The Effect of Docosahexaenoic Acid on t 10,c 12-Conjugated Linoleic Acid-Induced Changes in Fatty Acid Composition of Mouse Liver,Adipose,and Muscle

Abstract

Background:

Concomitant supplementation of 1.5% docosahexaenoic acid (22:6 n-3; DHA) with 0.5% t10, c12-conjugated linoleic acid (18:2 n-6; CLA) prevented the CLA-induced increase in expression of hepatic genes involved in fatty acid synthesis and the decrease in expression of genes involved in fatty acid oxidation. The effect of CLA on fatty acid compositions of adipose tissue and muscle and whether DHA can prevent those CLA-induced changes in fatty acid composition is not known.

Methods:

We investigated if DHA fed concomitantly with CLA for 4 weeks will prevent the CLA-induced changes in fatty acid compositions of liver, adipose, and muscle lipids in C57BL/6N female mice. We also examined changes in expression of adipose tissue genes involved in fatty acid synthesis, oxidation, uptake, and lipolysis.

Results:

CLA supplementation increased liver fat and decreased total n-3 polyunsaturated fat (PUFA) concentration. DHA not only prevented the CLA-induced changes in liver fat, but also increased n-3 PUFA by >350% as compared with the control group. CLA decreased adipose weight and the expression of genes involved in fatty acid synthesis, oxidation, and uptake and increased that of uncoupling protein 2 (UCP2). Supplementing DHA along with CLA increased adipose n-3 PUFA by >1000% compared with control group, but did not prevent the CLA-induced changes in mass or gene expression. Both CLA and DHA were incorporated into muscle lipids, but had minor effects on fatty acid composition.

Conclusions:

Liver, adipose tissue, and muscle responded differently to CLA and DHA supplementation. DHA prevented CLA-induced increase in liver fat but not loss of adipose mass.

Introduction

N onalcoholic fatty liver disease (NAFLD) is a rapidly growing health problem affecting adults and children in developed countries and is now considered the hepatic component of metabolic syndrome. NAFLD encompasses a spectrum of liver damage ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), and eventually can lead to advanced fibrosis and cirrhosis.^1,2 NAFLD is the most common liver disease worldwide and affects about 20% of the adult population.^3,4 Approximately 20%–30% of NAFLD patients have signs of NASH and are at higher risk of developing cirrhosis, liver failure, and hepatocellular carcinoma.⁵ Insulin resistance (IR) is a central factor in the development of NAFLD. It is a condition in which normal amounts of insulin fail to maintain normal blood glucose levels because of decreased responsiveness of muscle (glucose uptake), adipose tissue (glucose uptake and inhibition of lipolysis), and liver (inhibition of gluconeogenesis).^6,7 In 2005 over 40% of people aged 20 and over in the United States were hyperglycemic [diabetics 12.9 %; impaired fasting glucose (IFG) 25.7%; impaired glucose tolerance (IGT) 13.8 %].⁸ It is now recognized that increased fat deposition in the muscle can induce skeletal muscle IR.⁹

NAFLD and IR can be caused by an assortment of factors, including nutrition, genetics, inflammation, and lifestyle. Diets high in SFA and trans-fatty acids (TFA) with an increased ratio of n-6 to n-3 polyunsaturated fatty acids (PUFA) have been demonstrated to induce IR and NAFLD in humans and animals.^10

–13 One TFA of concern in the diet is conjugated linoleic acid (CLA), linoleic acid isomers having conjugated double bonds. Although there are more than two dozen positional and geometric isomers of CLA, two isomers, trans-10,cis-12-CLA (t10,c12-CLA) and cis-9,trans-11-CLA (c9,t11-CLA), have been studied most extensively. t10,c12-CLA is primarily found in processed foods and partially hydrogenated oils, and c9,t11-CLA is naturally found in dairy products and ruminant meats.¹⁴ It has been reported by our laboratory, as well as others, that t10,c12-CLA induces NAFLD and IR in mice and other animals, including humans.^{4,11,12,15

–18} On the other hand, n-3 PUFA have been shown to prevent IR, enhance fatty acid oxidation, and inhibit fatty acid synthesis.^19,20 Altered fatty acid metabolism in NAFLD decreased concentrations of hepatic total n-3 long-chain PUFA, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), which increased the ratio of n-6 to n-3 PUFA.^21

–24

Recently, we reported that the concurrent feeding of DHA with CLA in mice prevented the CLA-induced increase in liver fat and attenuated IR. Decrease in liver fat was associated with parallel decreases in expression of hepatic genes involved in fatty acid synthesis [acetyl-CoA carboxylase-α (ACCA), fatty acid synthase (FASN), stearoyl-CoA desaturase-1 (SCD1), and peroxisome proliferator-activated receptor-γ (PPARγ)] and increases in expression of key genes regulating fatty acid oxidation [acyl-coA oxidase 1 (ACOX1) and carnitine palmitoyl transferase 1 (CPT1A)].¹⁶ In that study, we did not investigate the effects of concomitant supplementation of CLA and DHA on the amount of fat in adipose tissue and muscle or the fatty acid composition of liver, adipose tissue, and muscle, nor did we monitor the changes in the expression of genes involved in fatty acid metabolism in adipose tissue and muscle. We have discussed above that these changes contribute to the development of NAFLD and IR. Therefore, in this report we extend our earlier findings by investigating the amount and fatty acid composition of liver, adipose tissue, and muscle, and the adipose tissue expression of genes involved in fatty acid metabolism.

Methods

Animals and diets

The animal protocol was approved by the Animal Use Committee at the University of California, Davis (UCD). Eight-week-old, pathogen-free C57BL/6N female mice (Charles River, Raleigh, NC) were maintained at the animal facility in the Genome and Biomedical Sciences Facility at UCD. The temperature in the room was maintained at 25°C with dark and light cycles of 12 h each. A modified AIN93G diet (soybean oil replaced with corn oil) was used as the control diet. The remaining three diets had a portion of the corn oil replaced with equivalent amounts of highly enriched DHA and/or t10,c12-CLA in the form of free fatty acids (FFA). Concentrations of fatty acids added were 0.5% CLA, 1.5% DHA, and 0.5% CLA+1.5% DHA. Both DHA and CLA were greater than 90% purity and were obtained from Larodan Fine Chemicals (Malmo, Sweden). Composition of the diets is given in Table 1. While mixing, diets were constantly flushed with nitrogen gas, packaged into 20-gram aliquots, flushed with nitrogen gas, and stored at −20°C. The doses and duration of CLA and DHA treatments were based on our previous studies.^16,18,25

Table 1.

Composition of Experimental Diets

Ingredients	Control ^a	CLA ^a	CLA+DHA ^a	DHA ^a
Cornstarch	417.5	417.5	417.5	417.5
Casein	200	200	200	200
Dextrose	132	132	132	132
Sucrose	100	100	100	100
Corn oil	50	45	30	35
Cellulose	50	50	50	50
Mineral mix^b	35	35	35	35
Vitamin mix^c	10	10	10	10
L-Cysteine	3	3	3	3
Choline bitartrate	2.5	2.5	2.5	2.5
CLA	0	5	5	0
DHA	0	0	15	15

grams/kg of diet.

Containing the following (grams/kg diet): calcium 5.0, phosphorous 1.56, potassium 3.6, sodium chloride 1.02, chlorine 1.57, sulfur 0.3, magnesium 0.51, ferric citrate 0.04, copper 0.01, manganese 0.01, zinc 0.03, chromium 0.001, iodine 0.0002, selenium 0.0002, fluoride .001, boron 0.0005, molybdenum 0.0002, silica 0.005, lithium chloride 0.0001, nickel 0.000, vanadium 0.0001.

Containing the following (mg/kg diet): thiamin HCl 6, riboflavin 6, pyridoxine HCl 7, niacin 30, calcium pantothenate 16, folic acid 2, biotin 0.2, cyanocobalamin 25, vitamin A palmitate 4000, vitamin E acetate 75, vitamin D3 1000, vitamin K1 0.75.

CLA, conjugated linoleic acid; DHA, docosahexaenoic acid.

Animals were acclimated to the laboratory chow diet before they were randomly assigned to one of the four experimental diets for the following 28 days, with 12 animals per group and 4 animals per cage. Fresh diet (20 grams/cage) was served daily, and the food left in the jars from the previous day was weighed and recorded. Body weight was recorded every 7 days. Animals were terminated by CO₂ asphyxiation after withholding food for 10–12 h. Blood was collected by cardiac puncture into a syringe and centrifuged in serum separator tubes (BD Microtainer SST, Franklin Lakes, NJ). Liver, periuterine adipose, and rectus femoris muscles from both pelvic limbs were collected, weighed, frozen in liquid nitrogen, and stored at −80°C for future analysis.

Fatty acid analysis

Approximately 25 mg of liver, adipose, or muscle tissue was pulverized mechanically on dry ice and transferred to a methanol- and hexane-rinsed microcentrifuge tube containing methanol and hexane-rinsed 3.0-mm diameter stainless steel balls (Retsch, Newtown, PA), and spiked with 5 μL of 0.2 mg/mL butylated hydroxytoluene/EDTA in 1:1 methanol/water and 5 μL of triglyceride surrogate solution. Total lipids were extracted using two liquid–liquid methods based on a modifications of the published protocols of Smedes.²⁶ A stepwise methanol/isopropanol/cyclohexane protocol was used for liver and adipose lipid extraction, and a 2-propanol/cyclohexane/ammonium acetate protocol was used for muscle lipid extraction. Solvents were removed by evaporation under vacuum and residues were reconstituted in 1 mL 1:1 methanol/toluene. Fatty acid methyl esters (FAMEs) were prepared by sequential ester transesterification/acid methylation. A 100 μL aliquot of the hexane extract was spiked with methyl tricosanoic acid (C23:0) as an internal standard. FAMEs were separated on an Agilent 6890 gas chromatograph (Agilent, Santa Clara, CA) equipped with a 30 m×0.25 mm×0.2 μm DB-225ms column and detected on an Agilent 5973 Mass Spectral Detector, operated in selected ion monitoring mode with electron impact ionization. Analytes were quantified against a 7- or 8-point calibration curve. All data were corrected for either 19:1n9 or 16:0-d31 surrogate recoveries. The comparability of data generated using slight procedural modifications was confirmed through replicate analyses. All data analysis and quantification were performed using Agilent Chemstation Software v. E.02.

RNA isolation and quantitative real-time PCR analysis

Total RNA was isolated from the periuterine adipose using the PureLink Micro-to-Midi purification kit according to manufacturer's instructions (Invitrogen, Carlsbad, CA). RNA quality and integrity were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Purified total RNA (1 μg) from each sample was used for cDNA synthesis (Superscript III First Strand Synthesis System; Invitrogen, Carlsbad, CA). Adipose RNA abundance for liver X receptor beta (LXRβ), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), PPARγ, sterol regulatory element binding protein-1c (SREBP1C), hormone sensitive lipase (HSL), ACOX1, CD36, and uncoupling protein 2 (UCP2) by using gene-specific Taqman^® primers and FAM-MGB labeled probes per manufacturer's instructions (Applied Biosystems, Foster City, CA). The cDNA (15 ng) was air dried in each well prior to adding PCR reagents (4 μL 2X Taqman Gene Expression Mastermix, 0.4 μL specific primer-probe set and 3.6 μL water). Real-time quantitative (qRT)-PCR reactions were conducted on a 384-well plate in triplicate and were carried out using an ABI 7900HT Fast Real-Time PCR System and documented by SDS software v.2.2.2 (Applied Biosystems). Relative gene expression was calculated using the 2^−ΔCt method, which normalizes against the endogenous control gene and the control group computed average ΔCt.

Statistical analysis

SAS version 9.3 statistical software was used for all analyses (SAS Institute Inc. 2011. SAS OnlineDoc^® 9.3. Cary, NC: SAS Institute Inc.). Levene's test was used to test for heterogeneity of variance, and, when significant, the heterogeneity was incorporated in the model using the “group=” option with the SAS MIXED procedure. Diet was the fixed effect and cage within diet was the random effect. Single degree of freedom contrasts provide significance tests for the primary hypotheses of interest. Data shown are least squares means and standard errors. Differences were considered significant at p<0.05.

Results

Effect of CLA and DHA on physical characteristics and tissue lipids

Least square mean (LSM)±standard error of the mean (SEM) for initial body weights of mice in the control, 0.5% CLA, 1.5% DHA, and 0.5% CLA+1.5% DHA were 20.9±0.23 gram, 20.1±0.22 gram, 20.3±0.17 gram, and 20.6±0.15 gram, respectively; those weights did not differ between any of the groups. Also there were no significant differences between the groups for the amount of diets consumed (data not shown). After 4 weeks of feeding the experimental diets, LSM body weight of mice in the CLA group was significantly less than the corresponding value in the control group (Table 2).

Table 2.

Effect of CLA and DHA on Physical and Biochemical Variables of Mice Fed Experimental Diets for 4 Weeks

	Dietary group
	Control		CLA		CLA+DHA		DHA
Variable	LSM	SEM	LSM	SEM	LSM	SEM	LSM	SEM
Final body weight (grams)	22.6^*	0.53	20.9^**	0.53	19.9	0.53	22.9	0.53
Liver weight (grams)	0.92^*	0.05	1.80^** ^a	0.22	0.97^b	0.05	0.82	0.05
Periuterine adipose weight (grams)	0.57^*	0.04	0.20^**	0.04	0.26	0.04	0.79	0.09
Muscle weight (grams)	0.22	0.01	0.21	0.01	0.21	0.01	0.22	0.01
Liver total lipids (mg/grams)	99.1	6.36	171	37.6	73.1	37.6	73.4	6.36
Adipose total lipids (mg/grams)	529	76.1	581	195	695	188	563	76.1
Muscle total lipids (mg/grams)	18.0	3.23	12.5	3.23	10.7	3.23	22.3	3.23

Data shown are LSM±SEM (n=6–12). Different number of asterisks within a row indicate a significant difference (P<0.05) between the control and CLA group. Different letters within a row indicate a significant difference between the CLA and CLA+DHA group.

CLA, conjugated linoleic acid; DHA, docosahexaenoic acid; LSM, least squares mean; SEM, standard error of the mean.

Feeding the CLA diet significantly increased liver weight compared to the control group; this increase was completely prevented by the concurrent feeding of DHA. In contrast to the increase in liver weight, CLA significantly decreased periuterine adipose tissue weight, and this loss was not prevented by concurrent feeding of DHA (Table 2).¹⁶ The adipose depot weights did not differ between the CLA and the CLA+DHA group. Neither CLA nor DHA alter rectus femoris muscle weight or its lipid content (Table 2). Mean liver lipids (mg/gram tissue) was 1.7 times greater in the CLA group compared with those in the control group; however, the differences did not attain statistical significance because of the large variances within the groups. Again, because of large variances within the groups, differences between the groups did not attain statistical significance for the adipose tissue and the muscle lipids.

Effect of CLA and DHA on liver fatty acid composition

CLA increased total SFA and MUFA when compared to the control group, but these differences did not attain significance due to large variations within groups (Table 3). CLA significantly decreased the total n-3 PUFA in liver when compared with those in the control group, whereas the decrease in n-6 PUFA did not attain statistical significance. Concomitant feeding of DHA along with CLA reversed the CLA-induced changes in liver total SFA, MUFA, and n-3 PUFA, but only the change in n-3 PUFA was significantly different from that in the CLA group.

Table 3.

Liver FA Composition (μmol/gram)

	Control		CLA		CLA+DHA		DHA
Fatty acid	LSM	SEM	LSM	SEM	LSM	SEM	LSM	SEM
SFA
16:0	102	8.28	197	47.6	86.7	47.6	77.6	8.28
18:0	16.3	3.3	20.2	3.2	18.9	3.2	17.1	3.3
MUFA
16:1n7	20.3	1.97	22.7	7.76	4.85	1.97	10.6	1.97
18:1n9	110	23.5	282	81.9	65.7	22.3	58.4	23.5
18:1n7	8.29	1.14	26.7	7.83	3.42	1.05	3.87	1.14
n-6 PUFA
18:2n6	70.4	9.2	51.6	9.0	35.2	9.0	44.0	9.2
CLA	0.02^*	0.02	1.43^**	0.32	1.06	0.32	0.01	0.01
18:3n6	3.95	0.26	1.12	0.26	0.25	0.10	0.39	0.11
20:4n6	18.5^*	1.39	13.1^** ^a	1.39	4.69^b	0.17	5.04	0.17
22:4n6	0.89	0.15	0.73^a	0.14	0.06^b	0.01	0.06	0.01
22:5n6	2.11	0.52	1.61	0.50	0.19	0.03	0.23	0.03
n-3 PUFA
18:3n3	0.51^*	0.1	0.20^**	0.1	0.16	0.1	0.51	0.1
20:5n3	0.11^*	0.01	0.02^** ^a	0.01	4.57^b	1.0	9.67	1.02
22:5n3	0.25^*	0.03	0.13^** ^a	0.03	1.94^b	0.41	2.22	0.42
22:6n3	8.48^*	0.45	5.69^** ^a	0.45	36.8^b	6.0	34.6	6.30
Total SFA	121	8.39	222	52.9	107	52.9	96.4	8.39
Total MUFA	143	27.1	347	103	76.5	25.6	74.7	27.1
Total n6 PUFA	95.9	10.0	69.6	9.80	41.5	9.80	49.7	10.0
Totaln3PUFA	9.33^*	0.48	6.04^** ^a	0.48	43.4^b	7.02	46.9	7.30
n6:n3 ratio	10.3 ^a	1.07	11.5^a	1.02	0.97^b.	0.14	1.06	0.14
16 desat index	0.20^*	0.01	0.11^** ^a	0.01	0.05^b	0.01	0.14	0.01
18 desat index	6.92^*	0.81	13.8^** ^a	0.81	2.88^b	0.81	3.41	0.81

Data are LSM±SEM (n=6). Different numbers of asterisks within a row indicate a significant difference (P<0.05) between the control and CLA group. Different letters within a row indicate a significant difference between the CLA and CLA+DHA group.

CLA, conjugated linoleic acid; DHA, docosahexaenoic acid; LSM, least squares mean; SEM, standard error of the mean; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; desat index, desaturation index.

Individual fatty acids whose concentrations were significantly altered in liver lipids by CLA compared with those in the control group include an increase in CLA and decreases in 20:4n6, 18:3n3, 20:5n3, 22:5n3, and 22:6n3. Compared with the CLA group, concomitant feeding of DHA with CLA significantly decreased liver concentrations of 20:4n6 and 22:4n6 and increased those of 20:5n3, 22:5n3 and 22:6n3. Consistent with the changes in individual n-6 and n-3 PUFA, DHA significantly decreased the n6:n3 ratio. The 16:1/16:0 and 18:1/18:0 desaturation indexes were also calculated by product-to-precursor fatty acid ratios. CLA significantly decreased the 16:1/16:0 desaturation index compared to the control group and increased the 18:1/18:0 desaturation index. In the CLA+DHA group, both desaturation indexes were significantly lower when compared to the CLA group.

Effect of CLA and DHA on adipose tissue fatty acid composition and gene expression

Adipose fatty acid composition. Neither CLA nor DHA had an effect on adipose total SFA, MUFA, or n-6 PUFA concentration (Table 4). CLA decreased adipose tissue total n-3 PUFA compared to the control group, but it did not attain significance (P<0.1). When fed along with CLA, DHA significantly increased adipose tissue total n-3 PUFA concentration as compared to corresponding values in the CLA group.

Table 4.

Adipose Fatty Acid Composition (μmol/gram)

	Control		CLA		CLA+DHA		DHA
Diet fatty acid	LSM	SEM	LSM	SEM	LSM	SEM	LSM	SEM
SFA
16:0	535	170.4	633	182	832.4	175	657	170
18:0	52.5	6.71	35.7^a	7.34	60.17^b	6.96	72.7	6.71
MUFA
16:1n7	134^*	21.3	61.6^**	22.4	45.9	21.8	158	21.3
18:1n9	702	81.0	867	275	797	259	586	81.0
18:1n7	45.8	5.70	43.5^a	5.83	24.6^b	5.76	33.4	5.70
n-6 PUFA
18:2n6	463	55.0	479	219	659	214	425	55.0
CLA	0.06^*	0.02	19.5^** ^a	2.36	52.0^b	2.36	0.02	0.02
18:3n6	1.63	0.37	1.28^a	0.11	0.91^b	0.11	1.19	0.11
20:4n6	6.42	1.63	5.22^a	0.44	1.30^b	0.38	3.07	0.34
22:4n6	1.09	0.19	0.93	0.19	0.37	0.19	0.64	0.19
22:5n6	1.38	0.18	0.55	0.18	0.93	0.18	1.38	0.18
n-3 PUFA
18:3n3	5.56^*	1.29	2.79^**	0.45	3.77	0.45	9.61	1.29
20:5n3	0.08	0.06	0.02^a	0.06	2.42^b	0.77	5.13	0.70
22:5n3	0.23	0.07	0.08^a	0.08	5.84^b	0.49	7.33	0.49
22:6n3	1.35^*	0.19	0.57^** ^a	0.21	65.9^b	17.5	99.5	17.2
Total SFA	615	172	689	183	921	177	770	172
Total MUFA	903	192	1003	207	897	198	799	192
Total n6 PUFA	473	153	511	160	715	156	431	153
Totaln3PUFA	7.23	1.20	3.39^a	1.30	77.7^b	18.7	122	18.3
n6:n3 ratio	71.6^*	31.3	184^** ^a	31.3	8.88^b	1.26	3.63	1.25
16 desat index	0.25^*	0.02	0.11^**	0.02	0.07	0.02	0.24	0.02
18 desat index	13.7	0.79	26.2	7.08	14.3	6.30	8.12	0.79

Data are LSM±SEM (n=6). Different number of asterisks within a row indicate a significant difference (P<0.05) between the control and CLA group. Different letters within a row indicate a significant difference between the CLA and CLA+DHA group.

Individually, CLA significantly decreased concentrations of 16:1n7, 18:3n3, and 22:6n3 and increased its own concentration in adipose tissue lipids when compared with the control group. DHA supplementation along with CLA completely reversed the CLA-induced reduction in n-3 PUFA (20:5n3, 22:5n3, and 22:6n3), whereas the changes in 18:3n3 and 16:1n7 were not fully restored. Concentrations of 20:5n3, 22:5n3, and 22:6n3 were not only reversed by DHA, but reached several folds greater than those in the control group. CLA significantly increased the n6:n3 ratio compared with that found in the control group, and DHA supplemented with CLA significantly decreased this ratio compared with that in the CLA group. CLA significantly decreased the 16:1/16:0 desaturation index compared to control group, which was not restored by DHA. Neither fatty acid altered the 18:1/18:0 desaturation index.

Adipose tissue gene expression

To investigate how CLA and DHA may be affecting fatty acid composition, we determined the changes in the expression of genes regulating fatty acid synthesis (LXRβ, PGC1α, PPARγ, and SREBP1C), fatty acid oxidation (ACOX1), fatty acid uptake (CD36), and lipolysis (HSL) in the periuterine adipose tissue. CLA significantly decreased the expression of LXRβ, PGC1α, PPARγ, SREBP1C, ACOX1, and CD36 adipose mRNA when compared to the control group (Table 5). We also observed a trend for CLA to decrease the expression of HSL (P=0.08). DHA was not able to prevent any of these decreases in gene expression. CLA significantly increased UCP2 mRNA expression when compared to control group; DHA again had no effect.

Table 5.

Effects of CLA and DHA on Adipose Gene Expression

	Control		CLA		CLA+DHA		DHA
Diet Gene	LSM	SEM	LSM	SEM	LSM	SEM	LSM	SEM
LXRβ	100^*	6.31	56.5^**	7.01	66.5	7.01	95.6	6.31
PGC1α	100^*	9.67	65.0^**	10.8	67.1	9.67	91.0	9.67
PPARγ	100^*	23.0	33.9^**	8.84	35.2	8.84	83.2	8.07
SREBP1c	100^*	6.48	77.2^**	6.95	75.0	6.95	95.1	6.48
ACOX1	100^*	15.6	48.5^**	6.57	65.1	6.57	87.8	6.04
HSL	100	22.7	26.5	5.48	35.1	5.48	101	4.94
CD36	100^*	16.3	55.5^**	7.53	69.9	7.53	92.3	7.13
UCP2	100^*	16.7	196^**	18.3	191	18.3	109	16.7

Data are LSM±SEM (n=6). Different number of asterisks within a gene represent a significant difference (P<0.05) between the control and CLA group.

CLA, conjugated linoleic acid; DHA, docosahexaenoic acid; LSM, least squares mean; SEM, standard error of the mean; ACOX1, acyl-CoA oxidase 1; HSL, hormone sensitive lipase; LSM, least squares mean; LXRβ, liver X receptor β; PGC1α, PPAR gamma coactivator-1 βalpha; PPARγ, peroxisome proliferator activated receptor; SREBP1C, sterol regulatory element binding protein 1c; UCP2, uncoupling protein.

Effect of CLA and DHA on muscle fatty acid composition

Muscle fatty acid composition

In the muscle, CLA did not alter concentrations of total SFA, MUFA, n-6 PUFA, and n-3 PUFA when compared with those in the control group (Table 6). Concomitant feeding of DHA along with CLA significantly decreased total n-6 PUFA and increased total n-3 PUFA concentrations when compared with the corresponding values in the CLA group. CLA did not alter concentrations of any other fatty acids despite a significant increase in its own concentration when compared to those in the control group. Concomitant feeding of DHA along with CLA significantly decreased the muscle concentrations of 18:1n7 and 20:4n6 when compared with corresponding values in the CLA group and increased that of 22:6n3 when compared with the CLA group. CLA alone had no significant effect on the n6:n3 ratio compared to the control group, whereas DHA fed with CLA significantly lowered the n6:n3 ratio compared to the CLA group. No significant differences were seen in the 16:1/16:0 and 18:1/18:0 desaturation indexes.

Table 6.

Muscle Fatty Acid Composition (μmol/gram)

	Control		CLA		CLA+DHA		DHA
Diet fatty acid	LSM	SEM	LSM	SEM	LSM	SEM	LSM	SEM
SFA
16:0	13.9	1.80	12.2	1.80	11.6	1.80	21.9	1.80
18:0	3.87	0.39	3.32	0.39	2.66	0.39	4.02	0.39
MUFA
16:1n7	4.40	1.63	2.82	1.48	2.17	1.53	9.48	1.63
18:1n9	12.9	3.14	9.86	3.14	5.88	3.14	20.9	3.14
18:1n7	1.48	0.20	1.26^a	0.20	0.51^b	0.20	1.43	0.20
n-6 PUFA
18:2n6	6.97	1.55	5.93	1.55	2.94	1.55	9.25	1.55
CLA	0.0	0.0	0.13	0.02	0.14	0.02	0.0	0.0
18:3n6	0.01	0.01	0.0	0.0	0.0	0.0	0.01	0.01
20:4n6	3.31	0.10	3.53^a	0.10	0.34^b	0.01	0.44	0.01
22:4n6	0.28	0.02	0.35	0.02	0.0	0.0	0.01	0.01
22:5n6	0.87	0.10	0.64	0.05	0.0	0.0	0.01	0.0
n-3 PUFA
18:3n3	0.08	0.04	0.04	0.04	0.02	0.04	0.19	0.04
20:5n3	0.0	0.0	0.0	0.0	0.04	0.01	0.12	0.03
22:5n3	0.16	0.07	0.16	0.06	0.18	0.07	0.18	0.07
22:6n3	4.89	0.49	4.73^a	0.49	11.1^b	0.49	12.2	0.49
Total SFA	18.4	1.51	15.8	2.56	14.6	1.51	27.2	2.56
Total MUFA	19.5	4.77	14.6	4.77	9.11	4.77	32.8	4.77
Total n6 PUFA	11.4	1.76	10.6^a	1.63	3.42^b	1.67	9.68	1.76
Total n3PUFA	5.13	0.51	4.93^a	0.51	11.31^b	0.51	12.7	0.51
n6:n3 ratio	2.47	0.53	2.15^a	0.52	0.30^b	0.13	0.73	0.13
16 desat index	0.30	0.05	0.18	0.05	0.17	0.05	0.41	0.05
18 desat index	3.56	0.96	2.73	0.96	2.16	0.96	5.72	0.96

Data are LSM±SEM (n=6). A different letter within a row indicate a significant difference (P<0.05) between the CLA and CLA+DHA group.

Discussion

In this study, we have investigated the relative contributions of three insulin-sensitive tissues (liver, adipose, and muscle) to CLA-induced NAFLD and IR and their prevention by DHA. Our results show that the effects of these fatty acids varied on total tissue fat and their fatty acid compositions. Both CLA and DHA were incorporated into lipids in all three tissues, and their concentrations were highest in the adipose tissue lipids and lowest in the muscle lipids. These concentrations may simply be a reflection of the adipose tissue, but not muscle, being the primary reservoir for fat storage and the liver as the storage site for FFA during NAFLD. CLA increased total fat in the liver, decreased it in the adipose tissue, and had no effect on the muscle. DHA prevented the CLA-induced increase in liver fat, but did not prevent the loss of adipose tissue fat. Previously, we reported that 1.5% DHA prevented the CLA-induced increases in hepatic expression of genes involved in fatty acid synthesis (ACCA, FASN, and SCD1) and decreases in expression of genes involved in fatty acid oxidation (ACOX1 and CPT1A).¹⁶ Those changes in hepatic gene expression were consistent with the changes in the amount of liver fat and its fatty acid composition. Changes in hepatic fatty acid composition caused by CLA and DHA in our study are consistent with those of studies showing that NAFLD is associated with a decrease in liver n-3 PUFA and an increase in the n6:n-3 ratio^17,21 and that supplementation with n-3 PUFA increases hepatic n-3 PUFA concentration and reverses NAFLD.^27,28

An increase in DHA concentration in adipose tissue lipids was inadequate to reverse the CLA-induced changes in gene expression (Table 5), and it also failed to restore adipose tissue mass. Because the expression of genes involved in fatty acid synthesis, oxidation, and uptake were all decreased in the CLA group, each of these mechanisms may have contributed to the loss of adipose tissue fat. It has been reported that about 59% of hepatic triglyceride (TG) in NAFLD patients come from an excess of circulating FFA from adipose tissue lipolysis, 26% from de novo lipogenesis (DNL), and 15% from diet.²⁹ CLA inhibits human preadipocyte differentiation and causes delipidation of differentiated human adipocytes and adipose tissue in mice.^30,31 An increased flux of FFA to the liver can be caused by insulin-resistant adipose tissue increasing TG lipolysis within adipose depots, while also inhibiting adipocyte uptake of glucose and FFA.³² The accompanying hyperinsulinemia promotes hepatic DNL and inhibits hepatic FAO. DHA has also been shown to have antiadipogenic effects in 3T3-L1 cells and in rodents through altering gene expression and fatty acid metabolites.^33,34 The combined antiadipogenic effects of CLA and DHA may explain why DHA failed to restore the adipose depots but prevented the increase in liver fat. Furthermore, adipose tissue CLA concentration in the CLA+DHA group was almost three times than that in the CLA group (Table 4), suggesting that DHA increased the uptake of CLA or decreased its metabolism. With a threefold increase, effects of CLA may override the effects of DHA. Increased expression of UCP2 may have also contributed to the depletion of adipose tissue by CLA, and DHA failed to reduce the CLA-induced expression of UCP2.

We did not study the changes in muscle gene expression because muscle fat content was not changed by CLA or DHA. Concentrations of 18:1n-7, 18:2n-6, and 20:4n-6 were significantly lower and that of DHA was significantly higher in the CLA+DHA group than in the CLA group. Failure of CLA and DHA to change the amount of muscle fat suggests that muscle may not be a major player in the CLA-induced NAFLD and IR and their reversal by DHA.

Our study had several limitations, including that we did not investigate the changes in gene expression and fatty acid composition of tissues at earlier time points leading up to 4 weeks. We were also not able to measure circulating adiponectin and leptin levels due to prioritization of analyses with limited serum samples. We did not measure gene and protein expression related to insulin signaling. Some of the variables measured in our study had a much higher variance than what we have found in our previous studies conducted for 8 weeks in this model.^18,25 This may be due to any one of a number of reasons; however, we believe the 4 week duration of the study was the most likely reason. Effects of CLA on NAFLD, IR, and fatty acid composition generally begin to be noticeable within 2 weeks of its feeding and attain the maximum response within approximately 12 weeks. Response time varies with different animals. Thus, in regard to fatty acid composition, termination of this study at 4 weeks may have been premature because some mice may have been just beginning to respond to the fatty acids in their diets and that the lack of significant differences seen in this study can be attributable to the variance in response times. Nevertheless, our results paralleled the significant changes we reported in our earlier studies involving 8 weeks of CLA and DHA feeding.

In conclusion, our results show that both dietary CLA and DHA were preferentially incorporated into adipose tissue lipids, followed by liver and muscle, respectively. Despite their highest incorporation into adipose tissue, these fatty acids had only modest effects on the overall fatty acid composition of adipose tissue. The largest changes in fatty acid composition were seen in the liver and smallest in muscle lipids. When fed concomitantly with CLA, DHA prevented the effects of CLA on liver total lipids and their fatty acid composition. Even if concentrations of most fatty acids in muscle did not change in response to CLA, feeding of DHA alone or with CLA increased DHA and decreased arachidonic acid concentrations in muscle lipids. Out of the three tissues we tested, adipose tissue seems to be most responsive to CLA and liver seems to be most responsive to DHA. Future studies are needed to determine the dose of DHA that will restore the CLA-induced depletion of adipose tissue and the mechanisms involved.

Footnotes

Acknowledgments

D.M.F, Y.A., and D.S.K all contributed to planning of the study, data interpretation, and preparation of the manuscript; D.M.F. and Y.A. participated in execution of the study and analysis and J.N. in fatty acid analysis and manuscript preparation; D.S.K. has primary responsibility for final content. We thank Theresa Pedersen and William Keyes for support with the gas chromatography/mass spectrometry and analysis.

Author Disclosure Statement

Funds for this study were provided by the U.S. Department of Agriculture (USDA). No competing financial interests exist. Reference to a company or product name does not imply approval or recommendation of the product by the USDA to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.

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