t10,c12-CLA decreases adiposity in peripubertal mice without dose-related detrimental effects on mammary development, inflammation status, and metabolism

Abstract

The trans 10, cis 12-conjugated linoleic acid (10,12-CLA) isomer reduces adiposity in several animal models. In the mouse, however, this effect is associated with adipose tissue inflammation, hyperinsulinemia and hepatic lipid accumulation. Moreover, 10,12-CLA was recently shown to promote mammary ductal hyperplasia and ErbB2/Her2-driven mammary cancer in the mouse. Reasons for detrimental effects of 10,12-CLA on the mouse mammary gland could relate to its effect on the mammary fat pad (MFP), which is essential for normal development. Accordingly, we hypothesized that mammary effects of 10,12-CLA were mediated through the MFP in a dose-dependent manner. Female FVB mice were fed 10,12-CLA at doses of 0%, 0.1%, 0.2%, or 0.5% of the diet from day 24 of age, and effects on mammary development and metabolism were measured on day 49. The 0.5% dose reduced ductal elongation and caused premature alveolar budding. These effects were associated with increased expression of inflammatory markers and genes shown to alter epithelial growth (IGF binding protein-5) and alveolar budding (TNF-α and receptor of activated NF-κB ligand). The 0.5% dose also caused hyperinsulinemia and hepatic lipid accumulation. In contrast, the 0.1% 10,12-CLA dose had no adverse effects on mammary development, metabolic events, and inflammatory responses, but remained effective in decreasing adipose weights and lipogenic gene expression. These results show that a low dose of 10,12-CLA reduces adiposity in the mouse without negative effects on mammary development, inflammation, and metabolism, and suggest that previously reported detrimental effects relate to the use of excessive doses.

Fig. 1.

Effect of trans 10, cis 12-conjugated linoleic acid (10,12-CLA) dose on plasma concentrations of insulin, glucose, and adiponectin. Female FVB mice received 10,12-CLA at doses of 0%, 0.1%, 0.2%, or 0.5% of the diet from day 24 to 49 of age (n = 8 to 10 per treatment). Plasma was collected on day 49 of age. Each bar represents the mean ± SE of 5 to 9 mice. ^a,b,cBars with different letters are significantly different, P < 0.05.

Fig. 2.

Effect of 10,12-CLA dose on mammary development in FVB mice. Female FVB mice received 10,12-CLA at doses of 0, 0.1, 0.2, or 0.5% of the diet from day 24 to 49 of age (n = 8 to 10 per treatment). The right abdominal mammary gland was collected at 49 days of age. A: mammary gland was subjected to whole mount staining, and ductal length was quantified by measuring the distance from the center of the lymph node to the most distal duct. B: whole mounts were photographed at 2× and 10× magnification. In the 10× magnification panels, arrows correspond to terminal end buds, and arrowheads to alveolar-like structures. Representative images are shown. Right, top: number of budding structures was calculated for each animal as described in the materials and methods. Right, bottom: total RNA was extracted from the fourth mammary gland and analyzed by real-time PCR for keratin 18 mRNA abundance. Data were normalized to 18S expression. Each bar represents the mean ± SE of 8 to 10 mice. ^a,bBars with different letters are significantly different, P < 0.05.

Fig. 3.

Effect of 10,12-CLA dose on mammary gland expression of genes regulating ductal growth. Female FVB mice received 10,12-CLA at doses of 0, 0.1, 0.2, or 0.5% of the diet from day 24 to 49 of age (n = 8 to 10 per treatment). The left abdominal mammary gland was collected at 49 days of age. Total RNA was analyzed by real-time PCR for the mRNA abundance of indicated genes. A: estrogen receptor (ERα) and amphiregulin (ARG). B: IGF-I and IGF binding protein 5 (IGFBP5). C: chemokine monocyte chemoattractant protein-1 (MCP-1) and the macrophage marker EGF-like module containing mucin-like hormone receptor-like sequence 1 (EMR1), TNF-α, and IL-6. D: receptor of activated NF-κB (RANK) and RANK ligand (RANKL). Data were normalized to 18S expression except for genes expressed predominantly in the epithelium (ARG, RANK, and RANKL). Each bar represents the mean ± SE of 8 mice. ^a,bBars with different letters are significantly different, P < 0.05.

Fig. 4.

Effect of 10,12-CLA dose on mammary gland weight and expression of lipogenic genes. Female FVB mice received 10,12-CLA at doses of 0, 0.1, 0.2, or 0.5% of the diet from day 24 to 49 of age (n = 8 to 10 per treatment). A: mammary glands were collected from mice at 49 days of age and weighed. Each bar represents the mean ± SE of 8 to 10 mice. B: total RNA was extracted and analyzed by real-time PCR for mRNA abundance of sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FASN), and thyroid hormone responsive spot 14 (S14). Data are normalized to 18S expression. Each bar represents the mean ± SE of 8 mice. ^a,b,c,dBars with different letters are significantly different, P < 0.05.

Fig. 5.

Effect of 10,12-CLA dose on gonadal fat weight and expression of lipogenic and inflammatory genes. Female FVB mice received 10,12-CLA at doses of 0, 0.1, 0.2, or 0.5% of the diet from day 24 to 49 of age (n = 8 to 10 per treatment). Gonadal fat was collected at day 49 of age. Total RNA was extracted, and the expression of specific genes was analyzed by real-time PCR. A: mass of gonadal fat. Each bar represents the mean ± SE of 8 to 10 mice. B: expression of SREBP-1c, FASN, and S14. Data were normalized to 18S. Each bar represents the mean ± SE of 6 or 7 mice. C: expression of chemokine MCP-1, the macrophage marker EGF-like module containing EMR1, TNF-α, and IL-6. Data were normalized to 18S expression. Each bar represents the mean ± SE of 5 mice. ^a,b,cBars with different letters are significantly different, P < 0.05.