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Activities of Enzymes Involved in Fatty Acid Metabolism in the Colon
Epithelium of Piglets Fed with Different Fiber Contents Diets
Y. H. Zhu, T. Lundh and J. F. Wang*
Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, P.O. Box 7024
SE 750 07 Uppsala, Sweden
ABSTRACT : The present study was conducted to evaluate the influence of dietary fiber on the activities of malic enzyme and citrate
lyase involved in fatty acid metabolism in the colon epithelium of pigs. Thirty-six weaned 5 weeks old crossbred (Yorkshire×Swedish
Landrace) piglets originating from twelve litters were randomly assigned to either a low fiber diet containing 10% non-starch
polysaccharides (NSP), a control diet containing 14.7% NSP or a high fiber diet containing 20% NSP. The activity of malic enzyme in
the colonic epithelium of pigs significantly (p<0.05) increased with age during the suckling-weaning transition. There was a tendency
(p<0.10) of decreased malic enzyme activity in the colonic epithelium of pigs fed on the high fiber diet. At week 6, a lowered (p<0.01)
activity of malic enzyme in pigs fed on the low fiber diet compared with that in pigs fed on the high fiber and the control diets.
Nevertheless, there were no significant differences in the activity of citrate lyase observed either between pigs with different ages or
between pigs fed with various diets. The current data suggest that piglets during the suckling-weaning transition have a limited capacity
to synthesize fatty acids from carbohydrate derivatives in the coloncytes. In addition, lipogenesis in coloncytes was enhanced with age
during the suckling-weaning transition. A tendency (p<0.10) to an increased capacity to utilize acetyl-CoA in coloncytes of pigs has
been observed for the high fiber diet. Moreover, the present work indicated that dietary fiber resulted in a lowered rate of lipogenesis and
a reduced activity of malic enzyme. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 10 : 1524-1528)
Key Words : Colon Epithelium, Suckling-weaning Pigs, Lipogenic Enzyme, Dietary Fiber
INTRODUCTION
In recent years, dietary fiber (DF) has received
considerable attention with regard to its influence on
nutrient digestion and utilization. DF has been defined as
plant materials that are resistant to mammalian digestive
enzymes. With the exception of lignin, all of the materials
called DF are carbohydrates in nature (Kritchevsky, 1988).
It is well documented that dietary fiber is thought to
mediate protective effects on the colonic epithelium through
their fermentation products and fecal bulking capacity
(Bach Knudsen and Hansen, 1991; Wang et al., 2002; Wang
et al., 2003). Some metabolic characteristics of colonic
epithelial cells are closely related to the digestive events in
nutrition of the large intestine. Resistant starch, non-starch
polysaccharides and residual protein are broken down by
colonic microflora to produce short-chain fatty acids. The
major products are acetate, propionate and butyrate, which
are rapidly absorbed through the colonic mucosa
(Macfarlane and Cummings, 1991). Acetate serves
primarily as a fuel for muscle, and propionate has been
implicated in the control of both cholesterol and
carbohydrate metabolism in the liver. In general, butyric
acid, which has important effects on epithelial cell growth
* Corresponding Author: J. F. Wang, College of Veterinary
Medicine, China Agricultural University No. 2 Yuanmingyuan
West Road, Beijing 100094, P.R. China, Tel: +86-10-62893053,
Fax: +86-10-62891274, E-mail: [email protected]
Received February 3, 2002; Accepted April 22, 2003
and differentiation and the concentrations of long-chain
fatty acids, may control cell turnover in the gut (Lupton,
1995).
Gaiva et al. (2001) found that ATP citrate lyase and
malic enzyme activities are contributed to alterations of in
vivo lipogenesis rate. In the study of lipogenesis in adipose
tissue in pigs, malic enzyme was recognized to play a key
role in the lipogenic process and the activity of malic
enzyme is thought to be a good indicator of lipogenesis
(Lee and Kauffman, 1974; Leveille, 1970).
In addition, fatty acids are degraded by oxidation at the
β-carbon in mitochondria. Ketogenesis occurs mainly in the
hepatocytes, but also in the intestines of suckling mammals
(Thumelin et al., 1993). In pigs, ketone-body metabolism is
much lower than in other mammals. One earlier study
shows that the limitation of ketogenesis in newborn-pig
liver is due to a very low amount and activity of
mitochondrial 3-hydroxy-3-methylglutary-CoA synthase (3HMG-CoA synthase) (Duee et al., 1994). However, there is
little information on investigating the enzyme activities
involved in fatty acid metabolism in the colon mucosa of
pigs fed on fiber diets.
The aim of the present work was to study the activities
of enzymes involved in fatty acid metabolism in the colon
mucosa of piglets fed diets with various fiber levels. It was
of interest to investigate whether the activity of enzymes
involved in fatty acid metabolism in the colon mucosa of
pigs was influenced by either the different fiber diets
supplied or different ages between animals.
DIETARY FIBER AND ACTIVITIES OF ENZYMES FOR PIGLETS
Table 1. Composition of the experimental diets (g/kg DM as fed)
fed to examine the activities of enzymes involved in fatty acid
metabolism in the colon epithelium of piglets
Diet
Low fiber
Control
High fiber
Metabolizable
15.59
14.43
14.31
energy (MJ/kg)
DM (%)
92.0
86.0
87.3
Crude fat
41.1
27.0
63.6
Crude protein
179.6
171.3
171.8
Lysine
10.3
8.1
7.2
Methionine
3.8
3.2
2.8
Threonine
5.8
5.6
4.7
Ash
38.6
34.5
44.1
Calcium
9.1
8.5
9.0
Phosphorus
7.9
7.0
8.2
Starch
538.2
549.4
276.2
99.8
147.0
199.6
Total NSPa
I-NSPb
75.7
107.4
149.0
24.1
40.2
50.6
S-NSPc
a
NSP, non-starch polysaccharides; b I-NSP, insoluble non-starch
polysaccharides; c S-NSP, soluble non-starch polysaccharides.
MATERIALS AND METHODS
Animals
Thirty-six crossbred (Yorkshire×Swedish Landrace)
piglets originating from twelve litters were randomly
allotted by litter, weight and gender to three groups with
four individuals per group. Each group was fed on one of
the three experimental diets. One piglet from each litter was
selected in one of the different age groups: 5-week
(suckling), 6-week (1st week post-weaning) and 7-week (2
weeks post-weaning) old, respectively. The mean body
weight (BW) at birth (n=36) was 1.6±0.2 kg, and the mean
BW of piglets (n=12) in each age group, i.e. 5, 6, and 7
weeks old, was 9.9±0.2, 10.9±2.2 and 12.2±2.5 kg,
respectively.
Piglets were given the three different experimental diets
ad libitum from 3 weeks old until the study ended. The
piglets used for sample collection were weighed and killed
by an injection of 30-40 ml of sodium pentobarbitone (100
mg/ml, i.p.) per pig at 5, 6 and 7 weeks, respectively. The
gastrointestinal tract was exposed by a midline laparotomy
and removed immediately.
Piglets were housed in pens with rubber mats on the
concrete floor, but without straw bedding, in an
environmentally controlled building with a room
temperature of 18°C and with light from 07:00 to 09:00 h.
Water was available at all times through low-pressure
nipples.
The Ethical Committee for Animal Experiments in
Uppsala approved all procedures involving animals.
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containing a minimal amount of non-starch polysaccharides
(NSP), a control diet (C) and a high fiber diet enriched with
NSP (H) (Table 1). All of the diets had a similar calculated
content of crude protein. On a dry matter (DM) basis, the
calculated metabolizable energy content in diets L, C and H
was 15.6, 14.4 and 14.3 MJ ME/kg DM, respectively. Fat
was added to diet H in order to provide the same amount of
energy as diets L and C.
The gross energy content was measured with an
adiabatic bomb calorimeter (IKA Calorimeter C 400; Janke
and Kunkel, Germany). Dry matter (DM) contents were
measured by drying at 105°C to constant weight. Crude fat
was determined by the Stoldt (1952) method that HCl-fat
was extracted with diethyl ether after acid hydrolysis. Crude
protein (N×6.25) was assayed by the Kjeldahl method. Ash
was analyzed by the method of the Association of Official
Analytical Chemists (AOAC method 942.05, 1990). Starch
was measured enzymatically as described by Bach Knudsen
(1997). Total NSP and constituent sugars were determined
as alditol acetates by GLC for neutral sugars, and by a
colorimetric method for uronic acids (Bach Knudsen, 1997).
Chemicals
Micro BCATM Protein Assay Reagent Kit was
purchased from Pierce (Rockford, IL, USA). EDTA and
Triton X-100 were from Merck (Darmstadt, Germany) and
the other chemicals were from Sigma Chemical (St. Louis,
MO, USA).
Preparation of intestinal tissues samples
About 30 cm large intestine samples between the apex
of the colon spiral and the rectum were rapidly excised,
opened longitudinally and rinsed twice with ice-cold 0.9%
sodium chloride solution. Immediately, the segments were
divided finely into about 10 cm specimen and snap-frozen
on dry ice. All samples were stored at -80°C for further
analysis of enzyme activities.
Preparation of homogenates
After thawing, a 1/3 (w/v) mucosal homogenate was
made by gently scraping the luminal surface of the colon
tissue with a small spatula, transferring a small quantity of
mucosa to ice-cold homogenating buffer (1 g mucosa/3 ml)
and homogenizing in a Potter-Elvehjem all-glass
homogenizer. The homogenate was sonicated for 60
seconds using an ultrasonic bath (Bandelin Sonorex
RK100H, Germany) and then centrifuged at 15,800 g for 30
min. All operations were conducted at 4°C. Homogenating
buffer contained 50 mM triethanolamine/HCl, 2 mM
magnesium chloride and 1 mM EDTA, pH 7.5 (Crabtree et
al., 1979). The supernatant fluid was considered to contain
the total soluble protein of the cell (cytoplasm and
Experimental diets
The experimental diets included a low fiber diet (L) mitochondria matrix), and was assayed without further
ZHU ET AL.
Activity (nkat/g tissue)
Activity (nkat/g tissue)
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50
40
30
20
10
0
Week 5
Week 6
Control
4
2
0
Week 5
Week 7
Age
Low fiber
6
Low fiber
Week 6
Age
Week 7
Control
High fiber
High fiber
Figure 1. Effect of dietary fiber on the activity of malic enzyme in
the colon epithelium of pigs aged 5-7 weeks. The results are
expressed as least square means±SEM. a, b Mean values within the
same group with different superscript letters differ significantly
(p<0.05). A, B Mean values within the same age with different
superscript letters differ significantly (p<0.05).
Figure 2. Effect of dietary fiber on the activity of citrate lyase in
the colon epithelium of pigs aged 5-7 weeks. The results are
expressed as least square means±SEM.
Y=µ+Ti+Aj+TAij+eijk
Where µ is the mean value for all treatments, Ti (a=3) is
preparation. The resulting supernatant is referred to as
the effect of diet, Aj (b=3) is the effect of age, TAij is the
“homogenates”.
effect of interaction between diet and age and eijk is the
residual variance. If the interaction was not significant,
Assay of enzyme activities
pairwise comparisons were then used to compare treatment
Malic enzyme activity was assayed as described by
groups and age groups, respectively.
Wise and Ball (1964) by measuring the rate of NADPH
formation by monitoring at 340 nm and 37°C. One unit of
RESULTS
enzyme activity causes 1 µmol NADPH to be formed.
Citrate lyase activity was assayed as described by
Activity of enzymes and protein content in the
Bergmeyer (1974). Citrate lyase activity was determined by
epithelium of the large intestine
the malate dehydrogenase-catalysed reduction of
Pigs fed on the control diet showed a significantly
oxaloacetate. One unit of enzyme activity is defined as the
higher (p<0.05) activity (nkat/g tissue) of malic enzyme in
consumption of 1 µmol NADH.
week 6 (38.25±3.57) and week 7 (34.36±3.57) than in week
The activities of these enzymes were measured by using
5 (20.7±3.57) (Figure 1). Also with the high and low fiber
a spectrophotometer (UV-2101PC UV-VIS Scanning
diets, the activity tended (p<0.10) to increase in pigs aged 6
Spectrophotometer, SHIMADZU, Japan). All measurements
weeks and 7 weeks as compared with that in pigs aged 5
were performed at 37°C and the unit of the activity of
weeks. At week 6, the activity of malic enzyme in pigs fed
enzyme is nkat per g tissue. The relationship between nkat
on the low fiber diet (23.9 nkat/g tissue) was significantly
and unit is 1 unit =16.67 nkat.
(p<0.01) lower than in pigs fed on the high fiber (31.7
nkat/g tissue) and the control diets (38.3 nkat/g tissue),
Protein determination
respectively. Also, the activity of malic enzyme in pigs fed
The protein assay was based on the spectrophotometer
on the high fiber diet was lower than in pigs fed on the
on a microwell plate reader (Labsystems Multiskan RC)
control diet at week 6 and week 7, respectively, although
TM
method by using Micro BCA Protein Assay Reagent Kit
there were no significant differences in the activity of malic
(U.S. Patent No. 4,839,295). Protein concentration in the
enzyme observed.
homogenate was adjusted to 1-20 µg/ml before
After weaning the activity of citrate lyase tended to
measurement.
decrease at week 6 whereupon it gradually increased at
week 7 (Figure 2). However, there were no significant
Statistical analysis
differences between either pigs of different ages or pigs fed
The results were presented as the least squares means
on the different fiber diets.
value±standard error of the mean (SEM) and were tested
There were no differences (p>0.05) in the protein
statistically in an analysis of variance test using a General
concentration (mg/g wet tissue) at week 5 (43.5, 51.5 versus
Linear Model (GLM) procedure. The model used was:
52.3), week 6 (47.3, 56.5 versus 57.2) and week 7 (50.0,
57.5 versus 56.6) between diets based on low fiber, control
DIETARY FIBER AND ACTIVITIES OF ENZYMES FOR PIGLETS
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and high fiber. The protein content in the colonic epithelium reactions (Stryer, 1995). In the high energy state the
gradually increased with age for all experimental diets.
mitochondrion has large quantities of ATP and NADH.
Consequently, citrate is formed in the mitochondrial matrix
by the condensation of acetyl-CoA with oxaloacetate. When
DISCUSSION
citrate formed in the mitochondrial matrix arrives at high
Malic enzyme is now recognized to be one of the level, it is transported to the cytosol, and meanwhile is
important enzymes involved in supplying NADPH for the cleaved by ATP-citrate lyase in the cytosol to form
reductive biosynthesis of fatty acids (Mourot et al., 1995). oxaloacetate and acetyl-CoA. The latter is the precursor for
Oxaloacetate is reduced by cytosolic malate dehydrogenase fatty acid synthesis (Salway, 1994). Several studies have
and malate is formed. Malate is oxidatively decarboxylated shown that glucose-6-phosphate dehydrogenase and malic
by malic enzyme with the formation of NADPH (Wise and enzyme were the main enzymes involved in supplying
Ball, 1964; Bergmeyer, 1974). Thumelin et al. (1993) NADPH for biosynthesis of fatty acids (Mourot et al., 1995;
reported that small intestine mucosa of suckling rats had the Bee, 2000). In the current study, we could not find
capacity for ketone-body synthesis. Malic enzyme was significant differences in the activity of citrate lyase in the
recognized to play a key role in the lipogenic process and colon epithelium of piglets between either different ages or
the activity of malic enzyme is also a good indicator of different diets.
lipogenesis (Lee and Kauffman, 1974; Leveille, 1970).
Other studies (Cecilia et al., unpublished data) have
Malic enzyme activity remained high in areas where shown that the activity of citrate synthase in pigs fed on the
lipogenesis was high, and it decreased in adipose tissues high fiber diet tended to increase as compared with that in
where lipogenesis was low. The present data show that pigs fed on the low fiber and control diets. This indicated
malic enzyme activities in pigs fed on the high fiber diet that feeding high fiber caused a tendency of an increased
had a tendency to decrease as compared with that in pigs capacity to utilize acetyl-CoA in pigs. A high fiber content
fed on the control diet. These results indicate that in the in the diet resulted in an increased amount of short-chain
colonic epithelium of pigs given the high fiber diet a fatty acids absorbed by the coloncytes. Acetate, which is the
diminished rate of lipogenesis and concomitant decrease in predominant short-chain fatty acids produced, can be
the activity of malic enzyme took place. Similarly, earlier rapidly absorbed by the coloncytes. In order to get energy,
reports on liver metabolism by Arbeeny et al. (1992) and citrate synthase regulates the acetyl-CoA entry into the
Gibbons (1990) indicated that dietary fiber could decrease TCA-cycle by catalysing the condensation with
lipogenic flux in the liver.
oxaloacetate to citrate (Stryer, 1995).
In contrast, Raju et al. (2001) reported that the activities
Fatty acids are degraded by oxidation at the β-carbon in
of the lipogenic enzymes, especially the activity of malic mitochondria. The hydration of enoyl CoA is the prelude to
enzyme, were increased in the liver and kidney of diabetic the second oxidation reaction of fatty acids, which converts
rats treated with Trigonella seeds powder which is rich in the hydroxyl group at C-3 into keto groups and generates
fibers.
NADH. This oxidation is catalyzed by 3-hydroxyacyl CoA
The present study demonstrates that there were dehydrogenase. Villaverde et al. (unpublished data) clearly
significant differences in the activity of malic enzyme showed that the activity of 3-hydroxyacyl CoA
between different weeks, and that the activity of malic dehydrogenase in the colon of pigs was very low. Their data
enzyme was increased with age, irrespective of type of diet. suggested that long chain fatty acids were not the major
Earlier findings reviewed by Leskanich and Noble (1999) substrates for the energy metabolism in the colonocytes of
indicated that the capacity for in vivo lipogenesis at birth pigs.
was low.
In the present study, the activity of malic enzyme was
The present study shows that the activity of citrate lyase significantly lower in pigs fed on the low fiber diet than in
was very low, indicating that there was a limited capacity to pigs given the high fiber and control diets, respectively.
synthesize fatty acids from carbohydrate derivatives in This might be partly due to a lower food intake in the low
coloncytes of piglets during the suckling-weaning transition. fiber group, which could affect the colonic growth
This situation is similar to that described by Le Dividich et characteristics. It must also be mentioned that low activity
al. (1994) who found that suckling newborn piglets were of malic enzyme resulted from the low short-chain fatty
unable to synthesize fatty acids from carbohydrate in the acid concentrations in the colon of pigs fed on the low fiber
adipose tissue. The general apprehension is that fatty acid diet.
synthesis occurs in the cytosol. However, acetyl-CoA is
In conclusion, piglets during the suckling-weaning
produced by pyruvate dehydrogenase in the mitochondrion. transition had a limited capacity to synthesize fatty acids
Fatty acid synthesis consists of a set of reactions, and the from carbohydrate in coloncytes. In addition, lipogenesis in
general principle is consuming NADPH in biosynthetic coloncytes was enhanced with age during the suckling-
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ZHU ET AL.
enzyme activities of porcine intramuscular adipose tissue. J.
weaning transition. Moreover, the present work indicates
Anim. Sci. 38:538-544.
that dietary fiber resulted in a lowered rate of lipogenesis
Leskanich,
C. D. and R. C. Noble. 1999. The comparative roles of
and a reduced activity of malic enzyme. However we
polyunsaturated fatty acids in pig neonatal development. Br. J.
cannot completely explain how fiber constituents affect the
Nutr. 81:87-106.
enzyme activity in the colon mucosa. Further studies are
Leveille, G. A. 1970. Adipose tissue metabolism: influence of
required to investigate the mechanisms by which dietary
periodicity of eating and diet composition. Fed. Proc. 29:1294fiber influences the activities of enzymes involved in fatty
1301.
acid metabolism in the colon mucosa.
Lupton, J. R. 1995. Short-chain fatty acids and colon
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