Synergistic effect of synthetic conjugated linoleic acid & non fat milk on fat deposition & lipid metabolism in mice

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1 Indian J Med Res 131, March 20, pp Synergistic effect of synthetic conjugated linoleic acid & non fat milk on fat deposition & lipid metabolism in mice R. Sagwal & V. K. Kansal Division of Animal Biochemistry, National Dairy Research Institute, Karnal, India Received July 31, 2008 Background & objectives: Conjugated linoleic acid (CLA) reduces fat deposition in the body, but the mechanism of action is not clear. The present study was conducted to investigate the effects of CLA on body fat metabolism. Since milk fat is the best natural source of dietary CLA, intervention of non-fat milk constituents on CLA treatment was also investigated. Methods: Diets containing CLA (1%) with or without skim milk powder (SMP) was fed to male Swiss albino mice for 60 days. Adipose depots weight, faecal fat and the activities of selected enzymes of lipid metabolism were determined. Results: The mice on CLA and CLA+SMP diets gained weight similar to those on control diet, despite higher feed intake in the former two groups. Total fat pad mass was significantly (P<0.05) less in CLA group than in control group, and inclusion of SMP in the diet enhanced the fat reducing effect of CLA. Adiposity index was also less on CLA and CLA+SMP diets than on control diet, and CLA+SMP was more efficacious in reducing adiposity index. The weight of liver and spleen was increased by CLA, and this effect was eliminated by inclusion of SMP in the diet. The fatty acid synthase (FAS) activity in liver and retroperitoneal adipose tissue decreased substantially on CLA and CLA+SMP diets compared to that on control diet. Interpretation & conclusions: Our preliminary data show that dietary CLA reduces body fat mass by decreasing fatty acid biosynthesis, and the effect is enhanced by inclusion of SMP in the diet. Key words Adipose depot weight - body fat reduction - conjugated linoleic acid - fatty acid synthase - skim milk powder Obesity is a growing epidemic with subsequent health consequences. Obesity increases the risks of heart ailments, hypertension, diabetes, arthritis, depression and even cancer and therefore, its prevention has become a major public health target. Conjugated linoleic acid (CLA) is a trans fatty acid, though researches claim that it is not harmful in the same fashion as other trans fatty acids, but rather 449 is beneficial. Conjugated linoleic acid is a naturally occurring group of dienoic derivatives of linoleic acid (18:2) formed by bacteria in the forestomach of ruminants 1. Non ruminants, such as humans, may be able to produce some isomers of CLA from vaccenic acid by delta-9-desaturase 2. CLA in diet comes predominantly from food derived from ruminant animals, especially dairy products. Several prominent health benefits have been attributed to consumption of CLA. In rodents,

2 4 INDIAN J MED RES, March 20 dietary CLA stops cancer growth 3, improves glucose tolerance 4, increases oxygen consumption 5-7 and reduces body fat 5-8. Milk fat typically provides 3-6 mg CLA/g fat, in which the predominant isomer is c9, t11. However, research indicates that t, c12-cla is the isomer that influences body composition most 8. Despite well known physiological effects of CLA, the mechanism of action is not known. Further, the concept that dairy products modulate energy metabolism and obesity risk has been developed over the past few years. McCarron et al 9 first reported an association between dairy consumption and weight regulation in USA. An anti-obesity effect of dietary calcium and dairy foods is now evident from animal studies,11, observational and population studies 12,13, and clinical trials 14,15. The present study was conducted to investigate the effects of CLA on body fat metabolism. Since milk fat is the best natural source of dietary CLA, intervention of non fat milk constituents on CLA treatment was also investigated. Material & Methods Nu-Chek-Prep, Inc. (Elysian, MN, USA) was the source of conjugated linoleic acid (CLA) with the reported isomer content as follows: 39.1 per cent cis 9, trans 11 and trans 9, cis 11; 40.7 per cent trans, cis 12; 1.8 per cent cis 9, cis 11; 1.3 per cent cis, cis 12; 1.9 per cent trans 9, trans 11 and trans, trans 12; 1.1 per cent cis 9, cis 12 CLA and 14.1 per cent remainder. [ 14 C] palmitoyl coenzyme A, coenzyme A (CoA), acetyl- CoA, palmitoyl-coa, bovine serum albumin (BSA) and 1, 4-bis[2-(5-phenyloxazolyl)]-benzene (POPOP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and obtained from Sisco Research Laboratories (SRL), Mumbai and S.D. Fine Chemicals Ltd., Mumbai. Male Swiss albino mice (5 wk old), obtained from Small Animal House of the National Dairy Research Institute, Karnal, were maintained individually in polypropylene cages on basal diet for 1 wk, then divided into three groups each consisted of 14 mice and were allowed respective diet and water ad libitum. The control group received basal diet containing 0.6 per cent calcium. CLA group was fed diet containing CLA (1%) substituting for equal amount of soybean oil in the basal diet. The third group was fed with diet containing CLA (1%) plus skim milk powder (CLA+SMP) so as to increase calcium content to 1.2 per cent level. The three diets were isocaloric with calories derived 18 per cent from protein, 69 per cent from carbohydrate and 13 per cent from fat (Tables I, II). Table I. Composition of various diets (g/kg) used in the study Ingredients Control CLA CLA+SMP SMP Starch Casein Lactose Soyabean oil CLA Cellulose Mineral mixture Vitamin mixture DL-Methionine Choline chloride BHA CLA, conjugated linoleic acid; SMP, skim milk powder; BHA, butylated hydroxyanisole; BHA was added to CLA (0 mg/0 ml) as an antioxidant; Mineral and vitamin mixtures contents conformed to minimum requirements according to AOAC 16 Table II. Fatty acid composition of dietary lipids (wt %) Fatty acids Control CLA CLA+SMP Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid CLA *Computed from fatty acid composition of soybean oil 17 and CLA supplemented CLA, conjugated linoleic acid; SMP, skim milk powder; BHA, butylated hydroxyanisole; BHA was added to CLA (0 mg/0 ml) as an antioxidant; Mineral and vitamin mixtures contents conformed to minimum requirements according to AOAC 16 Food intake (daily) and body weights (wkly) were recorded for 60 days. At the end of experimental period, animals were sacrificed by cervical dislocation, and adipose depots (inguinal, epididymal, mesenteric and retroperitoneal), testes, kidneys, spleen, heart, liver and hind leg skeletal muscles were removed. The adiposity index was calculated 7 as ratio of the summed weight of seven excised adipose depots to the weight of eviscerated carcass, where, seven excised adipose depots included left and right inguinal fat, left and right epididymal fat, left and right retroperitoneal fat, and mesenteric fat. Eviscerated carcass comprised of the organs without the gastrointestinal tract and the seven adipose depots. Faeces collected daily were dried at 60 C in hot air oven for fat estimation 18. Assay of Fatty acid synthase (FAS): Liver (11 %) and retroperitoneal adipose tissue (25 %) homogenates, prepared in phosphate bicarbonate buffer (70 mm NaHCO 3, 9 mm KH 2 PO 4, 1 mm NaEDTA, 1 mm

3 Sagwal & Kansal: Synergistic effect of CLA & milk on body fat 451 dithiothreitol, ph 8.0), were centrifuged (4 C) at 30,000xg for 30 min. FAS activity was estimated in the supernatant 19 and calculated using an extinction coefficient of 6.22/mM/cm and expressed as units / mg protein (one unit is the amount of enzyme catalyzing the oxidation of 1 nmole of NADPH per min). Assay of β-oxidation: Liver (0.5 g) homogenized in 5 ml buffer (0.25 M sucrose, 1 mm EDTA, mm tris-hcl, ph 7.4) was centrifuged (4 C) at 3000 x g and for 15 min. Supernatant was then centrifuged at 12,000 x g for 30 min. Supernatant was decanted off in another tube for peroxisomal β-oxidation assay. The pellet washed twice with 5 ml homogenization buffer was resuspended in 8 ml assay buffer ( mm Tris-HCl buffer, ph 8.0) for use in mitochondrial β-oxidation assay. β-oxidation was assayed 20 and expressed as nmoles of palmitoyl-coa that have undergone at least one cycle of β-oxidation per minute. Protein in the samples was estimated with the Folin phenol reagent 21. Statistical analysis: All results are expressed as means ± SE. Analysis of covariance (ANCOVA) (MedCalc statistical software version.4.0) was used to test for differences in body weight change, adipose depot, organ weight and faecal fat content between the three groups. In each ANCOVA, the initial body weight of the animals was used as the covariate to control for pre-existing differences. Homogeneity of regression assumptions were tested and met in each analysis. The mean values, adjusted for pretreatment difference, for the three experimental groups were further compared using the Bonferroni s procedure. The data on feed intake and fatty acid metabolism were compared using the SAS general linear model procedure (SAS institute, Cary, NC) and the differences were inspected further by Duncan s multiple range test. The critical level of statistical significance for all tests was P < 0.05 unless otherwise stated. Results Animals in CLA or CLA+SMP groups consumed greater amounts of feed compared to control diet fed mice, but the difference in body weight gain was not significant among three groups (Table III). Adipose depot and organ weight: Conjugated linoleic acid diet decreased inguinal, epididymal and retroperitoneal adipose depot by 23, 45 and 61 per cent, respectively (P<0.05), in comparison to control group. Inclusion of non fat milk in the diet enhanced the body fat reducing effect of CLA. The decline in body fat on Diet Control CLA CLA + SMP Table III. Feed intake and body weight gain in mice Average feed intake (g / d) 4.45 a ± b ± b ± 0.35 CLA+SMP diet was per cent for inguinal, 69 per cent for epididymal, 35 per cent for mesenteric and 74 per cent for retroperitoneal adipose depots, compared to control group. The effect of CLA and CLA+SMP was maximum in epididymal and retroperitoneal adipose tissue and least in mesenteric adipose tissue. The total adipose depot weight decreased by 39 and 61 per cent in CLA and CLA+SMP groups, respectively, in comparison to the control group (Table IV). Treatment with CLA significantly increased the weight of liver by 28 per cent, and spleen by 33 per cent, however, there was no difference in organ weights between CLA+SMP and control groups. Adiposity index in CLA and CLA+SMP groups was reduced to 51.6 and 38.7 per cent of that of control mice, respectively (Table IV). Faecal fat content: Excretion of faecal matter increased by 32 and 35.6 per cent on CLA and CLA+SMP diets, respectively, compared to control mice. Total fat excretion over the entire duration of experiment was 30 per cent higher on CLA diet than on control diet, and there was no significant difference between control and CLA+SMP group (Table V). Fatty acid metabolism: Fatty acid synthase (FAS) activity (Fig. 1) in liver decreased by 64.6 and 90.0 per cent in CLA and CLA+SMP groups, respectively, in comparison with control group. In retroperitoneal adipose tissue, the decline in FAS activity was 81.5 and 92.3 per cent in CLA and CLA+SMP groups, respectively, when compared with control group. The rate of mitochondrial β-oxidation (Fig.2) decreased by 35.3 and 30.5 per cent and peroxisomal β-oxidation decreased by 59.0 and 29.8 per cent on CLA and CLA+SMP diets, respectively, in comparison to control diet fed mice. Discussion Body weight gain (g) * 11.8 a ± a ± a ± 1.7 Values are mean ± SE (n=12); * Means for body weight gain are adjusted from ANCOVA; Values with different superscript letters are significantly different (P<0.05) In the present study the body weight gain in CLA fed mice was similar to those fed control diet despite

4 452 INDIAN J MED RES, March 20 Table IV. Effect of feeding CLA or CLA+SMP on body adipose depot weight, adiposity index and organ weights in mice Adipose depot weight (g) 1. Inguinal 2. Epididymal 3. Mesenteric 4. Retroperitoneal Total adipose depot weight Adiposity index Organ weight (g) 1. Liver 2. Spleen 3. Kidney 4. Testis 5. Heart Control CLA CLA+SMP 0.26 a ± a ± a ± a ± a ± a ± a ± a ± a ± a ± a ± b ± b ± a ± b ± b ± b ± b ± b ± a ± a ± a ± c ± b ± b ± b ± c ± b ± a ± a ± a ± a ± a ± 0.00 Values are mean ± SE (n=12); Means are adjusted from the ANCOVA analysis; Values in rows with different superscripts are significantly different (P<0.05); Adiposity index = total adipose depot weight / total organ weight Table V. Effect of feeding CLA or CLA+SMP on excretion of faecal fat in mice Groups Total faeces(g) Faecal fat (Total, g) Control CLA CLA+SMP 33.1 a ± b ± b ± a ± b ± a ± 0.23 Values are mean ± SE (n=12); Means are adjusted from the ANCOVA analysis; Faeces were collected over entire experimental duration; Values in columns with different superscripts are significantly different (P<0.05) increased feed consumption in the former group, suggesting reduced feed conversion efficiency caused by CLA. Previous studies on rodents have shown diverse results. The decrease in body weight has been reported in CLA fed mice 22 and hamsters 23. In another study the consumption of CLA, despite reduced feed intake, did not cause loss of body weight in mice 24. In experiments with ICR mice, 0.5 per cent dietary CLA did not affect the body weight of male mice despite decrease in feed intake; however, there was a slight but insignificant decline in weight gain in female mice 5, 8. Our observation that CLA fed as a dietary admixture reduced body adipose depot weight in mice is consistent with the previous reports 5-7. Interestingly, different regional adipose depots responded differentially to CLA and CLA + SMP diets. Reports showed that CLA had major effect on retroperitoneal adipose depot and minimal on epididymal adipose depot 6,7. We found that the greatest effect of CLA was on retroperitoneal adipose depot and epididymal adipose depot, and mesenteric adipose depot was least affected. The specific regional metabolic differences that explain the differential regional response to CLA treatment are not Fig. 1. Effect of feeding CLA or CLA+SMP diets on fatty acid synthase (FAS) activity in mice. Values (mean ± SE for n=6) with different letters are significantly different (P<0.05). Mean value is shown at the top of each bar. One unit is the amount of enzyme that catalyzes the oxidation of 1 nmole of NADPH per min. Fig. 2. Effect of feeding CLA or CLA+SMP diets on b-oxidation of fatty acid in liver in mice. Values (mean ± SE for n=6) with different letters are significantly different (P<0.05). Mean value is shown at the top of each bar. One unit is equal to 1 nmole of palmitoyl-coa that has undergone at least one cycle of b-oxidation per min.

5 Sagwal & Kansal: Synergistic effect of CLA & milk on body fat 453 clear. The decrease in adiposity index in mice fed CLA or CLA+SMP is consistent with decreased adipose depot weight. Conjugated linoleic acid significantly increased liver and spleen weight, confirming the previous reports 6,7. Histological studies are required to obtain a clear picture of hepatomegaly and spleenomegaly in CLA fed animals, if any. Further, the adverse effect, if any, of CLA on organ weight was eliminated by inclusion of non fat milk in the diet. Therefore, a detailed investigation on synergistic effect of food components and CLA is essential before considering fortification of food with synthetic CLA. The increased liver weight caused by CLA treatment is due, partly, to lipid accumulation since the amount of fat in liver of CLA fed mice (156 ± 17 mg/ g tissue) was significantly higher (P<0.05) than that of control mice (6 ± 19 mg/g tissue). Conjugated linoleic acid has been reported to modulate immune function 25, perhaps through cytokines, hence it is not surprising that spleen weight was affected. We examined possible effect of CLA and CLA+SMP on faecal fat excretion. In our study, dietary CLA increased faecal fat excretion, but there was no difference in faecal fat between CLA+SMP and control group. This observation is surprising in that higher dietary calcium was suggested to form non digestible calcium soaps with fatty acids in the digestive tract thus decreasing their absorption and increasing their excretion in faeces 26. There are reports of increased faecal fat with increased calcium content of diet 26,27, however, there is no report to show that increasing dairy calcium would increase faecal fat excretion. Therefore, the increase in faecal fat observed by Papakonstantinou et al 26 might be caused by increased dietary calcium from inorganic sources, while dairy derived calcium does not increase faecal loss of fat. The amount of faecal matter excreted was increased on CLA and CLA+SMP diets, which may account for the extra energy excreted possibly from components other than fat, such as carbohydrates and proteins. Terpstra et al 18 reported that only 27 per cent of the increase in faecal energy excretion was accounted for by faecal fat in mice fed CLA, remaining was suggested to be attributed to other components like proteins and carbohydrates. It is not clear, however, how CLA affects the excretion of energy from dietary components such as proteins or carbohydrates. Our study showed the effect of CLA on the activities of enzymes responsible for fatty acid biosynthesis as well as fatty acid oxidation. Oku et al 28 reported CLA as an inhibitor of fatty acid synthase (FAS) in rat hepatocytes cultured in vitro. Conjugated linoleic acid decreased FAS activity by more than 60 per cent in comparison with linoleic acid, and c9, t11 isomer of CLA was more effective than t, c12 isomer. This modulation in vitro has been confirmed by our in vivo study wherein the decline in FAS activity both in liver and retroperitoneal adipose tissue was observed by feeding CLA. Contrary to our observations on mice, CLA did not affect the activity level of FAS in rats 29 and pigs 30. The diminution in β-oxidation rate in liver by feeding CLA and CLA + SMP diets could be due to increased insulin level, as reported in CLA fed mice 31. The increased insulin level inhibits the mitochondrial as well as peroxisomal β-oxidation in liver cells 32. Although fatty acid degradation is reduced by CLA feeding, the magnitude of decline in FAS activity was greater than the decline in β-oxidation rate, and the net result could be responsible for reduced adipose fat depot in the body. Future animal studies should be conducted to compare the natural sources of CLA with the synthetic commercial CLA for their beneficial effects. This is because the natural CLA may be more effective than synthetic CLA and also it may circumvent the side effects such as enlargement of liver and spleen observed in studies with synthetic CLA. Also along with the control, another group with soybean oil + SMP should be included to delineate the effects of SMP per se, and also to see if the SMP effect is independent of dietary fat or specific to CLA alone. Further, the approach should be to coin the methods to increase the amount of specific beneficial isoforms of CLA (c9, t11 and t, c12) in dairy products and other natural sources. References 1. 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Insulin inhibits peroxisomal fatty acid oxidation in isolated rat hepatocytes. Endocrinology 2001; 142 : Reprint requests: Dr V. K. Kansal, Principal Scientist & Head, Division of Animal Biochemistry, National Dairy Research Institute Karnal , Haryana, India vkk49karnal@yahoo.com