chick. J. Nutr. 100: 917-924.
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t h e rate of fatty acid synthesis (Tanaka et al, 1979a,b,c, 1 9 8 1 ; Leveille and Hanson, 1 9 6 6 ; Leveille and Yeh, 1 9 7 2 ; O'Hea and Leveille, 1 9 6 9 ; Frenkel et al, 1 9 7 2 ; Gibson et al, 1 9 7 2 ; R o m s o s and Leveille, 1 9 7 4 ) . F u r t h e r o m r e , if glucose p r o d u c t i o n is e n h a n c e d w h e n dietary protein is increased, as suggested by Yeh and Leveille ( 1 9 6 9 ) , this would utilize cytoplasmic N A D H and further reduce t h a t available for t r a n s h y d r o g e n a t i o n . In E x p e r i m e n t 2, t h e activity of NADP-MDH was significantly increased as t h e dietary ME was increased by t h e s u p p l e m e n t a t i o n of p r o t e i n . We would therefore suggest t h a t t h e availability of cytoplasmic reducing equivalents is p r o b a b l y the major factor limiting fatty acid synthesis in chicks fed high dietary protein.