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Hepatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese
Available online at www.sciencedirect.com
Agricultural Sciences in China
2006, 5 ( 5 ) : 390-396
May 2006
Hepatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese LIU Xiang-you1.2, HE Rui-guol, HUANG Chou-shen3, LI Xiang', ZHOU Qi-an', WANG Cheng', ZHAO Nal
and ZHOU Shi-xis' College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R.China Department of Animal Science and Technology, Yunnan Province Zhaotong Agricultural School, Zhaotong 657000, P.R.China 3
Hubei Province Poultry Associafion, Wuhan 430070, P.R.China
Abstract This experiment studied hepatic lipogenesis associated with biochemical changes in overfed Landaise Geese and China Xupu geese. Twenty healthy male Landaise geese and 20 healthy male Xupu geese, hatched on the same day under the same feeding conditions, were selected as experimental anima1.s. The animals were divided into two groups and each breed served as an experimental group. Per goose of per experimental group served for a repeat. Brown rice was selected as test diet. After overfeeding for 21 d and then slaughtering, the biochemical changes of hepatic lipogenesis in the genetic susceptibility to fatty liver were evaluated. These results showed that (1) the weight of fatty liver of the two breeds of geese were 801 and 375 g (P0.05) was found; ( 2 ) in these two breeds of geese, there were no differences on very-lowdensity lipoprotein (VLDL), cholesteryl esters (CE) (P<0.05), free cholesterol (FC), triglycerides (TG), phospholipids (PL) and protein (P<0.05); (3) there were no differences on activities of malic enzyme (ME), glucose-6-phosphatedehydrogenase (G6PDH), acetyl-CoA-carboxylas (ACX), fatty acid synthase (FAS), and mRNA level of ME in the two experimental breeds of geese groups (P<0.05); (4) test in Landaise geese group showed that there was no significant correlation with the specific enzymatic activities, while in Xupu geese group, the liver weight was negatively correlated to the specific activity of ACX and positively to that of ME; (5) in these overfed geese, ME activity appeared to be a major factor involved in the genetic susceptibility to hepatic steatosis and it determined the hepatic lipogenesis capacity.
Key words: goose, liver, hepatic lipogenesis, overfed
INTRODUCTION Fatty liver in the goose results from an increased hepatic lipogenesis in response to overfeeding, together with a deficient secretion of triacylglycerol as verylow-densitylipoprotein (VLDL) (Dominique et al. 1994). There are few natural animal populations in which we
can study the metabolic adaptations resulting in hepatic steatosis, as well as individual responsiveness. These populations include lactating cows (Reids and Roberts 1983), fasting suncus (Yasuhara et al. 1991), and wild migrating species of birds and fishes, in which hepatic steatosis occurs spontaneously as a consequence of energy storage before migration (Pilo and George 1983). This process is facilitated in these oviparous species,
Received 31 May, 2005 Accepted 24 March, 2006 LIU Xiang-you, Ph D; Correspondence HE Rui-guo, Mobile: 13986231290, Tel: +86-27-87281060, Fax: +86-27-87280479, E-mail: liuxiangyou99@ 163.com
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Hepatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese
especially waterfowl (geese and ducks), which are particularly susceptible to hepatic steatosis because the liver is the major site of de novo lipogenesis (Pearce 1977). In response to overfeeding with a carbohydrate-rich corn-based diet, Landaise geese liver weight may increase up to 10-fold in 2 weeks and account for up to 10% of the body weight (Hermier et al. 1991). Further, this fatty liver is totally reversible, the liver returning to its initial composition when overfeeding is interrupted (Babile et al. 1998; Benard et al. 1998). However, the susceptibility to fatty liver induction is, at least partly, of genetic origin, determining the channeling of newly synthesized lipids between intracytoplasmic storage and secretion as plasma lipoproteins (Fournier et al. 1997). More precisely, Landaise geese are worldwide famous for fatty liver production. However, Xupu goose is master breed of meat in China. Indeed, in Landaise geese the secretion of hepatic lipids is not efficient enough to prevent their intercellular storage (Fournier et al. 1997), despite being a similar food intake in both the breeds. De novo lipogenesis differs markedly between avian and mammalian species (Pearce 1977). The liver is not only the major site of fatty acid synthesis in birds, but also has been described in the chicken, that the supply of nicotinamide adenine dinucleotide phosphate (NADPH) required for this synthesis depends mainly on the activity of malic enzyme (ME) instead of glucose-6-phosphatedehydrogenase (G6PDH) (Mourot et al. 2000; Heathet et al. 2004). Very few data are available on the metabolic response to force feeding, especially the information about the overfeeding of Xupu geese, on biochechemical changes is not reported till now. Thus, the present study was designed to evaluate the capacity of hepatic lipogenesis in Landaise geese and Xupu white goose. This would shed light on biochemical changes of hepatic lipogenesis in liver in the Landaise goose and the Xupu goose.
MATERIALS AND METHODS Animals and experimentaldesign Twenty male France Landaise geese and 20 male Xupu geese, hatched on the same day, were grown under natural conditions of light and temperature at the experimental station for waterfowl nutrition of Huazhong
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Agricultural University (Wuhan, P.R.China). The two breeds were housed collectively, but were bred in separated cages. From 0 to 4 wk of age, they had free access to a starter diet containing 12.13 MJ kg-' and 200 g protein kg-'. From 4 to 22 wk, they were given a growing diet containing 10.79 MJ kg-' and 130 g protein kg-'. The daily intake of the growing diet was reduced to avoid excessive fatness. The daily amount of feed provided was 290 g between 4 and 5 wk, 330 g between 5 and 7 wk, 260 g between 7 and 8 wk, and 200 g between 8 and 22 wk. At 22 wk of age, they began the period of 'preoverfeeding' for 1 wk, during which the amount of food was progressively raised to 700 g d-' on average at the end of this week to increase the volume of the digestive tract and to initiate the adaptation to overfeeding.At the end of the preoverfeeding period, both groups were given metabolic average of five meals per day for 14 d of a carbohydrate-rich brown rice diet (13.59 MJ kg-', 84.9 g CP kg-'). After dunking in the water (20°C) for 2 h and addition of 0.4% NaC1, the final mixture consisted of 3/4 feed and 1/4 water. The overfeedingdiets and the overfeeding amount of diets were presented in Tables 1 and 2, respectively. During the overfeeding period, birds were housed in individual pens, and had free access to water at all times. In the overfeeding room, the temperature was 18-22°C and the hygrometry was 60-70%.
Blood and liver samples The last day of the overfeeding period (26 wk), the two groups of geese were deprived of feed for 18 h with the water provided. Then blood was withdrawn by puncture of the occipital venous sinus, collected in a vacuum tube containing 1.2 g L-' of ethylene diamine tetraacetic acid (EDTA) , and kept at 4°C during the subsequent procedures. Individual plasma samples were separated Table 1 Composition of overfeeding diets Ingredients and analvsis Brown rice') Water NaHCO, Salt Compound vitamin Analytical characteristics2) Crude protein (CP) Gross energy (GE)
Comnosition 74.0% 24.8% 0.5% 0.5% 0.2%
8.49% 13.59 MJ kg-'
"Brown rice was only dipped in water at room temperature for 2 h, not boiled. "Crude protein (CP) content and gross energy (GE) calculated by the data from analysis: brown rice (GE. 18.37 MJ kg.'; CP, 11.47%).
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Table 2 Overfeeding amount of diets Day 1 2 3-7 8-14 15-end
Overfeeding amount (g goose1 d-1) 120 210 300-360 400-600 610-950
Overfeeding times (times d-1) 2 3 4 4
Overfeeding time 1O:OO 19:30 1O:OO 19:OO 24:OO 1O:OO 14:30 19:OO 24:OO 4:30 1O:OO 1 4 3 0 19:OO 24:OO 4:30 1O:OO 14:30 19:OO 24:OO
5
mRNA
by centrifugation at 3 OOO x g-force for 20 min. Antibacterial agents (sodium a i d e 0.1 g L-' and EDTA 0.8 g L-l) were added to plasma samples. Samples were frozen at -20°C for further analyses. Immediately after blood sampling, the geese were killed in a slaughter house by exsanguinations while under electronarcosis. A 20 g sample was immediately taken from the ventromedial portion of the main lobe (right lobe) of each liver (n= 12 from each breed), through a limited incision of the abdominal wall. These samples were frozen in liquid nitrogen and stored at -80°C before determination of enzymatic activities. The corresponding carcasses were then kept at 4°C overnight before dissecting and weighing representative body compartments: liver, abdominal adipose tissue, fillet and muscle Pectoralis major and the part composed of the skin plus subcutaneous adipose tissue. Liver samples from other geese (15 Landaise geese and 15 Xupu white geese) were processed immediately for quantification of messenger level of ME. Only the liver weight was measured in these groups.
Total RNA was isolated from individual livers by the guanidium thiocyanate method (Chomczynski and Sacchi 1987). About 1 g from each liver were crushed in denaturing solution (4 M guanidium thiocyanate, 25 mM sodium citrate pH 7, 0.5% sarcosyl, 0.1M betamercaptoethanol) and stored at -20°C for ME mRNA analysis. Electrophoresis and northern blot analyses were performed with 10 mg of total RNA from each bird as described by Douaire et al. (1992) and Rodriguez et al. (Miguel et al. 2003). Hybridization procedures with chicken ME probe (Back et al. 1986) and with mouse 18s ribosomal RNA used as control of RNA loading (Raynal et al. 1984) were previously described by Chaillou et al. (1997). Individual accumulation of ME mRNA was expressed as grey levels in arbitrary units corresponding to 10 mg of total liver mRNA, corrected for the possible quantitative variations in loading onto membranes.
Enzymes activity
VLDL analyses
Activities of the lipogenic enzyme in liver tissues were determined. Weighed quantities (about 800 mg) of liver were homogenized in 0.25 M sucrose and centrifuged at 40 000 x g for 40 min. Supernatants were analyzed for ME and G6PDH using modifications (Gandemer et al. 1983) from the methods of Fitch et al. (1959) and Hsu and Lardy (1969), respectively. NADPH formation was measured at 37°C by absorbance at 340 nm. Acetyl-CoA-carboxylase (ACX) was assayed by the H14C0,-fixationmethod (Chang et al. 1967; Chakrabarty and Leveille 1969). Fatty acid synthase (FAS) was measured as described by the method of Lavau et al. (1982). ME, G6PDH and FAS activities were expressed as micromoles of NADPH produced or used per minute per total liver and milligram of protein. ACX activity was expressed as nanomoles of bicarbonate incorporated per minute per total liver and milligram of protein.
VLDL were separated from plasma by ultracentrifugation ( 1 . 9 4 ~lo5g) for 18 h at 10°C. Their concentration and composition were determined as described previously (Fournier et al. 1997).
Statistical analyses Results were expressed as mean & SD and their significance was analyzed by Student's t-test. Correlations were determined by linear regression.
RESULTS Body composition After 3 wk of force feeding, the food intake of brown
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HeDatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese
rice corresponded to 13.30 and 13.88 kg in Landaise geese and Xupu geese, respectively, the body weight was similar and identical in both breeds, respectively, (Table 1). These results showed that the weight of fatty liver was doubly higher in Landaise geese than in Xupu geese, respectively; the weight of fatty liver of Landaise geese and Xupu geese were 801 and 375 g (P<0.05). There were differences on the abdominal fat pat, filet total, VLDL, CE and filet pectoralis major in the experimental Landaise geese and Xupu geese (P<0.05) and no difference on body and filet skin plus S.C.adipose tissue ( P > 0.05) (Table 3).
VLDL analyses VLDL concentration and composition in two birds are presented in Table 4.The data in this table showed FC, TG, PL and protein ( P >0.05) in the tested Landaise geese and Xupu geese.
Activities of lipogenic enzymes The activities of the major hepatic lipogenic enzymes of two breeds of goose are given in Table 5. The data in this table showed that there were no difference in activities of ME, G6PDH, mRNA level of ME, ACX and FAS (per whole liver) in the experimentalLandaise geese and Xupu geese ( P < 0.0s). Table 3 Body composition') Breed Body Liver Abdominal fat pad Filet total Filet skin+s.c.adipose tissue Filet pectoralis major
Landaise geese (g) 8 650 2 720 801 t 143' 420 & 68.5' 440 f 60.2' 160 t 42.7 260 t 53.8'
Xupu geese (g) 8 820 f 336 375 f 159' 578 + 120' 520 f 55' 175 f 44.2 330 + 37.5'
]!Results are mean f SD of 15 geese in each breed. * means within a row differ significantly (P
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Table 4 VLDL concentration and composition') VLDL (g L-l) Free cholesterol (%) Cholesteryl esters (%) Triglycerides (%) Phospholipids (%) Proteins (%) "Results are mean
Landaise geese 3.92 f 1.06' 5.2 f 1.4 8.5 + 4.9' 66.5 f 8.7 10.8 + 2.3 5.3 t 1.7
Xupu geese 2.10f 1.21'
4.8+ 1.1 4.0 f 2.2' 72.6 t 3.9 10.4 t 1.7 4.8 + 1.2
+ SD of 15 geese in each breed.
' means within a row differ significantly (Pc0.05).
Correlations The correlations between enzymatic activities and the fatty liver weight differed markedly in the two breeds (Table 6). The data in this table showed that in Landaise geese, there was no significant correlation with the various specific activities. In China Xupu geese, the liver weight was negatively correlated to the specific activity of ACX and positively to that of ME. The correlations between enzymatic activities and plasma VLDL concentration are given in Table 7. The data in this table show that Plasma VLDL concentration correlates positively with the activity of ME (both specific activity and activity per whole liver) and the liver weight, and negatively with the specific activity of ACX.
DISCUSSION The present study revealed some of the biochemical aspects taking place in the liver and blood of geese during liver fattening. These data showed that the limiting factors in the susceptibility to hepatic steatosis differ in the two breeds. In Xupu geese, which is the best and used for meat production, there is a positive relationship between ME activity, VLDL concentration, and liver weight. The weight of fatty liver was double higher
Table 5 Enzyme activities and mRNA levels Enzyme
ME G6PDH ACX FAS
Activities PM NADPH formed x lo, min-*liver 1 pM NADPH formed min-I mg-1 protein pM NADPH formed x 103 min-1 liver' pM NADPH formed min-l mg-1protein NM HCO-, x 10 3 min-1 liver-' NM HCO-, min-1 rng-1 protein @ NADPH I fixed x 10-3min~'liver-' pM NADPH formed mi& mg-1 protein
The levels of ME mRNA
Landaise geese 460+110' 8.8 f 1.62' 135 i 41' 2.51 2 0.63 18.9 + 5.21' 0.382 + 0.099 51.3 + 15.7' 0.963 t 0.255 35.0 + 11.6'
Xupu geese 2102 101' 5.11 -C 1.57' 92-c31' 2.42 + 0.59 14.1 + 4.36' 0.376 k 0.082 30.8 -c 12.5'
0.785 2 0.223 22.8 + 9.8'
IIResults are mean -c SD of 15 geese in each breed. * means within a row differ significantly (Pi0.05).
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LIU Xiang-you er a1
Table 6 Correlations with liver mass1) Breed ME
G6PDH ACX FAS I)
Activity liver' Activity mg-1 protein Activity liver-' Activity mg-1 protein Activity liver' Activity m g ~ lprotein Activity liver-' Activity mg-1 protein
Landaise geese (g) +0.732; 0.0207 +0.017; 0.951 +0.185; 0.620 -0.288; 0.400 +0.408; 0.230 -0.207; 0.542 +0.881; 0.0007 +0.521; 0.130
Xupu geese (g) +0.951; 0.0001 +0.789; 0.0059 ~ 0 . 6 3 10.0532 ; ~0.155;0.650 ~ 0 . 1 8 50.621 ; -0.730; 0.0160 +0.460; 0.180 -0.048; 0.900
Landaise geese -0.145; 0.691 -0.144; 0.691 -0.857; 0.0014 +0.401; 0.250 +0.080; 0.833 +0.260; 0.474 -0.176; 0.632 +0.136; 0.710 -0.218; 0.553 +0.178; 0.625
Xupu geese +0.277; 0.435 +0.855; 0.0021 ~ 0 . 8 4 00.0021 ; ~ 0 . 5 4 00. ; I 10 +0.280; 0.440 -0.001; 0.997 -0.677; 0.0345 0 312; 0.339 -0.018; 0.969 +0.825; 0.0023
Results are r (1st value) and P (2nd value) for 15 geese in each group.
Table 7 Correlations with plasma VLDL concentration') Breed Liver mass ME
G6PDH ACX FAS Liver mass
Activity mg 1 protein Activity liver-' Activity mg-l protein Activity liver-' Activity mg I protein Activity liver Activity mg-1 protein Activity liver-' Activity mg-1 protein g
~
I)
Results are r (1st value) and P (2nd value) for 15 geese in each group.
in Landaise geese than in Xupu geese, whereas Xupu geese exhibited a better development of muscular and abdominal fat pad and filet total than Landaise geese. The same difference in fatty liver weights was found between the two groups by using the determination of ME mRNA levels. This was consistent with what was known of lipid metabolism in other avian species (Hermier 1997). Compared to Poland white breed, which was the best used for meat production, Landaise geese was the best breed for its higher susceptibility to fatty liver production (Poujardieu et al. 1994). There were high percentage of TG and low protein content in both the breeds. These data confirmed those obtained previously in the Landaise geese (Hermier et at. 1984). In contrast to the previous data (Arjen et al. 200l), there was no difference between the two breeds (P<0.05). In response to overfeeding, the hepatic steatosis associated with an imbalance between lipid synthesis and secretion, the fact that de novo hepatic lipogenesis from dietary carbohydrates was dramatically enhance in goose resulted in the accumulation of TG and other lipids (Mourot and Guy 2000). VLDL and EC were higher in the Landaise geese. The FC and PL were similar in both the groups (P>O.O5). This was consistent with previous studies done in Poland goose and Landaise geese (Mourot et al. 2000). The plasma TG and VLDL concentrations were positively correlated
with the fatty liver weight in Xupu geese, but not in the case in Landaise geese. These indicated that plasma VLDL concentration was a good indicator of hepatic lipogenesis (Stephanie et al. 2000) and it might be a limiting factor of the susceptibility to fatty liver in this breed. This factor might be resulting in a balance between hepatic lipogenesis and lipoprotein export to peripheral tissue (Kouba et al. 1995). Davail et al. (1998) suggested that VLDL concentration in the overfed Landaise goose would better reflect the catabolic capacity of extrahepatic tissues. All enzymes were higher in Landaise geese than in Xupu geese. This indicated that lipogenesis remains very active. There were significant differences in the levels of the enzymatic parameters in both the breeds. This was consistent with previous studies about the growing chicken and turkey (Goodridge et a l . 1996). Moreover, the liver weight (Table 1) in both the breeds had relation to the levels of enzymes activities, especially ME. The levels of ME were two-fold higher in Landaise geese than in that of Xupu goose. This was in agreement with the present literature showing that the activity of ME was the best indicator of hepatic lipogenic capacity in avian species (Mourot and Guy 2000). Interestingly, G6PDH activity was markedly lower than that of ME, but remained far from negligible. This was because the overfed goose differed from other avian
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Hepatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese
species (Pearce 1977) in that the pentose-phosphate pathway may contribute substantially to the generation of reducing power necessary for fatty acid synthesis. Moreover, a higher content of ME mRNA in the liver of the Landaise geese had relations with the difference in activity of ME (Table 5). Although protein content of the fatty liver in Landaise geese was higher than that of Xupu geese, the absolute amount of hepatic proteins was identical in both the breeds, irrespective of the degree of steatosis. These data showed that this enzyme activity would be mainly regulated at the transcriptional level in the goose. Moreover, specific activities of lipogenic (ME, G6PDH and ACX) were not influenced by the diet (Mossab et al. 2002), and the difference in ME activity between the two birds might be either ME gene structure or regulator factor activity at a higher level. De novo fatty acid synthesis needs NADPH (Heathet et al. 2004). That was to say that the capacity of fatty acid synthesis and that of subsequent hepatic steatosis in the goose had relation with NADPH. However, the capacity of ME is three-fold more than G6PDH, 24 times more than ACX and nine times more than FAS. It was possible that the ME was the major enzyme to provide H’ to the synthesis of fat. These results showed that ME plays a major role in determining the level of hepatic lipogenesis in the goose. Thus, the lower ME activity in the overfed Xupu geese might be a limiting factor of hepatic steatosis in this breed. In Landaise geese, there was no significant correlation with the various specific activities. However, the liver weight increased in parallel with the activities of FAS and ME in the whole liver. In Xupu geese, the liver weight was negatively correlated to the specific activity of ACX and positively to that of ME. For Landaise geese, we noticed a positive relationship between liver weight on one hand and the total activity (per whole liver) of FAS and ME on the other hand (the correlation with FAS was not significant). However, since enzymatic activities were determined at the end of the overfeeding period, it is difficult to distinguish between causes and consequences. Most probably, these correlations reflected the increase in hepatic protein content with liver weight (Salichon et al. 1998); which resulted in a parallel increase in the available amounts of enzyme proteins. However, one may wonder why these correlations were not significant for G6PDH and ACX, which
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may indicate that factors other than hepatic protein content may be involved. The correlations between enzymatic activities and plasma VLDL concentration were more mnfusing (Table 7). In Landaise geese, the only significant correlation was a negative relationship with the specific activity of ME. In contrast, Xupu geese breed, plasma VLDL concentration was correlated positively with the activity of ME (both specific activity and activity per whole liver) and the liver weight, and negatively with the specific activity of ACX.
Acknowledgements This work is a part from Programs of Science and Technology Project of Early Paddy Quality Improvement (99-022) funded by the Ministry of Science and Technology of China, and Introduction and Selection of Specialized Geese Breeds for Fatty Liver Production and Research on Technology of Their Overfeeding (2002200513202) supported by the Bureau of Science and Technology of Wuhan City, Hubei Province, China. The authors thank Prof. Alain Dauta (French expert) for his constructive advices.
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Agricultural Sciences in China
2006, 5 ( 5 ) : 390-396
May 2006
Hepatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese LIU Xiang-you1.2, HE Rui-guol, HUANG Chou-shen3, LI Xiang', ZHOU Qi-an', WANG Cheng', ZHAO Nal
and ZHOU Shi-xis' College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R.China Department of Animal Science and Technology, Yunnan Province Zhaotong Agricultural School, Zhaotong 657000, P.R.China 3
Hubei Province Poultry Associafion, Wuhan 430070, P.R.China
Abstract This experiment studied hepatic lipogenesis associated with biochemical changes in overfed Landaise Geese and China Xupu geese. Twenty healthy male Landaise geese and 20 healthy male Xupu geese, hatched on the same day under the same feeding conditions, were selected as experimental anima1.s. The animals were divided into two groups and each breed served as an experimental group. Per goose of per experimental group served for a repeat. Brown rice was selected as test diet. After overfeeding for 21 d and then slaughtering, the biochemical changes of hepatic lipogenesis in the genetic susceptibility to fatty liver were evaluated. These results showed that (1) the weight of fatty liver of the two breeds of geese were 801 and 375 g (P
Key words: goose, liver, hepatic lipogenesis, overfed
INTRODUCTION Fatty liver in the goose results from an increased hepatic lipogenesis in response to overfeeding, together with a deficient secretion of triacylglycerol as verylow-densitylipoprotein (VLDL) (Dominique et al. 1994). There are few natural animal populations in which we
can study the metabolic adaptations resulting in hepatic steatosis, as well as individual responsiveness. These populations include lactating cows (Reids and Roberts 1983), fasting suncus (Yasuhara et al. 1991), and wild migrating species of birds and fishes, in which hepatic steatosis occurs spontaneously as a consequence of energy storage before migration (Pilo and George 1983). This process is facilitated in these oviparous species,
Received 31 May, 2005 Accepted 24 March, 2006 LIU Xiang-you, Ph D; Correspondence HE Rui-guo, Mobile: 13986231290, Tel: +86-27-87281060, Fax: +86-27-87280479, E-mail: liuxiangyou99@ 163.com
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Hepatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese
especially waterfowl (geese and ducks), which are particularly susceptible to hepatic steatosis because the liver is the major site of de novo lipogenesis (Pearce 1977). In response to overfeeding with a carbohydrate-rich corn-based diet, Landaise geese liver weight may increase up to 10-fold in 2 weeks and account for up to 10% of the body weight (Hermier et al. 1991). Further, this fatty liver is totally reversible, the liver returning to its initial composition when overfeeding is interrupted (Babile et al. 1998; Benard et al. 1998). However, the susceptibility to fatty liver induction is, at least partly, of genetic origin, determining the channeling of newly synthesized lipids between intracytoplasmic storage and secretion as plasma lipoproteins (Fournier et al. 1997). More precisely, Landaise geese are worldwide famous for fatty liver production. However, Xupu goose is master breed of meat in China. Indeed, in Landaise geese the secretion of hepatic lipids is not efficient enough to prevent their intercellular storage (Fournier et al. 1997), despite being a similar food intake in both the breeds. De novo lipogenesis differs markedly between avian and mammalian species (Pearce 1977). The liver is not only the major site of fatty acid synthesis in birds, but also has been described in the chicken, that the supply of nicotinamide adenine dinucleotide phosphate (NADPH) required for this synthesis depends mainly on the activity of malic enzyme (ME) instead of glucose-6-phosphatedehydrogenase (G6PDH) (Mourot et al. 2000; Heathet et al. 2004). Very few data are available on the metabolic response to force feeding, especially the information about the overfeeding of Xupu geese, on biochechemical changes is not reported till now. Thus, the present study was designed to evaluate the capacity of hepatic lipogenesis in Landaise geese and Xupu white goose. This would shed light on biochemical changes of hepatic lipogenesis in liver in the Landaise goose and the Xupu goose.
MATERIALS AND METHODS Animals and experimentaldesign Twenty male France Landaise geese and 20 male Xupu geese, hatched on the same day, were grown under natural conditions of light and temperature at the experimental station for waterfowl nutrition of Huazhong
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Agricultural University (Wuhan, P.R.China). The two breeds were housed collectively, but were bred in separated cages. From 0 to 4 wk of age, they had free access to a starter diet containing 12.13 MJ kg-' and 200 g protein kg-'. From 4 to 22 wk, they were given a growing diet containing 10.79 MJ kg-' and 130 g protein kg-'. The daily intake of the growing diet was reduced to avoid excessive fatness. The daily amount of feed provided was 290 g between 4 and 5 wk, 330 g between 5 and 7 wk, 260 g between 7 and 8 wk, and 200 g between 8 and 22 wk. At 22 wk of age, they began the period of 'preoverfeeding' for 1 wk, during which the amount of food was progressively raised to 700 g d-' on average at the end of this week to increase the volume of the digestive tract and to initiate the adaptation to overfeeding.At the end of the preoverfeeding period, both groups were given metabolic average of five meals per day for 14 d of a carbohydrate-rich brown rice diet (13.59 MJ kg-', 84.9 g CP kg-'). After dunking in the water (20°C) for 2 h and addition of 0.4% NaC1, the final mixture consisted of 3/4 feed and 1/4 water. The overfeedingdiets and the overfeeding amount of diets were presented in Tables 1 and 2, respectively. During the overfeeding period, birds were housed in individual pens, and had free access to water at all times. In the overfeeding room, the temperature was 18-22°C and the hygrometry was 60-70%.
Blood and liver samples The last day of the overfeeding period (26 wk), the two groups of geese were deprived of feed for 18 h with the water provided. Then blood was withdrawn by puncture of the occipital venous sinus, collected in a vacuum tube containing 1.2 g L-' of ethylene diamine tetraacetic acid (EDTA) , and kept at 4°C during the subsequent procedures. Individual plasma samples were separated Table 1 Composition of overfeeding diets Ingredients and analvsis Brown rice') Water NaHCO, Salt Compound vitamin Analytical characteristics2) Crude protein (CP) Gross energy (GE)
Comnosition 74.0% 24.8% 0.5% 0.5% 0.2%
8.49% 13.59 MJ kg-'
"Brown rice was only dipped in water at room temperature for 2 h, not boiled. "Crude protein (CP) content and gross energy (GE) calculated by the data from analysis: brown rice (GE. 18.37 MJ kg.'; CP, 11.47%).
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Table 2 Overfeeding amount of diets Day 1 2 3-7 8-14 15-end
Overfeeding amount (g goose1 d-1) 120 210 300-360 400-600 610-950
Overfeeding times (times d-1) 2 3 4 4
Overfeeding time 1O:OO 19:30 1O:OO 19:OO 24:OO 1O:OO 14:30 19:OO 24:OO 4:30 1O:OO 1 4 3 0 19:OO 24:OO 4:30 1O:OO 14:30 19:OO 24:OO
5
mRNA
by centrifugation at 3 OOO x g-force for 20 min. Antibacterial agents (sodium a i d e 0.1 g L-' and EDTA 0.8 g L-l) were added to plasma samples. Samples were frozen at -20°C for further analyses. Immediately after blood sampling, the geese were killed in a slaughter house by exsanguinations while under electronarcosis. A 20 g sample was immediately taken from the ventromedial portion of the main lobe (right lobe) of each liver (n= 12 from each breed), through a limited incision of the abdominal wall. These samples were frozen in liquid nitrogen and stored at -80°C before determination of enzymatic activities. The corresponding carcasses were then kept at 4°C overnight before dissecting and weighing representative body compartments: liver, abdominal adipose tissue, fillet and muscle Pectoralis major and the part composed of the skin plus subcutaneous adipose tissue. Liver samples from other geese (15 Landaise geese and 15 Xupu white geese) were processed immediately for quantification of messenger level of ME. Only the liver weight was measured in these groups.
Total RNA was isolated from individual livers by the guanidium thiocyanate method (Chomczynski and Sacchi 1987). About 1 g from each liver were crushed in denaturing solution (4 M guanidium thiocyanate, 25 mM sodium citrate pH 7, 0.5% sarcosyl, 0.1M betamercaptoethanol) and stored at -20°C for ME mRNA analysis. Electrophoresis and northern blot analyses were performed with 10 mg of total RNA from each bird as described by Douaire et al. (1992) and Rodriguez et al. (Miguel et al. 2003). Hybridization procedures with chicken ME probe (Back et al. 1986) and with mouse 18s ribosomal RNA used as control of RNA loading (Raynal et al. 1984) were previously described by Chaillou et al. (1997). Individual accumulation of ME mRNA was expressed as grey levels in arbitrary units corresponding to 10 mg of total liver mRNA, corrected for the possible quantitative variations in loading onto membranes.
Enzymes activity
VLDL analyses
Activities of the lipogenic enzyme in liver tissues were determined. Weighed quantities (about 800 mg) of liver were homogenized in 0.25 M sucrose and centrifuged at 40 000 x g for 40 min. Supernatants were analyzed for ME and G6PDH using modifications (Gandemer et al. 1983) from the methods of Fitch et al. (1959) and Hsu and Lardy (1969), respectively. NADPH formation was measured at 37°C by absorbance at 340 nm. Acetyl-CoA-carboxylase (ACX) was assayed by the H14C0,-fixationmethod (Chang et al. 1967; Chakrabarty and Leveille 1969). Fatty acid synthase (FAS) was measured as described by the method of Lavau et al. (1982). ME, G6PDH and FAS activities were expressed as micromoles of NADPH produced or used per minute per total liver and milligram of protein. ACX activity was expressed as nanomoles of bicarbonate incorporated per minute per total liver and milligram of protein.
VLDL were separated from plasma by ultracentrifugation ( 1 . 9 4 ~lo5g) for 18 h at 10°C. Their concentration and composition were determined as described previously (Fournier et al. 1997).
Statistical analyses Results were expressed as mean & SD and their significance was analyzed by Student's t-test. Correlations were determined by linear regression.
RESULTS Body composition After 3 wk of force feeding, the food intake of brown
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HeDatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese
rice corresponded to 13.30 and 13.88 kg in Landaise geese and Xupu geese, respectively, the body weight was similar and identical in both breeds, respectively, (Table 1). These results showed that the weight of fatty liver was doubly higher in Landaise geese than in Xupu geese, respectively; the weight of fatty liver of Landaise geese and Xupu geese were 801 and 375 g (P<0.05). There were differences on the abdominal fat pat, filet total, VLDL, CE and filet pectoralis major in the experimental Landaise geese and Xupu geese (P<0.05) and no difference on body and filet skin plus S.C.adipose tissue ( P > 0.05) (Table 3).
VLDL analyses VLDL concentration and composition in two birds are presented in Table 4.The data in this table showed FC, TG, PL and protein ( P >0.05) in the tested Landaise geese and Xupu geese.
Activities of lipogenic enzymes The activities of the major hepatic lipogenic enzymes of two breeds of goose are given in Table 5. The data in this table showed that there were no difference in activities of ME, G6PDH, mRNA level of ME, ACX and FAS (per whole liver) in the experimentalLandaise geese and Xupu geese ( P < 0.0s). Table 3 Body composition') Breed Body Liver Abdominal fat pad Filet total Filet skin+s.c.adipose tissue Filet pectoralis major
Landaise geese (g) 8 650 2 720 801 t 143' 420 & 68.5' 440 f 60.2' 160 t 42.7 260 t 53.8'
Xupu geese (g) 8 820 f 336 375 f 159' 578 + 120' 520 f 55' 175 f 44.2 330 + 37.5'
]!Results are mean f SD of 15 geese in each breed. * means within a row differ significantly (P
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Table 4 VLDL concentration and composition') VLDL (g L-l) Free cholesterol (%) Cholesteryl esters (%) Triglycerides (%) Phospholipids (%) Proteins (%) "Results are mean
Landaise geese 3.92 f 1.06' 5.2 f 1.4 8.5 + 4.9' 66.5 f 8.7 10.8 + 2.3 5.3 t 1.7
Xupu geese 2.10f 1.21'
4.8+ 1.1 4.0 f 2.2' 72.6 t 3.9 10.4 t 1.7 4.8 + 1.2
+ SD of 15 geese in each breed.
' means within a row differ significantly (Pc0.05).
Correlations The correlations between enzymatic activities and the fatty liver weight differed markedly in the two breeds (Table 6). The data in this table showed that in Landaise geese, there was no significant correlation with the various specific activities. In China Xupu geese, the liver weight was negatively correlated to the specific activity of ACX and positively to that of ME. The correlations between enzymatic activities and plasma VLDL concentration are given in Table 7. The data in this table show that Plasma VLDL concentration correlates positively with the activity of ME (both specific activity and activity per whole liver) and the liver weight, and negatively with the specific activity of ACX.
DISCUSSION The present study revealed some of the biochemical aspects taking place in the liver and blood of geese during liver fattening. These data showed that the limiting factors in the susceptibility to hepatic steatosis differ in the two breeds. In Xupu geese, which is the best and used for meat production, there is a positive relationship between ME activity, VLDL concentration, and liver weight. The weight of fatty liver was double higher
Table 5 Enzyme activities and mRNA levels Enzyme
ME G6PDH ACX FAS
Activities PM NADPH formed x lo, min-*liver 1 pM NADPH formed min-I mg-1 protein pM NADPH formed x 103 min-1 liver' pM NADPH formed min-l mg-1protein NM HCO-, x 10 3 min-1 liver-' NM HCO-, min-1 rng-1 protein @ NADPH I fixed x 10-3min~'liver-' pM NADPH formed mi& mg-1 protein
The levels of ME mRNA
Landaise geese 460+110' 8.8 f 1.62' 135 i 41' 2.51 2 0.63 18.9 + 5.21' 0.382 + 0.099 51.3 + 15.7' 0.963 t 0.255 35.0 + 11.6'
Xupu geese 2102 101' 5.11 -C 1.57' 92-c31' 2.42 + 0.59 14.1 + 4.36' 0.376 k 0.082 30.8 -c 12.5'
0.785 2 0.223 22.8 + 9.8'
IIResults are mean -c SD of 15 geese in each breed. * means within a row differ significantly (Pi0.05).
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LIU Xiang-you er a1
Table 6 Correlations with liver mass1) Breed ME
G6PDH ACX FAS I)
Activity liver' Activity mg-1 protein Activity liver-' Activity mg-1 protein Activity liver' Activity m g ~ lprotein Activity liver-' Activity mg-1 protein
Landaise geese (g) +0.732; 0.0207 +0.017; 0.951 +0.185; 0.620 -0.288; 0.400 +0.408; 0.230 -0.207; 0.542 +0.881; 0.0007 +0.521; 0.130
Xupu geese (g) +0.951; 0.0001 +0.789; 0.0059 ~ 0 . 6 3 10.0532 ; ~0.155;0.650 ~ 0 . 1 8 50.621 ; -0.730; 0.0160 +0.460; 0.180 -0.048; 0.900
Landaise geese -0.145; 0.691 -0.144; 0.691 -0.857; 0.0014 +0.401; 0.250 +0.080; 0.833 +0.260; 0.474 -0.176; 0.632 +0.136; 0.710 -0.218; 0.553 +0.178; 0.625
Xupu geese +0.277; 0.435 +0.855; 0.0021 ~ 0 . 8 4 00.0021 ; ~ 0 . 5 4 00. ; I 10 +0.280; 0.440 -0.001; 0.997 -0.677; 0.0345 0 312; 0.339 -0.018; 0.969 +0.825; 0.0023
Results are r (1st value) and P (2nd value) for 15 geese in each group.
Table 7 Correlations with plasma VLDL concentration') Breed Liver mass ME
G6PDH ACX FAS Liver mass
Activity mg 1 protein Activity liver-' Activity mg-l protein Activity liver-' Activity mg I protein Activity liver Activity mg-1 protein Activity liver-' Activity mg-1 protein g
~
I)
Results are r (1st value) and P (2nd value) for 15 geese in each group.
in Landaise geese than in Xupu geese, whereas Xupu geese exhibited a better development of muscular and abdominal fat pad and filet total than Landaise geese. The same difference in fatty liver weights was found between the two groups by using the determination of ME mRNA levels. This was consistent with what was known of lipid metabolism in other avian species (Hermier 1997). Compared to Poland white breed, which was the best used for meat production, Landaise geese was the best breed for its higher susceptibility to fatty liver production (Poujardieu et al. 1994). There were high percentage of TG and low protein content in both the breeds. These data confirmed those obtained previously in the Landaise geese (Hermier et at. 1984). In contrast to the previous data (Arjen et al. 200l), there was no difference between the two breeds (P<0.05). In response to overfeeding, the hepatic steatosis associated with an imbalance between lipid synthesis and secretion, the fact that de novo hepatic lipogenesis from dietary carbohydrates was dramatically enhance in goose resulted in the accumulation of TG and other lipids (Mourot and Guy 2000). VLDL and EC were higher in the Landaise geese. The FC and PL were similar in both the groups (P>O.O5). This was consistent with previous studies done in Poland goose and Landaise geese (Mourot et al. 2000). The plasma TG and VLDL concentrations were positively correlated
with the fatty liver weight in Xupu geese, but not in the case in Landaise geese. These indicated that plasma VLDL concentration was a good indicator of hepatic lipogenesis (Stephanie et al. 2000) and it might be a limiting factor of the susceptibility to fatty liver in this breed. This factor might be resulting in a balance between hepatic lipogenesis and lipoprotein export to peripheral tissue (Kouba et al. 1995). Davail et al. (1998) suggested that VLDL concentration in the overfed Landaise goose would better reflect the catabolic capacity of extrahepatic tissues. All enzymes were higher in Landaise geese than in Xupu geese. This indicated that lipogenesis remains very active. There were significant differences in the levels of the enzymatic parameters in both the breeds. This was consistent with previous studies about the growing chicken and turkey (Goodridge et a l . 1996). Moreover, the liver weight (Table 1) in both the breeds had relation to the levels of enzymes activities, especially ME. The levels of ME were two-fold higher in Landaise geese than in that of Xupu goose. This was in agreement with the present literature showing that the activity of ME was the best indicator of hepatic lipogenic capacity in avian species (Mourot and Guy 2000). Interestingly, G6PDH activity was markedly lower than that of ME, but remained far from negligible. This was because the overfed goose differed from other avian
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Hepatic Lipogenesis Associated with Biochemical Changes in Overfed Landaise Geese and China Xupu Geese
species (Pearce 1977) in that the pentose-phosphate pathway may contribute substantially to the generation of reducing power necessary for fatty acid synthesis. Moreover, a higher content of ME mRNA in the liver of the Landaise geese had relations with the difference in activity of ME (Table 5). Although protein content of the fatty liver in Landaise geese was higher than that of Xupu geese, the absolute amount of hepatic proteins was identical in both the breeds, irrespective of the degree of steatosis. These data showed that this enzyme activity would be mainly regulated at the transcriptional level in the goose. Moreover, specific activities of lipogenic (ME, G6PDH and ACX) were not influenced by the diet (Mossab et al. 2002), and the difference in ME activity between the two birds might be either ME gene structure or regulator factor activity at a higher level. De novo fatty acid synthesis needs NADPH (Heathet et al. 2004). That was to say that the capacity of fatty acid synthesis and that of subsequent hepatic steatosis in the goose had relation with NADPH. However, the capacity of ME is three-fold more than G6PDH, 24 times more than ACX and nine times more than FAS. It was possible that the ME was the major enzyme to provide H’ to the synthesis of fat. These results showed that ME plays a major role in determining the level of hepatic lipogenesis in the goose. Thus, the lower ME activity in the overfed Xupu geese might be a limiting factor of hepatic steatosis in this breed. In Landaise geese, there was no significant correlation with the various specific activities. However, the liver weight increased in parallel with the activities of FAS and ME in the whole liver. In Xupu geese, the liver weight was negatively correlated to the specific activity of ACX and positively to that of ME. For Landaise geese, we noticed a positive relationship between liver weight on one hand and the total activity (per whole liver) of FAS and ME on the other hand (the correlation with FAS was not significant). However, since enzymatic activities were determined at the end of the overfeeding period, it is difficult to distinguish between causes and consequences. Most probably, these correlations reflected the increase in hepatic protein content with liver weight (Salichon et al. 1998); which resulted in a parallel increase in the available amounts of enzyme proteins. However, one may wonder why these correlations were not significant for G6PDH and ACX, which
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may indicate that factors other than hepatic protein content may be involved. The correlations between enzymatic activities and plasma VLDL concentration were more mnfusing (Table 7). In Landaise geese, the only significant correlation was a negative relationship with the specific activity of ME. In contrast, Xupu geese breed, plasma VLDL concentration was correlated positively with the activity of ME (both specific activity and activity per whole liver) and the liver weight, and negatively with the specific activity of ACX.
Acknowledgements This work is a part from Programs of Science and Technology Project of Early Paddy Quality Improvement (99-022) funded by the Ministry of Science and Technology of China, and Introduction and Selection of Specialized Geese Breeds for Fatty Liver Production and Research on Technology of Their Overfeeding (2002200513202) supported by the Bureau of Science and Technology of Wuhan City, Hubei Province, China. The authors thank Prof. Alain Dauta (French expert) for his constructive advices.
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