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Department of Veterinary Sciences and Technologies for Food Safety, University of Milan, 20133 Milan, Italy
3To whom correspondence should be addressed. E-mail: carlo.corino{at}unimi.it.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSLITERATURE CITED |
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KEY WORDS: • adipose tissue • conjugated linoleic acid • heavy pigs • histometry • immunofluorescence
Conjugated linoleic acids (CLAs)4 are geometric and positional diene isomers of linoleic acid [18:2(n-6)]. CLAs have attracted considerable attention because of their ability to reduce fat mass, an effect that appears to result from actions on the number of adipocytes in mice and their volume in rats (1). Adipocyte volume depends on the dynamic balance between lipolysis and lipogenesis, whereas adipose cell number is determined by rates of cell acquisition and cell loss (2). In this case, the antiadipogenic effect of CLA could develop through an oxidative stress pathway, which is supposed to cause apoptosis and inhibit growth in adipocytes (3). The possible antiadipogenic effects of CLA appear largely species-specific and postulated primarily on the basis of in vitro studies (1).
It is not fully understood whether CLAs act directly upon adipocytes or via other messengers. Nitric oxide (NO) release from endothelial cells was shown recently to be inhibited by CLA (4). NO is a neurotransmitter in gut intramural neurons; it modulates cell proliferation, differentiation, and apoptosis in various tissues, and regulates vascular tone (5). NO may also regulate lipid metabolism, as suggested by the findings that NO is present in rat white adipose tissue (6,7); inducible nitric oxide synthase (iNOS) occurs in human subcutaneous adipose tissue (8), whereas NOS inhibition causes increased lipolysis in this tissue (9). A neuronal modulation aimed at regulating lipolysis in vivo was suggested recently, in which cholinergic neurons act both per se and utilizing NO as an accessory mediator (9). It is therefore conceivable that the adipose tissue is both a source of NO and a target for NO regulatory activities.
We investigated in vivo effects of dietary CLA administration to heavy pigs. We assessed morphofunctional aspects and chemical characteristics of pig subcutaneous adipose tissue with the aim of clarifying the involvement of oxidative stress in the established antiadipogenic effect of CLA. A direct rather than an indirect role of CLAs upon adipocytes was also evaluated. On these bases, this study aims to clarify aspects useful for pigs per se as a species of food animal and as an animal model for human medical and biological aspects.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSLITERATURE CITED |
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155 ± 1.7 kg LW, when they were slaughtered. At slaughter, backfat thickness of each pig was measured in relation to the proximal end of the last rib, and values were recorded. The dressing percentage (hot carcass weight/live weight at slaughter) was also calculated for each pig. The pigs used in this study were cared for in accordance with European Union guidelines (No. 86/609/EEC) approved by the Italian Ministry of Health (L.116/92).
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For microanatomical analyses (histology, histometry, histochemistry, immunohistochemistry, and immunofluorescence), additional samples (1 x 1 cm) were removed from the same location on each pig (n = 36), promptly fixed in Bouin’s fixative for 24 h at 4°C, dehydrated in graded alcohols, cleared with xylene, and embedded in paraffin. Other 1 x 1 cm samples (n = 36) were frozen in isopentane, cooled in liquid nitrogen, and stored at –20°C pending additional analyses.
Chemical analyses. Chemical analyses (11) for dry matter, nitrogen, and ash were conducted on samples of subcutaneous adipose tissue from each of the 36 pigs. The iodine value, or quantity of iodine (g) bound/100 g of fat, was determined by the Wijs method (11). Iodine value is a measure of the degree of unsaturation.
Microanatomical analyses. The microanatomical observations were performed by one of the authors without knowledge of the treatment group of the pigs.
Histological and histometrical analyses. Using a microtome, each of paraffin-embedded specimens was divided into 3 serial sections (10 µm thick), which were stained with hematoxylin and eosin to assess morphology under a light microscope (Olympus BX51). For histometry, the size (area, µm2) of 100 adipocytes from each section was determined under the microscope (magnification X200) using the DP software for image analysis (Olympus). Mean adipocyte size per treatment group was calculated, and 5 different classes of areas were determined, in which the cell size distribution was expressed as a percentage. The number of adipocytes was also determined in 5 fields/section at magnification X200. The mean number per group was calculated.
Histochemistry: lipid droplets, lipid peroxidation, NADPH-diaphorase (NAPDH-d). Frozen specimens were used to obtain serial sections (20 µm thick) with a cryostat. Lipid staining with Sudan Black B (Merck) was used to evaluate lipid droplets within adipocytes according to Soldani et al. (12). Lipid droplet size and number were determined for 10 adipocytes/section (3 sections for each sample, number of evaluated adipocytes = 30/pig) at a magnification of X400. Lipid peroxidation values were assessed according to Pompella et al. (13). With this method, free aldehydes and carbonyl functions were detected after a peroxidative breakdown of unsaturated fatty acids. The density of the autofluorescent Schiff’s reaction products (absorbing at 544 nm), expressions of lipid peroxidation, was determined with a confocal laser microscope (Olympus FV300-IX). Other frozen sections were processed to detect NADPH-d activity according to He et al. (14). Both lipid peroxidation and NADPH-d activity were quantitated (at magnification X200) by image analysis as described below.
Immunohistochemistry: proliferating and apoptotic adipose cells. Paraffin-embedded sections were processed to reveal adipocytes, which were in the S-phase of the cell cycle by immunostaining (peroxidase anti-peroxidase method) with a monoclonal antiserum against proliferating cell nuclear antigen (PCNA; clone PC10, Sigma; dilution, 1:3000), as described elsewhere (15). The PCNA-immunoreactive nuclei are designated here as belonging to "proliferating" cells. The specificity of immunostaining was tested by incubating other sections with normal pig serum instead of the primary antiserum, and this procedure always gave negative results. As positive controls, alimentary canal samples from calves and dogs were tested; in all cases, the expected positive reactions occurred in the intestinal crypts.
Other sections were processed to identify adipocytes, which were in apoptosis. Apoptotic nuclei were identified using a modified TdT-mediated dUTP nick end labeling technique (DeadEndTM Colorimetric TUNEL System, Promega) and identified with streptavidin labeled with horseradish peroxidase as described previously (15). The TUNEL-reactive nuclei are designated here as belonging to "apoptotic" cells. As positive controls, alimentary canal samples from calves and dogs were tested; in all cases, the expected positive reactions occurred within the intestinal villi.
With the aid of the DP software for image analysis, apoptotic (A) and proliferating (P) cells were counted in at least 100 adipose cells/section (3 sections for each sample, number of evaluated adipose cells = 300/pig), and the results expressed as percentages of positive cells. Subsequently, the apoptotic cell/proliferating cell index (A:P index) was calculated as the proportion of apoptotic cells divided by the proportion of proliferating cells.
Fluorescence immunohistochemistry: NOS isoenzymes and macrophages. Frozen sections of adipose tissue (10 µm thick) from each specimen were processed using the fluorescence immunohistochemical procedures. To detect neuronal (nNOS), and endothelial nitric oxide synthases (eNOS), polyclonal rabbit either anti-nNOS or anti-eNOS antisera (1:100 both, Santa Cruz Biotechnology) were used according to the manufacturer’s protocols. Anti-nNOS had been raised against an epitope at the C-terminus of nNOS, and anti-eNOS against an epitope at the N-terminus of eNOS. Negative control sections were treated with the same procedures except they were incubated with 1% bovine serum albumin instead of specific antisera. These procedures gave negative results.
Double fluorescence labeling for iNOS (Santa Cruz Biotechnology; dilution 1:100) and macrophages (MAC 387, Serotec; dilution 1:400) was carried out on the same sections using established protocols (16). Sections from specimens of the 2 experimental groups were stained at the same time and examined through a confocal laser microscope (FV300-IX) with excitation and barrier filters set for fluorescein (iNOS) and rhodamine (macrophages).
Image analysis. For quantitation of Schiff’s reaction, nNOS, eNOS, iNOS, and macrophage immunofluorescence, adipose tissue sections were examined using a confocal laser microscope (Olympus FV300-IX, equipped with argon:helium:neon lasers) and the FluoView software for image analysis (Olympus). Excitation and barrier filters were set for fluorescein and rhodamine. The laser power and photomultiplier tube voltage were constant, so that fluorescence intensities of various samples could be compared. Images were digitized under constant gain and laser offset, with no postcapture modifications. Before quantification, the images were digitally zoomed 1.5 times according to Murphy et al. (17). Five section areas that contained the largest and brightest immunofluorescence for each sample were selected for measurement. The areas to be assessed were defined manually. For nNOS in blood vessel smooth muscle cells (VSMC, vascular smooth muscle cells), 5 arteries per section were chosen. For nNOS and eNOS in endothelial cells of vascular tunica intima, 5 arteries and 5 venules/section were defined. For eNOS and Schiff’s reaction in adipocytes, 10 adipocytes/section were defined, and the calculated mean fluorescence intensity was obtained for the selected structures according to Murphy et al. (14). Pixel intensity was determined using the histogram/area functions of the FluoView software, which assigned the gray levels (GL) within a 0–256 Gy scale. Data were presented as mean fluorescence intensity.
In sections stained for NADPH-d, optical densities, represented by GL, were measured with a computed image analyzer (Image Pro-Plus software, version 4.1; Media Cybernetics, LP), according to He et al. (14). Optical density measurements on sections from the 2 experimental groups were taken under identical illumination and magnification (X200) conditions.
Statistical analysis.
Statistical analysis of the data was performed using SAS statistical software (18). Data from the chemical analyses were analyzed using 2-way ANOVA with diet and sex as the main factors. Data from the histometric, histochemical, and immunofluorescent analyses were analyzed by mixed model ANOVA, which included the fixed effects of sex and treatment, and the random effect of each pig. Values from each pig were considered as the experimental unit of all response variables. The data are presented as least-square means ± SEM. Differences between means were considered significant at P 
0.05.
RESULTS
Final weight (154.8 ± 2.24 kg CTR vs. 156.8 ± 2.45 kg CLA), hot carcass weight (126.1 ± 1.98 kg CTR vs. 125.7 ± 2.25 kg CLA), dressing percentage (81.55 ± 1.14 CTR vs. 80.15 ± 0.31 CLA), and last rib backfat thickness (23.58 ± 1.23 mm CTR vs. 24.00 ± 0.85 mm CLA) were not affected by dietary CLA supplementation. Sex influenced last rib backfat thickness, which was lower (P < 0.01) in F than CM (20.27 ± 0.73 mm F vs. 25.54 ± 0.82 mm CM).
Chemical analyses. Dry matter, lipid content, and protein content were not affected by dietary CLA supplementation (Table 2). CLA supplementation decreased the ash content (P < 0.05) and iodine values (P < 0.01) of adipose tissue. Sex influenced the fat content, which was lower (P < 0.05) in F than CM.
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CLA supplementation decreased adipocyte size (P < 0.05), with a consequent increase in number of adipocytes per unit area (P < 0.01) (Table 3). Sex also affected adipocyte size and number per area in that F had smaller (P < 0.01) adipocytes than CM, and more (P < 0.01) cells per unit area. The size distribution of adipocytes was not affected by dietary CLA supplementation. By contrast, sex affected adipocyte size distribution (P < 0.05) in that CM had significantly more cells in the 11000–13000 µm2 size range than F (16.66 ± 3.31% of total cells CM vs. 7.33 ± 4.94% of total cells F).
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The products of the Schiff’s histochemical reaction were evident (and their optical densities were measured) at the level of adipocyte plasmalemma. Neither CLA supplementation nor sex affected the extent of lipid peroxidation, as was shown histochemically.
NADPH-d activity was present as shown histochemically in adipocyte cytoplasm and in blood vessel cells, particularly those of the tunica media. Neither CLA supplementation nor sex affected NADPH-d activity in adipose tissue.
Immunohistochemistry: proliferating and apoptotic adipose cells. PCNA-positive nuclei were found in preadipocytes only (Fig. 1a). TUNEL-positive nuclei were found in adipocytes (Fig. 1b). The number of proliferating preadipocytes was lower (P < 0.05), and the number of apoptotic adipocytes was higher (P < 0.05) in pigs fed CLA compared with CTR, so that the A:P index was significantly (P < 0.01) lower in CLA-fed pigs (Table 4). The preadipocyte proliferation rate was also higher (P < 0.05) in F than in CM, so that the A:P index was lower in F (P < 0.01). Apoptosis rates did not differ between the sexes.
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DISCUSSION
The data of the present study agree with those of our previous study (19), which reported no effect of dietary CLA on growth performances and only a slight effect on carcass characteristics in heavy pigs.
Chemical analysis. Adipose tissue chemical composition and backfat were not affected by CLA dietary treatment. Our present data agree with the results of Azain et al. (20), who reported a reduction in adipocyte size and fat pad weight in CLA-fed rats, but no differences in carcass chemical composition. The fat content of adipose tissue in the same species was lower in females than castrated males, according to Barton-Gade (21).
The iodine value is a measure of unsaturation, and therefore an indirect indicator of fat firmness. Because of their susceptibility to oxidation, high levels of PUFA can have detrimental effects on the sensorial and technical qualities of pork products, reducing acceptability to consumers (22,23). We found a mean iodine value of 64.3 in CLA pigs; this was significantly lower than in controls and below the maximum level of 70 recommended by the Parma Ham Production Consortium for pig subcutaneous adipose tissue to avoid fat quality problems (24,25). In our previous study (19) and the studies of Gatlin et al. (26) and Eggert et al. (27), dietary CLA supplementation reduced the iodine value of pig adipose tissue. Thus, CLA supplementation had positive effects on the technological quality of fat.
In fact, fat consistency/saturation is fundamental to obtain a dry cured product of high quality; often oils such as palma or copra oil, containing medium-chain SFA, were used in pig nutrition to reduce iodine values. The increase in saturation due to CLA is related principally to increases in palmitic (16:0) and stearic acid (18:0), with concomitant reductions in palmitoleic (16:1) and oleic acid (18:1), consistent with a reduction in stearoyl-CoA desaturase activity through suppression of either mRNA expression (28) or activity (29).
These 2 SFA, and stearic acid in particular, were shown to decrease the risk of coronary artery disease compared with medium-chain SFA such as lauric (12:0) and myristic acid (14:0) (30). In addition, CLA dietary supplementation is accompanied by an increase in the CLA content of fat (19), and CLA is considered to be a healthy dietary component with the potential to affect human health in the areas of cancer, obesity, diabetes, and cardiovascular disease (31).
Histological and histometrical analyses. Histology showed that CLA did not have detrimental effects on the structure of the adipose tissue of heavy pigs, including its vascular support, which did not differ in the tissues of the 2 experimental groups.
Histology associated with histometry showed that dietary CLA supplementation (0.75%) significantly reduced adipocyte size and increased adipocyte number per unit area. The smaller adipocyte size in pigs fed CLA confirmed that adipocyte size can be affected by dietary means. We can assume that the reduction in adipose tissue masses in response to CLA might be related to a reduction in the adipose tissue depots. Notwithstanding the effect on mean size, CLA did not affect adipocyte size distribution, although smaller (5–7 x 103 µm2) adipocytes tended to be more numerous in CLA-fed pigs (P = 0.09). Azain et al. (20) reported that CLA reduced adipocyte size in rats, whereas Poulos et al. (32) found that CLA increased the proportion of smaller cells and decreased the proportion of larger cells in rat pups. The different ages (and different sampling localizations) of the pigs treated in this study may explain such different results.
We found that sex also had a significant influence on adipocyte size, number per unit area, and size distribution. As reported by Hausman et al. (33), androgenic status affects cellularity in vivo, and decreases in testosterone due to castration alter fat cell size.
The mean area of adipocyte was significantly different between CLA vs. CTR group and F vs. CM; in contrast, cell size distribution did not differ. Smaller adipocytes and higher adipocyte numbers were detected in both CLA vs. CTR and F vs. CM, but the differences were not significant. Similar results were reported by Sisk et al. (34) in rats fed CLA; the authors observed a reduction in the adipocyte mean diameter (P < 0.05) without differences in cell size distribution.
Histochemistry: lipid droplets, lipid peroxidation, NADPH-d. Dietary CLA did not affect the size and number of cytoplasm lipid droplets, which were stained by Sudan Black. In each adipocyte, we observed the presence of a large central lipid droplet encircled by peripheral small lipid droplets. Because the larger ones accumulate and increase in size by coalescence of the smaller peripheral lipid droplets (35), we can assume that dietary CLAs in pigs do not interfere with the metabolism of the subcutaneous adipose tissue in terms of incorporation of the small within the large lipid droplets. It is noteworthy, however, that Kang et al. (36) in an in vitro study found a CLA-related reduction in numbers of fat droplets in differentiating 3T3-L1 preadipocytes. In contrast to Evans et al. (37), who reported that CLA increased fatty acid oxidation in cultured 3T3-L1 preadipocytes, our in vivo study in pigs did not reveal differences in lipid peroxidation between adipose tissue specimens of CTR and CLA-fed pigs. This result agrees with our previous study (19), which found differences in the production of TBARS in the muscle of CLA-fed pigs compared with controls after an exceptionally long incubation (300 min). These observations suggest that, the CLA-dependent decrease in adiposity in pigs is not related to an increase in lipid peroxidation.
The synthesis of the fatty acids requires NADPH (38). There are 2 major sources of NADPH, i.e., oxidative decarboxylation of malate to pyruvate and CO2, with simultaneous generation of NADPH from NADP, and the pentose phosphate shunt, which also generates NADPH (38). CTR and CLA-supplemented pigs did not differ in the NADPH-d levels of optical densities, indicating no differences in NADPH production. This finding is in accord with our previous study (19) in which we investigated effects of CLA supplementation on both glucose-6-phosphate dehydrogenase and malic enzyme pathways of the pig adipose tissue, and did not find differences associated with the dietary treatment.
NADPH-d is also a histochemical marker of all NOS (nitric oxide synthase, see below) isoforms. NADPH-d is a multicomponent enzyme that catalyzes the transfer of electrons from NADPH to molecular oxygen, a reaction that serves to maintain cell redox balance. This same metabolic pathway is also the major source of reactive oxygen species, which in some instances can be considered per se mediators of vasodilatation (39). The previously mentioned lack of demonstrated differences between the control and CLA-supplemented pigs in the NADPH-d optical densities enables us to hypothesize that NADPH does not interfere with local blood flow changes via a CLA-mediated mechanism.
Immunohistochemistry: proliferating and apoptotic adipose cells. Immunohistochemical analyses showed that the preadipocyte proliferation rate was lower in CLA-fed than CTR pigs. We also found a clear sex-related difference, with a proliferation rate higher in F than in CM, possibly due to links, at present not fully known, to sex hormone substrates. The effects of CLA on preadipocyte proliferation in vivo do not appear to have been studied previously in healthy animals. Indeed, previous studies were performed in pathologic or in vitro studies. Our data are consistent with the results of in vitro studies reporting that CLA inhibits the proliferation of 3T3-L1, a cell line much used to investigate adipocyte development (40,41). In addition, we found that the number of apoptotic nuclei was greater in the adipose tissue of CLA-treated pigs than CTR, as reported in previous studies and reviewed by Belury (42). Moreover, the adipocyte proliferation and apoptotic rate were very high in both treatment groups, particularly compared with another study in rats (32).
The timing of adipose tissue development depends on the species as well on the adipose depot. White adipose tissue expansion takes place rapidly after birth as result of the increased size of existing fat cells and proliferation of preadipocytes. New fat cells continue to be generated throughout life (1). We suggest that all of the adipose tissue metabolic pathways are high in this species at this age, and certainly in this anatomical location.
Fluorescence immunohistochemistry: NOS isoenzymes and macrophages. In interpreting the results of fluorescence immunohistochemistry, we must consider that NOS isoenzymes are related to the synthesis of NO, which is a potent vasodilator, particularly when released by neurons innervating VSMCs and endothelium (39). Indeed, the local availability of NO in arteries and venules seems to depend on distinct enzymes such as nNOS and eNOS, respectively, as observed in rat mesentery by Kashiwagi et al. (43). In addition, when released and targeting the VSMCs, NO plays regulative roles in contractile function and energy production. Via its vasodilator action, NO may regulate blood flow to adipose tissue, which in turn may influence body adiposity. We found that VSMC of small arteries expressed only neuronal NOS. Nitrergic neurons, which are responsible for endogenously generated NO are presumably sympathetic neurons, in which NO acts as a neuromodulator (or a cotransmitter). Indeed, the existence of sympathetic noradrenergic nerve terminals was documented in mammalian adipose tissue, where their activation stimulates lipolysis (44). Lipolysis is also facilitated by stimulation of adipocyte ß-adrenoreceptors (45), and NO might act in this respect by inhibiting ß-adrenergic receptor–stimulated lipolysis (7). nNOS immunofluorescence did not differ between CLA-fed and CTR pigs, suggesting that local changes in blood flow in small arteries and ß-adrenergic–stimulated lipolysis were not influenced by CLA treatment.
Another important source of endogenous NO in adipose tissue is the endothelial cells, which form the vascular tunica intima. As expected, we found that endothelial cells from both arteries and venules reacted to the eNOS isoform; however, the expression of this enzyme did not differ between the 2 treatment groups, providing further evidence that CLA does not induce blood flow changes in pig adipose tissue.
Weisber et al. (46) showed that adipose tissue macrophages are responsible for all adipose tissue iNOS expression in obese humans. NO can also be produced by adipocytes of specific adipose tissue depots of human and rats (9,47) in which this synthesis and release depend on the presence of iNOS. In our study, we detected macrophages in pig adipose only rarely (so that a quantitation was not possible), and they were always negative for iNOS, suggesting that macrophages in pig subcutaneous adipose do not synthesize NO, in particular the inducible form of NO. We found that iNOS-immunofluorescence was present at the peripheral cytoplasmic rim of adipocytes, as previously observed by Fruhbeck et al. (48). Quantitation revealed that adipocyte iNOS was significantly lower in CLA-treated than CTR pigs. We suggest that the greater apoptotic rate in the adipose tissue specimens of CLA-treated pigs (see above) might be related to the reduced presence of adipocyte iNOS, in line with the findings of Andersson et al. (9), who showed that iNOS inhibition resulted in increased lipolysis in humans. Interestingly, NO produced by adipocytes may function as an autocrine regulator of catecholamine-stimulated lipolysis, as suggested by Gaudiot et al. (7) and Eliazalde et al. (8).
We conclude from our in vivo study of pig subcutaneous adipose tissue that dietary CLA antiadipogenic effects are likely not mediated by an oxidative stress–related mechanism, or by alterations in local blood flow, at least those regulated by noradrenergic innervation through the action of NO as either a vasodilator messenger or an accessory neuromodulator. It seems likely, however, that endogenous NO exerts CLA-related actions only when coming from adipocytes. In addition, CLA supplementation improves the technological quality of adipose tissue, increasing the degree of saturation, which in the heavy pig industry is important as a source of quality for Italian dry-cured ham.
These effects were demonstrated in vivo and were not accompanied by structural alterations of the adipose tissue of the heavy pigs studied; both of these aspects increase the value of the present study. Thus CLA has a potential usefulness as a dietary supplement in heavy pigs, a species of food animal that is important for a typical Italian product. These results may also be useful in resolving human disorders associated with body fat composition.
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FOOTNOTES |
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2 Supported by grants from the University of Milan (FIRST 2004).
4 Abbreviations used: A, apoptotic; CLA, conjugated linoleic acid; CM, castrated males; CTR, control; eNOS, endothelial nitric oxide synthase; F, female; GL, grey level; iNOS, inducible nitric oxide synthase; LW, live weight; NAPDH-d, NADPH-diaphorase; nNOS, neuronal nitric oxide synthase; P, proliferating; PCNA, proliferating cell nuclear antigen; TUNEL, TdT-mediated dUTP nick end labeling; VSMC, vascular smooth muscle cells.
Manuscript received 3 January 2005. Initial review completed 7 February 2005. Revision accepted 25 March 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSLITERATURE CITED |
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