Conjugated linoleic acids inhibit lipid deposition in subcutaneous adipose tissue and alter lipid profiles in serum of pigs

Abstract

Conjugated linoleic acids (CLAs) have served as a nutritional strategy to reduce fat deposition in adipose tissues of pigs. However, the effects of CLAs on lipid profiles in serum and how these lipid molecules regulate fat deposition are still unclear. In this study, we explored the effects of CLAs on regulating lipid deposition in adipose tissues in terms of lipid molecules and microbiota based on a Heigai pig model. A total of 56 Heigai finishing pigs (body weight: 85.58 ± 10.39 kg) were randomly divided into two treatments and fed diets containing 1% soyabean oil or 1% CLAs for 40 d. CLAs reduced fat deposition and affected fatty acids composition in adipose tissues of Heigai pigs via upregulating the expression of the lipolytic gene (hormone-sensitive lipase, HSL) in vivo and in vitro. CLAs also altered the biochemical immune indexes including reduced content of total cholesterol (TChol), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) and changed lipids profiles including decreased sphingolipids especially ceramides (Cers) and sphingomyelins (SMs) in serum of Heigai pigs. Mechanically, CLAs may decrease peroxisome proliferator-activated receptorγ (PPARγ) expression and further inhibit adipogenic differentiation in adipose tissues of pigs by suppressing the function of Cers in serum. Furthermore, Pearson's correlation analysis showed HSL expression was positively related to short-chain fatty acids (SCFAs) in the gut (P ≤ 0.05) but the abundance of Cers was negatively related to the production and functions of SCFAs (P ≤ 0.05). CLAs altered the distribution of the lipid in serum and inhibited adipogenic differentiation by suppressing the function of Cers and further decreasing PPARγ expression in adipose tissues of Heigai pigs. Besides, the HSL expression and the abundance of Cers are associated with the production and functions of SCFAs in the gut.

Keywords: ceramide; conjugated linoleic acids; fat deposition; lipidome; pig.

Figure 1.

Morphological structure and the expression of lipid metabolism relative genes in adipose tissues of Heigai pigs after CLAs treatment. (A) Carcass weight after feeding CLAs. (B) Backfat thickness after CLAs treatment. (C) H&E staining of SAT sections from Control and CLAs pigs. Scale bars, 500 μm and 100 μm (n = 3). (D) Cell count and cell size percentage of SAT sections from control and CLAs pigs (n = 3). (E) The expression of lipid metabolism relative genes in SAT from control and CLAs pigs (n = 5). (F) Protein levels of FABP4 and PLIN1 were detected by western blot. (G) H&E staining of VAT sections from control and CLAs pigs. Scale bars, 500 μm and 100 μm (n = 3). (H) Cell count and cell size percentage of VAT sections from control and CLAs pigs (n = 3). Error bars represent SEM. ^*P < 0.05, ^**P < 0.01, two-tailed Student’s t-test.

Figure 2.

Changes in the overall lipid composition and distribution in the serum of Heigai pigs after feeding CLAs. (A) Distribution of lipid classes that were considered for subsequent analysis in all of the samples detected by LC‒MS/MS. (B) The content of total lipids in LDM of Heigai pigs. (C–H) The contents of glycerophospholipids (C), glycerolipids (D), sterol lipids (E), sphingolipids (F), fatty acyls (G), and prenol lipids (H) in LDM from control and CLAs pigs. Error bars represent SEM. ^*P < 0.05, two-tailed Student’s t-test.

Figure 3.

Comparison of Cers acyl chain composition between control and CLAs group. (A) The total intensity fold changes of individual fatty–acyl chains associated with Cers are sorted by degree of saturation. The transparency of each bar is proportional to the significance values, which are displayed as –Log10 (P-value). (B) Cers acyl chain content at different saturation levels. Box indicates IQR; whisker indicates min or max; plus shows mean. (C) Cers acyl chain percentage at different saturation levels. (D) Cers with different numbers of carbon atoms. (E) Cers with different numbers of double bond content. SFA, saturated fatty acyls; MUFA, monounsaturated fatty acyls; PUFA, polyunsaturated fatty acyls. n = 6. Error bars represent SEM.

Figure 4.

Comparison of SMs acyl chain composition between control and CLAs group. (A) The total intensity fold changes of individual fatty acyl chains associated with SMs are sorted by the degree of saturation. (B) The total intensity fold changes of odd-numbered fatty acyl chains associated with SMs. Odd, odd-numbered fatty acyls. The transparency of each bar is proportional to the significance values, which are displayed as –Log 10 (P-value). (C) SMs acyl chain content at different saturation levels. Box indicates IQR; whisker indicates min or max; plus shows mean. (D) SMs acyl chain percentage at different saturation levels. (E) SMs with different numbers of carbon atoms. (F) SMs with different numbers of double bond content. SFA, saturated fatty acyls; MUFA, monounsaturated fatty acyls; PUFA, polyunsaturated fatty acyls. n = 6. Error bars represent SEM. ^*P < 0.05, two-tailed Student’s t-test.

Figure 5.

Cer inhibited the adipogenic differentiation of primary SAT cells in vitro. (A) Oil red O staining of total lipids in differentiated SAT cells after treatment with control and CLAs. Scale bars, 100 μm. (B) OD490 levels of total lipids in differentiated SAT cells after different treatments (n = 4). (C) Differentiated SAT cells stained with Nile red and DAPI in different groups. Scale bars, 100 μm. (D) The expression of lipid metabolism relative genes in differentiated SAT cells in different groups (n = 5). (E) Protein levels of FABP4 were detected by western blot. (F) The expression of adipogenesis and lipolysis relative genes in differentiated SAT cells after 6 h lipolysis in different groups (n = 6). Error bars represent SEM. ^*P < 0.05, two-tailed Student’s t-test.

Figure 6.

Correlations among fatty acid, differential lipids, gene expression, and SCFAs in colonic digesta of Heigai pigs. (A) Correlations between fatty acid, differential lipids, and gene expression with SCFAs of Heigai pigs (n = 6). Each square represents a Pearson correlation coefficient between a genus and an index, while the gradation of color represents the size of each correlation coefficient. (B) The expression of adipogenesis and lipolysis relative genes in differentiated SAT cells after 6 h lipolysis after 1 mM sodium butyrate treatment (n = 6). (C) Scheme illustrating a working model of CLAs in regulating fat deposition in adipose tissue. CLAs altered the biochemical immune indexes and decreased lipid deposition in SAT. CLAs inhibited adipogenic differentiation by suppressing the function of Cers further decreasing PPARγ expression and promoting lipolysis through enhancing butyrate function in adipose tissues. CLAs, conjugated linoleic acids; Cer, ceramide; HDL-C, high-density lipoprotein; LDL-C, low-density lipoprotein; PPARγ, peroxisome proliferator-activated receptorγ; SAT, subcutaneous adipose tissue; SCFAs, short chain fatty acids; SFAs, saturated fatty acids; TChol, total cholesterol; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; UFAs, unsaturated fatty acids. Error bars represent SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, two-tailed Student’s t-test.