Improving obesity and lipid metabolism using conjugated linoleic acid

Abstract Background Conjugated linoleic acid (CLA) can prevent fatty acid accumulation induced by a high‐fructose diet and improve lipid metabolism disorders in patients. Objectives We aimed to investigate the effect of CLA on obesity and lipid metabolism and its possible mechanism. Methods Eight‐month‐old male BKS.Cg‐Dock7m +/+ Leprdb/JNju (db/db) mice (n = 12) were fed a CLA mix composed of equivalent c9, t11‐CLA and t10, c12‐CLA for 1 month. The effect of CLA on body weight, water and food intake, and triglyceride (TG) and total cholesterol (TC) levels was investigated. PPARα, PPARγ and CD36 expression was determined by quantitative PCR and western blotting. Additionally, the expression of these three genes was studied in HepG2 cells treated with CLA and linoleic acid. Results CLA treatment notably reduced the dietary and water intake of db/db mice, effectively reduced body weight, and decreased serum TG and TC levels (p < 0.05). Increased expression of PPARα (p < 0.05) and decreased expression of CD36 (p < 0.001) were observed in the liver of mice that were fed CLA. CLA increased PPARα expression (p < 0.001) and decreased PPARγ (p < 0.001) and CD36 expression (p < 0.01) in HepG2 cells. Conclusions Our results showed that CLA can improve lipid metabolism in obese mice through upregulation of PPARα expression and downregulation of CD36 expression.

Recently, the FDA has approved CLA as 'Generally Recognised as Safe'; therefore, it can be used in various food and beverage items. CLAs can prevent fatty acid accumulation induced by a high-fructose diet, indicating that CLA can improve lipid metabolism disorders in patients (Maslak et al., 2015).
In this study, we investigated the effect of CLA on the expression of lipid metabolism-associated genes in db/db mice.

Blood Biochemistry
At the end of the experiment, blood was collected from the heart after anaesthesia. Next, the serum was collected and total cholesterol (TC) and total triglycerides (TG) were measured using an automated biochemical analyser (HITACHI 7150, Japan).

HepG2 cell treatment
HepG2 cells were treated with c9, t11-CLA and t10, c12-CLA (Cayman Chemical Company, USA) at a concentration of 50 µl in the CLA group.
The negative control group was treated with LA (Macklin, Shanghai, China).

Quantitative reverse-transcription (qRT)-PCR
Total RNA was extracted from liver tissues or HepG2 cells using the Trizol method and then quantified using a spectrophotometer (NanoDrop 1000, NanoDrop Technologies, Wilmington, DE, USA).

Western blotting
Total protein was extracted from liver tissues or HepG2
The cells were inoculated into a six-well plate, and 3.75 µl of Lipo3000(ThermoFisher Scientific, USA) and 40 pmol of siRNA (Shang-

Statistical analysis
Results are shown as the mean ± standard error of the mean. Based on the data obtained, a two-way analysis of variance, Kruskal-Wallis or Fisher exact test was performed. p < 0.05 was considered statistically significant. All data were analysed using the Statistical Program for Social Sciences version 21.0.

F I G U R E 1
Water and food intake amount of the control and CLA groups. (a) Water intake amount (g). (b) Food intake amount (g) F I G U R E 2 Body weight changes (g) in the control and CLA groups.

Food and water intake and body weights
After 1 month of treatment, the water and food intake markedly decreased in the CLA group compared with that in the control group ( Figure 1a and b). Furthermore, the body weight decreased to normal after treatment with CLA ( Figure 2).

TC and TG levels
The TC and TG levels were markedly lower (p < 0.001) in the CLA group than in the control group (Figure 3).

Gene expression analyses (quantitative PCR)
In  (Figure 5a), while that of the PPARα gene increased (Figure 5b) and the CD36 gene decreased (Figure 5c).

Western blotting
The expression level of each protein obtained from the in vivo experiments was ascertained (Figure 4b). PPARα expression increased, while CD36 expression decreased, in the CLA group compared with that in F I G U R E 5 mRNA and protein expression levels of CD36, PPARα and PPARγ after treatment with linoleic acid, c-9, t11-CLA, or t10, c12-CLA.

siRNA interference and immunofluorescence
After siRNA transfection, PPARγ and CD36 protein expression decreased significantly in the HepG2 cells (Figure 6a-c). The PPARγ knockout group showed weaker green fluorescence signal (CholEsteryl Bodipy TM FL C12) and red fluorescence signal (CD36) than the control group. However, the CLA group showed weaker green and red fluorescence signals than the PPARγ knockout group (Figure 7).

DISCUSSION
Dietary CLA can markedly reduce body fat mass (Hayman et al., 2002;Blankson et al., 2000). CLA comprises several positional and geometric isomers of which c9 t11-CLA and t10 c12-CLA are the main components. In this study, db/db mice were fed a CLA mix, and our results showed that CLA can significantly reduce body weight and improve TG and TC levels. Previous studies have also shown that CLA mix can significantly decrease body weight and fat mass (Park et al., 1997;Park et al., 1999;West et al., 1998). In our study, the CLA supplement markedly affected food and water intake in addition to body weight; these findings were consistent with those of prior investigations (Park et al., 1999;Hargrave et al., 2004).
Furthermore, to elucidate the mechanism underlying the effects of CLA on lipid metabolism, we investigated the expression of three genes associated with lipid metabolism: PPARγ, PPARα and CD36.
The qRT-PCR and western blotting results showed that CD36 expression significantly decreased, while that of PPARα increased in the CLA group. PPARα is highly expressed in mitochondria-rich and active βoxidation tissues such as the liver, renal cortex, intestinal mucosa and heart. PPARα comprises both natural and synthesised ligands. Natural ligands include polyunsaturated fatty acids (PUFAs), unsaturated fatty acids, and inflammatory mediators, among which PUFAs have the highest affinity. PPARα can promote fatty acid oxidation and ketogenesis, decrease blood glucose and regulate the uptake and storage of lipids