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Departments of
*
Foods and Nutrition and
Animal and Dairy Sciences, The University of Georgia, Athens, GA 30602 and
**
U.S. Department of Agriculture/ARS Russell Research Center, Athens, GA 30605
2To whom correspondence should be addressed. E-mail: ghausman{at}saa.ars.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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expression in inguinal
adipose tissue from females, whereas there was no difference in
expression in males. Significant differences in size distribution of
inguinal adipocytes at weaning and retroperitoneal adipocytes at 11 wk
of age were observed. In general, CLA increased the proportion of
smaller cells and decreased the proportion of larger cells. The main
long-term effect of the dams’ diet was the significantly heavier
gastrocnemius and soleus muscles, and significantly longer tail
lengths, an indication of skeletal growth, of male pups whose dams were
fed CLA. Postweaning diet reduced fat pad weights in female but not
male pups fed CLA. This response was due to differences in cell size
rather than number. Response to CLA treatment may depend on the sex and
age of the animal as well as duration of feeding.
KEY WORDS: • conjugated linoleic acid • gestation • lactation • adipogenesis • imprinting • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3)
. This suggests that fetal life has far-reaching
consequences influencing chronic diseases such as cardiovascular
disease, hypertension, diabetes and obesity. Numerous studies have also
correlated birth weight and size with maternal nutrition. Combined,
these studies indicate that maternal diet may affect size at birth and,
therefore, alter risks for chronic disease in adulthood. One particular
chronic disease, obesity, is of special interest because of its high
prevalence and its correlation with adverse health status. This
far-reaching problem is not limited to humans; decreasing profit
margins in animal production and increased health concerns are creating
demands for improved production of high quality meat products. Nutrient
partitioning during the rapid growth phases of gestation and lactation
may be key to understanding how growth can be manipulated to achieve a
more favorable body composition for animal and human health.
Unfortunately, the development and methods of management of
obesity are still debated. Fat tissue develops during fetal life and
its development can be manipulated. The intricate regulation of
adipocyte development is a result of the actions of many factors
including hormones, glucocorticoids, growth factors and transcription
of various genes (4)
. Dietary manipulation can alter the
activity of these factors fetally and postnatally, thus altering
adipose development (5
,6)
. There are also numerous in vivo
and in vitro studies showing that fetal and neonatal adipocyte
development is affected by exposure to these factors
(7
,8)
. Thus, it is possible that maternal diet during
gestation and lactation may alter adipose tissue development in the
offspring, which may have long-lasting effects on the growth of
this tissue and therefore, the development of obesity. This would have
substantial implications for both human health and animal production.
The next obvious prerequisite is to determine which dietary
manipulations favorably alter adipose development.
One nutrient that has been receiving much attention on the basis of its
potential to repartition body mass in growing animals is conjugated
linoleic acid
(CLA).3
Recent studies with CLA indicate that it alters body composition
in growing animals. CLA is a group of positional and geometric isomers
of linoleic acid. Although naturally occurring, CLA content in foods
and feeds can be manipulated through processing, diet alteration and
supplementation with CLA in both milk products and meat
(9
–12)
. Initial interest in this group of compounds seems
to have stemmed from its anticarcinogenic activity (13)
.
Subsequent studies have shown that feeding CLA can also improve
atherosclerosis (14)
and glucose tolerance
(15)
, alter immune function (16)
and vitamin
storage (17)
, change body composition in mice
(18)
, rats (19)
and pigs (20)
,
and alter milk composition in several species
(9
,10
,21
,22)
. Some of these changes may be a result of
incorporation into adipose tissue and subsequent specific changes in
the activity of this dynamic tissue. Recent studies have noted
significant changes at both the tissue and cellular levels. In vitro
studies of cell lines exposed to CLA suggest that adipocyte development
may be limited by decreasing proliferation and/or differentiation of
these cells (23
,24)
. Altering adipose tissue with small
amounts of a naturally occurring compound has therapeutic potential in
humans and offers opportunities to improve efficiency in animal
production. Critical development occurs during fetal and neonatal
growth. Therefore, increased CLA during pregnancy and lactation could
alter body composition and adipocyte development in both the mother and
the offspring.
This experiment was designed to determine whether prenatal effects of CLA on fat and growth in Sprague-Dawley rats are associated with previously reported postnatal effects such as decreased adiposity and increased growth rate. More specifically, the goal of this study was to determine whether exposure to CLA during the time of adipocyte development in the fetus and neonate reduced the potential for adiposity later in life.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Timed pregnant Sprague-Dawley rats (Harlan, Madison, WI) were
obtained on d 7 of gestation, housed in individual plastic shoebox
cages with free access to both food and water, and were maintained at
22°C with a 12-h light:dark cycle. Dams were allocated to one of two
dietary treatment groups, control (n = 11) or
CLA (n = 12). Diet treatments were as follows
(Table 1
): the control diet (CON) consisted of AIN-93
(25)
growth diet containing 20% casein and 7%
soybean oil; the CLA diet (CLA) consisted of AIN-93 growth diet
containing 20% casein, 6.5% soybean oil and 0.5% CLA supplement
(Nu-Chek-Prep, Elysian, MN). The maternal dietary treatment was begun
on d 7 of pregnancy and continued through 21 d of lactation when
the pups were weaned.
|
. One hour before killing, a
minimum of two pups per litter were injected with
5-bromo-2'-deoxy-uridine (BrdU) (30 mg/kg body) for determination of
cell proliferative activity. Pups were killed via cervical dislocation
and whole-body cross sections containing the inguinal adipose depot
(posterior end) were collected. One whole-body cross section per
litter was frozen in liquid nitrogen and maintained at -80°C; one
cross section was fixed in Bouin’s fixative for 24 h and embedded
in paraffin. At weaning on d 21, one male and one female pup from each litter were killed with sodium pentobarbital and blood was collected via cardiac puncture. One inguinal fat pad was frozen and one fixed in Bouin’s fixative from each pup. Of the four pups remaining from each litter (two male, two female), two (one male, one female) were fed the CON diet, and two were fed the CLA diet until 11 wk of age. The pups consumed food from containers designed to minimize spillage, and access to water was unlimited. The growing pups were housed singly in wire-mesh hanging cages at an ambient temperature of 22°C with a 12-h light:dark photocycle. Feed intake and body weight were determined twice each week until the pups were 11 wk old, at which time all were killed with carbon dioxide. Dams were killed after the litters had been weaned and fat pads collected.
Immediately after carbon dioxide administration (d 77), trunk blood was collected from each pup. Rat carcasses were then weighed and tail length was determined. Tissues removed and weighed included the liver, soleus muscle, gastrocnemius muscle, retroperitoneal (RP) fat pads and parametrial (PM)/epididymal fat pads.
All animal procedures were conducted in accordance with established guidelines of the University of Georgia Institutional Animal Care and Use Committee.
Fatty acid analysis.
Fatty acid profiles of whole-body cross sections of newborns,
inguinal adipose tissue of weanlings, and RP and inguinal adipose
tissue of mature rats (11 wk old) were determined by gas chromatography
with a flame ionization detector (Shimadzu, Model 14A, Columbia, MD).
Whole-body cross sections (
1–1.5 g) from newborn pups and
inguinal adipose tissue (100 mg) from pups at weaning were frozen at
-20°C until evaluation. In duplicate, tissue was saponified using 1
mL of 4 mol/L NaOH and 2 mL of methanol as previously described
(26)
. Heptadecanoic acid (2 mg) was used as an
internal standard (2 g/L in methanol). Samples were acidified and
extracted with hexane and methylated with methanol and methanolic boron
trifluoride. Fatty acid methyl esters were separated on a Supelcowax-10
fused capillary column (60 m x 0.53 mm, 0.50-µm
film thickness; Supelco, Bellefonte, PA) under isothermal conditions.
Sample size was 0.5 µL and helium was the carrier gas.
Peaks were identified via comparison of retention times of known
standards including pure samples of CLA isomers (Matreya, Pleasant Gap,
PA). Quantification was corrected for recovery of the internal standard
and is based on the reference standard.
BrdU proliferative activity.
In vivo proliferation of various cell types was determined by quantification of 5-bromo-2-deoxyuridine (BrdU) incorporation into tissue of newborn and weanling pups. Briefly, 30 mg/kg body of BrdU (Boehringer Mannheim, Indianapolis, IN), a thymidine analog, was injected intraperitoneally into two pups from each litter 1 h before killing. During this hour, BrdU was incorporated into the DNA of replicating cells. After killing, tissue was collected, fixed in Bouin’s fixative and embedded in paraffin. Immunohistological evaluation of BrdU incorporation was performed on 7-µm tissue sections using a streptavidin-biotin detection kit containing a monoclonal anti-BrdU biotinylated antibody (Zymed, San Francisco, CA). Whole-body cross sections of newborn rats were used to determine the proliferation of epidermal cells directly above the vertebrae and on the distal side of each femur, cells of the abdominal muscles and longissimus dorsi muscle, and adipocytes and blood vessel endothelial cells within the inguinal adipose depot. Inguinal adipose tissue of weanlings was used to determine the proliferation of adipocytes. Controls included in the staining system and the staining of epidermal cells served as positive controls, whereas tissues from pups not injected with BrdU served as negative controls. Relative numbers of cell nuclei staining positive for BrdU incorporation and unstained cell nuclei are reported.
Adipose tissue cellularity.
Duplicate samples of 50 mg of inguinal adipose tissue were collected
at weaning and 50 mg of RP adipose tissue were collected at d 77 for
determination of total cell number and cell size distribution as
described by Hirsch and Gallian (27)
. Although previous
work in rats fed CLA showed minimal changes in inguinal adipose depot
cellularity, this is the only depot large enough to dissect at weaning,
and thus was used to determine cellularity at weaning
(26)
. The RP adipose depot was used to determine adipose
cellularity because previous work has shown this depot to be responsive
to dietary CLA. Briefly, adipose tissue samples were placed in
scintillation vials containing 1 mL collidine and 1.5 mL osmium
tetroxide for immediate fixation. Postfixation, connective tissue and
debris smaller than 20 µm were removed via filtering
through 20-µm nylon mesh screens with 9 g/L saline.
Cells were resuspended in 8 mol/L urea and 9 g/L saline for further
digestion of connective tissue. Samples were rinsed onto 240- and
20-µm screens, suspended in 40 g/L filtered saline,
and counted and sized using a Coulter multisizer (Beckman, Fullerton,
CA).
Western blotting.
Inguinal fat depots (d 21) were homogenized and lysed with 1X lysis
buffer containing 60 nmol/L Tris (pH 6.8) and 10 g/L SDS. The lysate
was centrifuged (12,000 x g, 10 min) and the protein
concentration was determined using the Bio-Rad protein assay
(Bio-Rad Laboratories, Hercules, CA). Protein (100 µg)
was diluted in SDS-sample buffer and boiled for 5 min before
electrophoresis and separation on a 12.5% SDS-polyacrylamide gel
at 100 V. This was followed by protein transfer to Immobilon-P
protein sequencing membrane (Millipore, Bedford, MA) at 30 V overnight
and 60 V for 2 h. A 50 g/L powdered milk solution was used to
block nonspecific binding sites. Membranes were probed for
CCAAT/enhancer binding protein (C/EBP; Santa-Cruz, Santa
Cruz, CA) for 1 h and incubated with horseradish
polypeptidase-conjugated secondary anti-rabbit immunoglobulin G
(Santa-Cruz; Amersham, Arlington Heights, IL) antibody for 1 h.
The antibodies were diluted to 1:500 and 1:4000, respectively.
Immunoreactive polypeptides were visualized using ECL chemiluminescence
reagents (Amersham). Protein band density, determined with a
densitometer, was used to compare C/EBP protein levels (Molecular
Dynamics, Sunnyvale, CA). To ensure the specificity of these results,
Western blots were exposed only to the secondary antibody and used as
negative controls.
Oil-Red-O lipid filling.
Lipid staining with Oil-Red-O on 24-µm frozen sections (d 0) was used to determine in utero lipid filling within the inguinal adipose depot. Frozen whole-body cross sections of newborn pups were used to obtain 24-µm sections. These sections were fixed with Baker’s Formalin (10% Formalin) for 30 min and stained with a 60% Oil-Red-O solution for 10 min. Lipid accretion and the percentage of lipid area were determined using image analysis quantification (Image Pro-Plus, Media Cybernetics, Silver Springs, MD) of three 20X microscope fields.
Serum assays.
Blood was collected and kept at 4°C for 12 h before
centrifugation. Serum was frozen at -80°C until assayed. Serum from
weanling pups was used to measure insulin and insulin growth factor-1 (
IGF-1) via RIA. Serum insulin was determined using an insulin RIA kit
containing precoated tubes and used as directed (ICN Pharmaceuticals,
Costa Mesa, CA); the IGF-1 RIA was performed using rabbit IGF-1
antiserum UBK487 (distributed by the National Hormone and Pituitary
Program, Bethesda, MD) (28)
. Recombinant human
IGF-1 (Amgen Biologicals, Thousand Oaks, CA) was used as the standard.
The specific radioactivity of the 125I-IGF-1 was 12.8
µBq/g.
Serum from pups at d 77 was assayed colorimetrically for cholesterol (Chol), triglycerides (TG) (352; INT 336; Sigma Diagnostics, St. Louis, MO) and free fatty acids (FFA) (NEFA-C; WAKO Chemicals, Dallas, TX). Insulin was measured as described above.
Statistical analysis.
Data were analyzed using the PROG GLM procedure of SAS (Cary, NC).
Least-square means ± SEM are reported. Data from
newborn pups were analyzed using a model that contained the main effect
of maternal diet. Data from weanling pups were analyzed using maternal
diet, sex of the pup and their interaction in the model. Data for adult
female and male progeny (d 77) were analyzed separately. The separate
analysis of the sexes was performed because although female rats
reached a plateau in growth, the males continued to exhibit relatively
linear growth at the time the study ended (8 wk postweaning). A similar
analysis was used previously (21)
. Within each sex,
analysis was based on maternal diet, postweaning diet and their
interactions. The main effect of maternal diet was tested against dam
within diet as the error term. Differences were considered significant
when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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CLA, at a level of 0.5 g/100 g diet, did not affect body weight of the dams at any time during gestation or lactation. There were no differences in the number of pups born per litter in CLA-fed (14.25 ± 0.82) and CON-fed (13.13 ± 0 0.59) dams. Weights of whole litters (88.15 ± 3.2 g, CLA; 81.11 ± 2.3 g, CON) and litters normalized to 6 pups were not different between groups (38.06 ± 0.63 g, CLA; 37.64 ± 0.91 g, CON) and remained this way until weaning. There was no difference in litter weight gain (g/d) or litter efficiency (litter weight gain per dam feed intake) between treatment groups. Feed intake was not different in dams fed CON and CLA diets. Body and liver weights of dams fed CLA were not different from those of dams fed the CON diet (P < 0.05, data not shown), but PM fat pad weight (2.92 g, CON; 2.08 g, CLA; 0.22 g pooled SEM; P < 0.02) and RP fat pad weight (0.84 g, CON; 0.49 g, CLA; 0.08 g pooled SEM; P < 0.01) were less in dams fed CLA.
Food intake and growth.
Dietary CLA did not alter absolute adipose depot weight at weaning. However, female pups exposed to CLA were significantly heavier than those of dams fed CON at weaning. As expected, male pups were significantly heavier than female pups as early as d 21 regardless of dietary treatment, and they continued to have significantly faster rates of growth throughout the study.
Dams’ diet had no effect on average daily gain or food intake in
either male (Table 2
) or female pups (Table 3
). CLA in the maternal diet increased gain:feed in males but not
females. CLA had no effect on liver weight in either sex. In male
progeny, CLA in the maternal diet increased the weights of the soleus
and gastrocnemius muscles (P < 0.01) and increased the
tail length (Table 2)
(P < 0.001). Males exposed to
CLA throughout gestation and lactation, as well as postweaning, were
the heaviest, fastest growing and most feed efficient of all treatment
groups, whereas males never exposed to CLA in any of these phases of
development were smallest. Rats exposed to CLA in some phases but not
in others, were intermediate in these growth variables.
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. Fatty acid composition.
Dietary CLA did not alter total fatty acid amounts in inguinal adipose tissue cross sections of newborns or in tissue of weanling rats. Whole-body cross sections from CON-fed newborn pups contained 0.75 ± 0.06 g/100 g tissue and CLA-fed pups contained 0.85 ± 0.06 g/100 g tissue (P > 0.1). Inguinal adipose tissue collected on d 21 of lactation contained 63.5 ± 3.3 g/100 g lipid in CLA-fed females (CLA-F), 60.4 ± 3.6 g/100g in CLA-fed males (CLA-M), 66.9 ± 4.5 g/100 g in control females (CON-F), and 61.2 ± 4.1 g/100 g fatty acids in control males (CON-M). Although total extractable saponafiable lipid content of tissue was not affected, the amounts of certain fatty acids were altered by sex, age or diet.
Overall, the most identifiable fatty acids were not significantly altered in tissues of newborns. A significant increase in 16:0 of whole-body cross sections was observed with dietary CLA (25.35% in CLA; 23.19% in CON), but substantial incorporation of CLA was not observed in these tissues. This may have been due to the use of whole-body cross sections which contain little lipid.
Incorporation of both the 9-cis, 11-trans and the
10-trans, 12-cis CLA isomers into the inguinal
adipose tissue of CLA-exposed weanling rats (d 21) was evident
(P < 0.0001). There were significant differences in
the total amount of fatty acids recovered in male and female pups,
independent of dietary treatment. CLA-exposed pups had
significantly lower relative amounts of 16:1, 18:1 and 18:3 than
controls. Regardless of treatment, there was more CLA
10-trans,12-cis isomer recovered than CLA
9-cis, 10-trans isomer. Results are summarized in
Table 4
.
|
). In general, males had higher percentages of
monounsaturated fatty acids than females (males, 39.0 vs. females,
37.3%, P < 0.01), but there were few other consistent
differences between sexes. Thus, results for both genders are pooled.
There were no sex x diet or maternal diet x postweaning
diet interactions. The only significant effect of maternal diet
remaining at 77 d of age was an increase in the relative
proportion of palmitate in progeny of dams fed CLA during gestation and
lactation (Control 21.9 vs. CLA 23.0 g/100 g palmitate, P
< 0.05). Inguinal pad content of CLA was low (<0.1 g/100 g) and
not different from controls in progeny of dams that had been fed CLA
and that were weaned onto the control diet. In contrast, there were
significant postweaning diet effects on the fatty acid profile. CLA
content was elevated in progeny fed the CLA diet postweaning (Table 5)
.
There was no difference in content between those fed CLA only during
the postweaning period and those that had been exposed to CLA
constantly beginning in gestation. Consistent with an inhibition of
desaturase activity, the content of total saturated fatty acids was
greater and that of total monounsaturates lower in pads from rats fed
CLA.
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Of the cell types analyzed in body cross sections of newborns, the percentage of cells replicating was highest in epidermal cells (24.3 ± 3.1% CLA, 24.2 ± 1.1% CON), followed by adipose tissue vascular endothelium (13.5 ± 2.3% CLA, 18.0 ± 2.8% CON), abdominal muscles (9.2 ± 1.2% CLA, 9.6 ± 0.7% CON), inguinal adipose tissue (8.2 ± 1.5% CLA, 10.0 ± 1.3% CON) and longissimus dorsi muscle (7.9 ± 1.1% CLA, 8.7 ± 1.4% CON) and was unaffected by treatment. There were no differences in rates of replication in these cells types from newborn CLA-exposed pups and controls. There also were no differences in proliferation rates of inguinal adipocyte tissue from weanling rats (d 21). Rates were 4.4 ± 1.5% in CLA-F, 5.9 ± 0.9% in CON-F, 6.4 ± 0.9% in CLA-M and 5.8 ± 1.9% in CON-M pups.
Adipose cell size.
At the time of weaning (d 21), there was no difference in the number of
fat cells > 30 µm in diameter per inguinal adipose
depot (CLA-F: 1.41 ± 0.17 x 106;
CLA-M: 1.50 ± 0.14 x 106; CON-F: 1.76
± 0.17 x 106; CON-M: 1.41 ± 0.16 x 106). However, the distribution of
fat cells was significantly different in CLA-exposed pups compared
with controls (Fig. 1
). CLA-exposed pups had a greater proportion of cells that were
40–60 µm (P < 0.05) and fewer cells that
were 80–140 µm in diameter (P < 0.05).
These differences were independent of sex. The pups’ sex significantly
affected the percentage of cells in the 20–40 µm
ranges, (P < 0.05) in which male pups had
fewer cells in the smaller diameter ranges.
|
).
|
Dietary CLA significantly increased expression of both the 42- and
29-kDa isomers of C/EBP in inguinal adipose tissue of weanling rat
pups by sixfold in female pups relative to controls (6.0 ± 0.9).
C/EBP expression in CLA-exposed male pups was not affected (0.9
± 0.7) (Fig. 2
).
|
Image analysis of frozen inguinal adipocyte sections showed no difference in lipid accretion in CLA-fed litters compared with CON-fed litters (P > 0.05). Lipid droplet diameters expressed in arbitrary units were 0.64 ± 0.07 in the former and 0.68 ± 0.06 in the latter.
Serum metabolites.
Serum insulin concentrations of pups at weaning were not affected by
diet or sex (625.1 ± 138.2 pmol/L for CLA-F, 515.3 ± 94.4 pmol/L for CLA-M, 509.1 ± 95.8 pmol/L for CON-F and
477.1 ± 38.2 pmol/L for CON-M). Serum IGF-1 concentrations did
not differ among groups of weanling pups (CLA-F: 1.19 mg/L, CLA-M: 1.09
mg/L, CON-F: 1.27 mg/L and CON-M: 1.25 mg/L; 0.52 mg/L pooled
SEM). Serum insulin concentrations in 77-d-old rats were
not affected by postweaning diet. Males had significantly greater
levels of insulin regardless of diet. Progeny of dams fed CLA had serum
concentrations of Chol, TG, FFA and insulin that were not different
from those of progeny of dams fed the control diet (Tables 2
and 3)
.
Similarly, postweaning diet did not affect serum concentrations of
Chol, TG or FFA, except Chol in 77-d-old males, which was greater in
those fed CLA (Table 2)
. There was no significant effect of sex on
serum Chol or FFA, but males, regardless of treatment, had
significantly higher serum concentrations of TG than did females.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Changes in adipose cell size, but not number, were evident in
CLA-exposed litters by weaning (21 d) with a significant reduction
in fat pad mass of females, but not males at 11 wk of age (Tables 2
and 4)
. Inguinal adipose tissue from pups in CLA-exposed litters at
21 d of age contained significantly greater levels of both the
9-cis,11-trans and the
10-trans,12-cis isomers of CLA. At the same time,
adipocytes from the inguinal adipose tissue of CLA-exposed pups
showed distinct differences in cell size distributions, C/EBP
expression and fatty acid composition, but no differences in the rate
of cell proliferation. A dramatic sixfold increase in C/EBP protein
levels of CLA-F suggests that these adipocytes have an increased
capacity for differentiation. These results are in contrast to a lack
of change in C/EBP mRNA in 3T3-L1 adipocytes exposed to varying
levels of the trans-10, cis-12 CLA isomer
(29)
. This may be due to inherent differences between in
vivo and in vitro studies or to the use of pure isomers in the in vitro
studies. C/EBP activates the transcription of several genes
expressed when preadipocytes differentiate, resulting in the adipocyte
phenotype. In addition to its transcription activator role, one isoform
of C/EBP (42 kDa) may also suppress clonal expansion because it has
been shown to limit mitotic activity in 3T3L1 adipocyte cell lines
(30)
. However, this phenomenon was not apparent in the
present experiment. Proliferation of inguinal adipose cells and total
fat cell number per inguinal pad were not different in CLA- and
CON-fed pups. Initially, these results seem contradictory. How
could CLA increase the adipocyte phenotype, the expression of C/EBP,
while decreasing total body fat? A possible answer lies in the size
distribution of adipocytes. At weaning, CLA-exposed rats had
markedly fewer "large adipocytes" in the size range of 80–140
µm with an increased number of "smaller adipocytes" in
the range of 40–60 µm. These results are similar to those
recently reported in growing Sprague-Dawley rats fed CLA
(19)
. Collectively, these results suggest that
dietary CLA shifts the pattern of adipocyte differentiation and results
in earlier differentiation of adipocytes, thereby limiting their size
and storage of triacylglycerides, resulting in a decrease in body fat.
This is supported by the suppression of triacylglyceride accumulation
in 3T3-L1 adipocyte cell cultures exposed to CLA isomers
(31)
. It is of interest that the changes in C/EBP
expression occurred in females but not in males at weaning. This change
in a cellular protein, which is partly responsible for adipocyte
differentiation, seems to predict the differential size distribution in
RP adipocytes at 77 d of age when, again, there was a decrease in
larger adipocytes and an increase in smaller adipocytes. These changes,
however, were not limited to the cellular level. When littermates were
compared at 77 d of age, differences in adipose tissue mass were
observed in females but not in males.
After feeding of the pups through 11 wk of age, remarkable sex
differences were noted in regards to the main effects of the maternal
and postweaning CLA diets. In females, the main effect of CLA in the
postweaning diet was to reduce the weight of the RP and PM fat pads.
The changes in body composition and growth in females were markedly
different from those in males where the main effect of CLA in the
maternal diet was to increase the weights of the soleus and
gastrocnemius muscles, as well as increase the tail length. CLA also
increased the weight of the soleus muscle when present in the
postweaning diet. This indicates that CLA increased lean tissue mass
and skeletal growth in male but not female progeny. Previous work
(21)
has indicated that progeny of Fisher dams fed CLA
during gestation and lactation had improved feed efficiency. This
observation was confirmed in male offspring of Sprague-Dawley dams
in the present study.
This study is the first to demonstrate that long-term effects of
dietary CLA on body composition changes are due in part to
sex-dependent changes in fat mass and lean tissue mass. These
results raise the possibility that CLA may interact, either directly or
indirectly, with sex-dependent characteristics, resulting in body
composition changes. This is supported by several other studies, which
have indicated that response to CLA may be sex dependent. One in vitro
study showed that CLA added to MCF-7 human breast cancer cells
inhibited proliferation of cells with estrogen receptors more than in
those with no receptors (32)
. Moya-Camarena
et al. (33)
also reported that hepatic peroxisome
proliferator-activated receptor (PPAR)-responsive genes of female
rats responded less to CLA than did those of male rats.
This paper and other previously published studies report that dietary
CLA changes the fatty acid composition of various tissues in different
animal models including neonatal chicks (34
,35)
. It has
been concluded that due to its structural similarity to linoleic acid,
CLA decreases steroyl-CoA desaturase enzyme activity and gene
expression, thus changing membrane fatty acid composition
(29
,36)
. It has been shown that changes in dietary fatty
acids can alter the fatty acid composition of adipocyte membranes
(37)
, adipocytes and fibroblasts (38)
.
Furthermore, the changes in membrane fatty acids are associated with
significant changes in insulin responsive cellular activities including
glucose transport and lipogenesis (39)
. A previous example
of this is the report of improved insulin-stimulated glucose
transport in adipocytes from rats fed a fish oil diet high in
polyunsaturated fats (40)
. These responses are also
similar to those seen by Houseknecht et al. (15)
who
reported that in diabetic Zucker rats, dietary CLA improves glucose
tolerance. Although serum insulin levels of rats in the present study
were not altered by dietary CLA, they were not deprived of food before
blood collection and showed no overt signs of diabetes.
Both the changes in adipocyte membrane fatty acid composition and increased differentiation may result in varied responses of cell receptors to their ligands, potentially altering adipocyte response to sex hormones, IGF and insulin, all of which have been shown to influence adipocyte development.
Although the role of sex hormones in adipose tissue development is
poorly understood, regional differences between sexes have been
described, suggesting a role for sex hormones. Several methods,
including ligand blotting and sequencing, have verified that receptors
for both androgens and estrogens are present in adipose tissue
(41
,42)
. Pederson et al. (41)
reported that
mature adipocytes have estrogen, glucocorticoid and androgen receptors
but not progesterone receptors, whereas only glucocorticoid and
androgen receptors are present in preadipocytes. This may be one of the
reasons why sex hormones elicit very different responses in vitro where
addition of androgens antagonized adipogenesis and estrogens enhanced
adipogenesis (43)
. However, it has also been shown that
the lack of estrogen is adipogenic in ovariectomized rats and may
suppress lipoprotein lipase (44)
. The activity of estrogen
receptors on rat adipocytes has been characterized intensively and
shows interesting results. Studies have shown that insulin increases
estradiol binding to these receptors (45)
. In turn,
estrogen treatment decreases lipoprotein lipase and cell size while
increasing insulin receptor binding (46)
. It has also been
suggested that the effects of androgens and estrogens on adipogenesis
may be related to the IGF-1 receptor and PPAR-
(43)
,
further supporting the idea that fatty acid composition can regulate a
complex interrelationship involved in the regulation of adipocyte
development. The increased level of differentiated adipocytes in
CLA-fed females suggests that CLA may increase the number of
estrogen-responsive cells, which could alter metabolism in these
cells. The inherently low levels of estrogen present in prepuberty may
limit this response. The evidence of cellular changes without changes
in tissue mass may, therefore, be due to timing, in that the
interaction between CLA and steroid hormones is not maximal at this
age.
In vivo and in vitro studies show that the IGF system is a critical
regulator of adipogenesis and the lack of IGF-1 or IGF-binding
protein (IGFBP) 1 significantly inhibits adipogenesis
(47
,48)
. It has been concluded that IGF may act through
differential glucose uptake by adipose tissue and muscle cells in
female Sprague-Dawley rats (49)
. A previous study by
Li et al. (50)
showed that CLA increases IGFBP-3 and serum
IGF-1 levels, suggesting that CLA may be modulating body mass through
mechanisms involving the IGF system. Although our results showed that
serum IGF-1 was not altered in weanling rats, it is possible that other
elements of the IGF system, including IGF-II and the IGFBP, may be
influenced.
If the mode of action of CLA is through mediating growth, the response
in females may have been masked by the timing of killing the rats (11
wk of age) in this study. It may be necessary to study males and
females at various ages when similar stages of growth have been
reached. Studies of CLA-supplemented adult humans have
produced conflicting results. Zambell et al. (51)
did not
find significant changes in body composition or energy expenditure in
healthy women consuming 3 g CLA/d, whereas Blankson et al.
(52)
observed reductions in weight of overweight men and
women given 3.4 or 6.8 g CLA/d. These results suggest that changes
in adiposity with CLA may occur only during certain stages of growth,
further demonstrating the need for studies during different stages of
growth. It is also important to note that conflicting results
concerning the extent of body composition changes may also be due to
species differences. There are a number of studies in mice that have
shown great reductions in body fat mass and improved feed efficiency
and growth (18
,53
–56,)
. However, studies in rats have
repeatedly failed to show similar extents of change in variables
including food intake, feed efficiency, growth and weight gain
(15
,16
,19
,57
–60)
.
Collectively, these results suggest that CLA consumption during
gestation and lactation may imprint development and result in changes
in adulthood similar to the results seen in other fetal programming
studies. Contrary to results in in vitro studies suggesting effects of
CLA on adipocyte proliferation (23
,24)
, there was no
evidence of an in vivo effect of CLA on fat cell number in rats. It
seems that the effects of CLA on adipose tissue development, muscle
development, growth and body composition are influenced by the
animal’s sex and that this influence may play a key role in
development before the onset of puberty. These effects may be a result
of direct or indirect action by androgens and/or estrogens or a result
of growth during different phases. The growth of female
Sprague-Dawley rats reaches a plateau several weeks earlier than
that of males, which may be a confounding factor if the actions of CLA
are dependent on growth of the animal. The present results justify
future studies to determine the effects of CLA at various stages of
growth and development and the differential effects of CLA on males and
females.
|
FOOTNOTES |
|---|
3 Abbreviations used: BrdU; 5-bromo-2'-deoxy-uridine; C/EBP, CCAAT/enhancer binding protein ; Chol, cholesterol; CLA, conjugated linoleic acid; CLA-F, CLA female; CLA-M, CLA male; CON; control; CON-F, control female; CON-M, control male; FFA; free fatty acids; IGF-1, insulin-like growth factor 1; IGFBP, insulin-like growth factor binding proteins; PM, parametrial; PPAR, peroxisome proliferator-activated receptor; RP, retroperitoneal; TG, triglycerides.
Manuscript received January 29, 2001. Initial review completed February 28, 2001. Revision accepted June 27, 2001.
|
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|---|
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