Synthesis of long-chain polyunsaturated fatty acids in lactating mammary gland: role of D5 and D6 desaturases, SREBP-1, PPARa, and PGC-1

The purpose of this work was to study whether rat lactating mammary gland can synthesize PUFAs through the expression of D5 and D6 desaturases (D5D and D6D), whether these enzymes are regulated by the transcription factors sterol-regulatory element binding protein 1 (SREBP-1) and peroxisome proliferator-activated receptor a (PPARa) and the coactivator peroxisome proliferator-activated receptor g coactivator 1b (PGC-1b), and whether these desaturases are regulated by the lipid concentration in the diet. The results showed that on day 12 of lactation,z35% of the linoleic acid in the diet, which is the precursor of PUFAs, is transferred to the mammary gland. There was expression of D5D andD6D inmammary gland, and it was regulated by the corn oil content in the diet. The higher the corn oil content in the diet, the lower the expression of both desaturases. Induction of D5D and D6D was associated positively with similar changes in SREBP-1 and PGC-1b. Expression of PPARa was barely detected and was not affected by the corn oil content in the diet, whereas PGC-1b expression increased as the corn oil in the diet increased. These results indicate that the lactating mammary gland has the capacity to synthesize PUFAs and can be regulated by the lipid content in the diet.—Rodriguez-Cruz, M., A. R. Tovar, B. PalaciosGonzález, M. del Prado, and N. Torres. Synthesis of longchain polyunsaturated fatty acids in lactating mammary gland: role ofD5 andD6 desaturases, SREBP-1, PPARa, and PGC-1. J. Lipid Res. 2006. 47: 553–560. Supplementary key words sterol-regulatory element binding protein 1 . peroxisome proliferator-activated receptor a . peroxisome proliferator-activated receptor g coactivator 1 D5 and D6 desaturases (D5D and D6D) are microsomal enzymes that form part of a three enzyme system that includes NADH-cytochrome b5 reductase, cytochrome b5, and D6D or D5D (1). Mammals are unable to synthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from acetyl-CoA; thus, two PUFAs, linoleic acid (LA; 18:2 n-6) and a-linolenic acid (18:3 n-3), called essential fatty acids, must be supplied from the diet. Mammals are able to synthesize LC-PUFAs from their precursor PUFAs. Fatty acid desaturases introduce a double bond in a specific position of long-chain fatty acids and are required for the synthesis of highly unsaturated fatty acids (LC-PUFAs). The conversion of LA and a-linolenic acid to g-linolenic acid (18:3 n-6) and stearidonic acid (18:4 n-3) is determined by the enzymatic activity of D6D. After desaturation and elongation by D6D and elongase, respectively, D5D introduces another double bond at the D5 position of the 20 carbon fatty acids 20:3 n-6 and 20:4 n-3 to synthesize 20:4 n-6 (arachidonic acid; AA) and 20:5 n-3 (eicosapentanoic acid), respectively. The highly unsaturated fatty acids are mainly esterified into phospholipids and contribute to the maintenance of membrane fluidity (2). These desaturases are expressed in liver, heart, and brain and are regulated by hormonal and nutritional manipulation in rodents (3, 4). In addition, studies in rats indicate that hepatic D6D mRNA expression and enzymatic activity are highly dependent upon the composition of dietary fat. The ingestion of diets low in essential fatty acids or LC-PUFAs results in high levels of D6D enzyme activity and mRNA expression (5, 6). Diets high in PUFAs are negative regulators of lipogenesis that exert their effects primarily at the level of transcription. Two transcription factors, sterol-regulatory element binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor a (PPARa), are transcription factors that play a key role in the regulation of desaturases by PUFAs in liver. SREBP-1c is synthesized as a larger precursor protein that is anchored to the endoplasmic reticulum. After proteolytic cleavage, the Manuscript received 13 September 2005 and in revised form 10 November 2005. Published, JLR Papers in Press, December 6, 2005. DOI 10.1194/jlr.M500407-JLR200 1 To whom correspondence should be addressed. e-mail: [email protected] Copyright D 2006 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 47, 2006 553 by gest, on O cber 3, 2017 w w w .j.org D ow nladed fom N-terminal domain migrates to the nucleus and activates target genes by binding to sterol-regulatory elements (2). Expression of D6D and D5D mRNA is positively regulated by SREBP-1 in liver as a result of the presence of a sterolregulatory element in the promoter region of D6D and probably D5D (7, 8). On the other hand, PPARa is a member of the steroid receptor family that contains a hydrophobic ligand binding site and a DNA binding domain. Binding of a ligand causes a conformational change of PPARa, which then forms a heterodimer with retinoid X receptor and binds to peroxisome proliferator response elements located in the promoter region of the target genes. Hypolipidemic compounds called peroxisome proliferators, such as fibrates, which are ligands of PPARa, induce the expression of enzymes involved in fatty acid oxidation as well as hepatic D5D and D6D (7). The increase in expression of D6D is associated with the presence of peroxisome proliferator response elements in the promoter region of this gene (9). In addition, these transcription factors are regulated by specific coactivators that enhance or repress their activity. The family of peroxisome proliferator-activated receptor g coactivators (PGC1) is involved in the expression of multiple genes that are regulated by SREBP-1 and PPARa. There are two members of the PGC-1 family, PGC-1a and PGC-1b. PGC-1a is associated with the activation of fatty acid oxidation, whereas PGC-1b is associated with lipogenesis, among other functions. During lactation, there is an increase in the use of metabolic fuels to sustain high rates of lipid and protein synthesis. In the lactating mammary gland, there is a high demand of the LC-PUFAs docosahexanoic acid (DHA) and AA to incorporate them into the milk. These LCPUFAs are essential for the normal growth and development of the brain and retina of the newborn. Lactating mothers lose z70–80 mg DHA/day in breast milk, in addition to the amount lost by oxidation or used to fulfill the mother’s own requirement (10). Thus, the maternal organism synthesizes them when they are not provided by dietary lipids. Studies in Mexican women with low intake of AA showed adequate levels of AA in their milk (11), suggesting that during lactation, there are some metabolic adaptations to synthesize adequate amounts of LC-PUFAs. However, it is not known whether these adaptations include the participation of extra hepatic tissues, such as mammary gland and/or adipose tissue, to synthesize LCPUFAs by regulating the expression of D5D and D6D. The purpose of the present study was to investigate the presence of D6D and D5D mRNA in mammary gland and whether or not this expression was regulated by dietary LA present in corn oil (55% LA). In addition, we explored the possible mechanism of regulation of these desaturases by SREBP-1 and PPARa during lactation. Our results indicate that D6D and D5D mRNAs are present in lactating mammary gland, liver, and adipose tissue. Also, Northern blot analysis showed that a low corn oil diet increased the mRNA abundance of these desaturases in a tissue-specific manner in liver and lactating mammary gland but not in adipose tissue. Furthermore, our results indicate that expression of these desaturases was regulated by dietary LA only in mammary gland and liver. This effect could be attributable in part to SREBP-1, because SREBP-1 mRNA expression followed the same pattern of expression of D5D and D6D and was also regulated by the presence of LA in the diet. The higher the LA content in the diet, the lower the expression of SREBP-1 in liver and mammary gland. Contrary to that, liver PPARa mRNA increased with the LA content in the diet, and there was little expression and no change of PPARa in mammary gland and adipose tissue. These results indicate that mammary gland participates in the synthesis of LC-PUFAs by increasing D6D and D5D expression when the LA content in the diet is low through reduction in the expression of SREBP-1, whereas in situations in which the presence of corn oil in the diet is high, there is an inhibition of D5D, D6D and SREBP-1 and an increase in the fatty acid oxidation through an increase in PPARa expression. MATERIALS AND METHODS Female Sprague-Dawley rats (8 weeks old) obtained from the Animal Care Facilities at the Centro Médico Nacional, Instituto Mexicano del Seguro Social, were housed at 22 6 28C with a 12 h light/dark cycle and free access to water. From weaning until 8 weeks of age, rats were fed a commercial diet (Purina, Guadalajara, México). When rats reached 8 weeks old, they were randomly assigned and adapted to one of the specific diets described below for 6 weeks before mating. When animals reached 14 weeks of age (220–280 g), they were mated. Pregnant females were housed individually and continued on the same diet until day 12 of lactation. The day of birth was considered day 0 of lactation, and litters were weighed and adjusted to eight pups per dam. No gender differentiation was done. Food intake and body weight were assessed daily. The protocol was approved by the Animal Care Committee of the Instituto Mexicano del Seguro Social.