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Endogenous synthesis of n-3 polyunsaturated fatty acids in Fat-1 mice is associated with increased mammary gland and liver syndecan-1.

Sun H, Hu Y, Gu Z, Wilson MD, Chen YQ, Rudel LL, Willingham MC, Edwards IJ - PLoS ONE (2011)

Bottom Line: Fatty acid analysis of plasma lipoproteins showed that total n-6 PUFA reflected dietary intake similarly in both genotypes (VLDL, 36.2±2.2 and 40.9±3.9; LDL, 49.0±3.3 and 48.1±2.0; HDL, 54.6±1.2 and 58.2±1.3, mean ± SEM percent of total fatty acids for Fat-1 and wt animals respectively).Lipoprotein percent n-3 PUFA was also similar between groups.However, phospholipids and triglycerides extracted from mammary and liver tissues demonstrated significantly higher n-3 PUFA and a corresponding decrease in the ratio n-6/n-3 PUFA in Fat-1 compared to wt mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.

ABSTRACT
Long chain n-3 PUFA have been shown to have chemopreventive properties against breast cancer through various mechanisms. One pathway, studied in human breast cancer cell lines, involves upregulation of the proteoglycan, syndecan-1 (SDC-1) by n-3 PUFA-enriched LDL. Using Fat-1 mice that are able to convert n-6 to n-3 PUFA, we tested whether SDC-1 level in vivo is elevated in mammary glands due to endogenously synthesized rather than LDL-derived n-3 PUFA. Female Fat-1 and wild type (wt) mice were fed an n-6 PUFA- enriched diet for 7 weeks. Fatty acid analysis of plasma lipoproteins showed that total n-6 PUFA reflected dietary intake similarly in both genotypes (VLDL, 36.2±2.2 and 40.9±3.9; LDL, 49.0±3.3 and 48.1±2.0; HDL, 54.6±1.2 and 58.2±1.3, mean ± SEM percent of total fatty acids for Fat-1 and wt animals respectively). Lipoprotein percent n-3 PUFA was also similar between groups. However, phospholipids and triglycerides extracted from mammary and liver tissues demonstrated significantly higher n-3 PUFA and a corresponding decrease in the ratio n-6/n-3 PUFA in Fat-1 compared to wt mice. This was accompanied by higher SDC-1 in mammary glands and livers of Fat-1 mice, thus demonstrating that endogenously synthesized n-3 PUFA may upregulate SDC-1 in the presence of high dietary n-6 PUFA.

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Expression of proteoglycans in tissues of Fat-1 and wild type (wt) mice.A. transcript levels of proteoglycans in mammary gland. B, transcript levels of SDC-1 in liver. Total RNA was isolated from tissues and mRNAs were measured by real-time RT-PCR. Numbers represent the level of expression relative to that of a housekeeping gene, peptidyl prolyl isomerase B (PPIB) using triplicate samples of each animal (n = 5). The expression of SDC-4 relative to PPIB in mammary tissue was <0.001 and similar between Fat-1 and wt tissues. C and D. Western analysis using a SDC-1 core protein-specific antibody of heparinase-treated protein extracts of C, mammary tissues and D, liver. Bars are means ± SEM (n = 5) of SDC-1 protein adjusted to β actin. Insert shows representative samples of wt and Fat-1bands. * P<0.05 Fat-1 versus wt by Student's t test.
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pone-0020502-g002: Expression of proteoglycans in tissues of Fat-1 and wild type (wt) mice.A. transcript levels of proteoglycans in mammary gland. B, transcript levels of SDC-1 in liver. Total RNA was isolated from tissues and mRNAs were measured by real-time RT-PCR. Numbers represent the level of expression relative to that of a housekeeping gene, peptidyl prolyl isomerase B (PPIB) using triplicate samples of each animal (n = 5). The expression of SDC-4 relative to PPIB in mammary tissue was <0.001 and similar between Fat-1 and wt tissues. C and D. Western analysis using a SDC-1 core protein-specific antibody of heparinase-treated protein extracts of C, mammary tissues and D, liver. Bars are means ± SEM (n = 5) of SDC-1 protein adjusted to β actin. Insert shows representative samples of wt and Fat-1bands. * P<0.05 Fat-1 versus wt by Student's t test.

Mentions: SDC-1 is up-regulated when mammary cells are treated in vitro with n-3 PUFA [20], [22]. We investigated whether the modest increase in n-3 PUFA measured in the mammary tissues of the Fat-1 mice was sufficient to modify the expression of SDC-1 in vivo. As shown in Fig. 2A, mRNA for SDC-1 was 50% higher in mammary tissue of Fat-1 compared to wt mice. This effect was specific for SDC-1 since expression of the other proteoglycans, SDC-4, perlecan, decorin and biglycan were similar between Fat-1 and wt tissues (Fig. 2A). It is of interest to note that expression of the chondroitin sulfate proteoglycan, versican, which is generally thought to have a tumor-promoting effect, trended lower (p = 0.057) in the n-3 PUFA-enriched Fat-1 tissues. No previous studies have reported regulation of versican by n-3 PUFA. To determine whether SDC-1 expression was elevated in tissues other than mammary gland of Fat-1 mice, we examined liver where SDC-1 is known to function in the clearance of triglyceride-rich lipoprotein remnants [38]. As shown in Fig. 2B, SDC-1 mRNA was > two-fold higher in the livers of Fat-1 compared to wt animals. To confirm that the increased SDC-1 mRNA in Fat-1 tissues was translated into increased protein, Western analysis of tissue protein extracts demonstrated a six-fold higher SDC-1 protein level in mammary tissue (Fig. 2C) and a 50% higher SDC-1 protein in liver (Fig. 2D) of Fat-1 compared to wt mice. Finally, since whole mount preparations of mammary glands of Fat-1 mice were previously shown to display a more differentiated phenotype than wt animals [39], we examined SDC-1 distribution in the mammary tissues (Fig. 3). Immunostaining for SDC-1, localized primarily to epithelial components, was consistently more intense in Fat-1 than in wt tissues but no significant morphological differences were observed between the genotypes.


Endogenous synthesis of n-3 polyunsaturated fatty acids in Fat-1 mice is associated with increased mammary gland and liver syndecan-1.

Sun H, Hu Y, Gu Z, Wilson MD, Chen YQ, Rudel LL, Willingham MC, Edwards IJ - PLoS ONE (2011)

Expression of proteoglycans in tissues of Fat-1 and wild type (wt) mice.A. transcript levels of proteoglycans in mammary gland. B, transcript levels of SDC-1 in liver. Total RNA was isolated from tissues and mRNAs were measured by real-time RT-PCR. Numbers represent the level of expression relative to that of a housekeeping gene, peptidyl prolyl isomerase B (PPIB) using triplicate samples of each animal (n = 5). The expression of SDC-4 relative to PPIB in mammary tissue was <0.001 and similar between Fat-1 and wt tissues. C and D. Western analysis using a SDC-1 core protein-specific antibody of heparinase-treated protein extracts of C, mammary tissues and D, liver. Bars are means ± SEM (n = 5) of SDC-1 protein adjusted to β actin. Insert shows representative samples of wt and Fat-1bands. * P<0.05 Fat-1 versus wt by Student's t test.
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pone-0020502-g002: Expression of proteoglycans in tissues of Fat-1 and wild type (wt) mice.A. transcript levels of proteoglycans in mammary gland. B, transcript levels of SDC-1 in liver. Total RNA was isolated from tissues and mRNAs were measured by real-time RT-PCR. Numbers represent the level of expression relative to that of a housekeeping gene, peptidyl prolyl isomerase B (PPIB) using triplicate samples of each animal (n = 5). The expression of SDC-4 relative to PPIB in mammary tissue was <0.001 and similar between Fat-1 and wt tissues. C and D. Western analysis using a SDC-1 core protein-specific antibody of heparinase-treated protein extracts of C, mammary tissues and D, liver. Bars are means ± SEM (n = 5) of SDC-1 protein adjusted to β actin. Insert shows representative samples of wt and Fat-1bands. * P<0.05 Fat-1 versus wt by Student's t test.
Mentions: SDC-1 is up-regulated when mammary cells are treated in vitro with n-3 PUFA [20], [22]. We investigated whether the modest increase in n-3 PUFA measured in the mammary tissues of the Fat-1 mice was sufficient to modify the expression of SDC-1 in vivo. As shown in Fig. 2A, mRNA for SDC-1 was 50% higher in mammary tissue of Fat-1 compared to wt mice. This effect was specific for SDC-1 since expression of the other proteoglycans, SDC-4, perlecan, decorin and biglycan were similar between Fat-1 and wt tissues (Fig. 2A). It is of interest to note that expression of the chondroitin sulfate proteoglycan, versican, which is generally thought to have a tumor-promoting effect, trended lower (p = 0.057) in the n-3 PUFA-enriched Fat-1 tissues. No previous studies have reported regulation of versican by n-3 PUFA. To determine whether SDC-1 expression was elevated in tissues other than mammary gland of Fat-1 mice, we examined liver where SDC-1 is known to function in the clearance of triglyceride-rich lipoprotein remnants [38]. As shown in Fig. 2B, SDC-1 mRNA was > two-fold higher in the livers of Fat-1 compared to wt animals. To confirm that the increased SDC-1 mRNA in Fat-1 tissues was translated into increased protein, Western analysis of tissue protein extracts demonstrated a six-fold higher SDC-1 protein level in mammary tissue (Fig. 2C) and a 50% higher SDC-1 protein in liver (Fig. 2D) of Fat-1 compared to wt mice. Finally, since whole mount preparations of mammary glands of Fat-1 mice were previously shown to display a more differentiated phenotype than wt animals [39], we examined SDC-1 distribution in the mammary tissues (Fig. 3). Immunostaining for SDC-1, localized primarily to epithelial components, was consistently more intense in Fat-1 than in wt tissues but no significant morphological differences were observed between the genotypes.

Bottom Line: Fatty acid analysis of plasma lipoproteins showed that total n-6 PUFA reflected dietary intake similarly in both genotypes (VLDL, 36.2±2.2 and 40.9±3.9; LDL, 49.0±3.3 and 48.1±2.0; HDL, 54.6±1.2 and 58.2±1.3, mean ± SEM percent of total fatty acids for Fat-1 and wt animals respectively).Lipoprotein percent n-3 PUFA was also similar between groups.However, phospholipids and triglycerides extracted from mammary and liver tissues demonstrated significantly higher n-3 PUFA and a corresponding decrease in the ratio n-6/n-3 PUFA in Fat-1 compared to wt mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.

ABSTRACT
Long chain n-3 PUFA have been shown to have chemopreventive properties against breast cancer through various mechanisms. One pathway, studied in human breast cancer cell lines, involves upregulation of the proteoglycan, syndecan-1 (SDC-1) by n-3 PUFA-enriched LDL. Using Fat-1 mice that are able to convert n-6 to n-3 PUFA, we tested whether SDC-1 level in vivo is elevated in mammary glands due to endogenously synthesized rather than LDL-derived n-3 PUFA. Female Fat-1 and wild type (wt) mice were fed an n-6 PUFA- enriched diet for 7 weeks. Fatty acid analysis of plasma lipoproteins showed that total n-6 PUFA reflected dietary intake similarly in both genotypes (VLDL, 36.2±2.2 and 40.9±3.9; LDL, 49.0±3.3 and 48.1±2.0; HDL, 54.6±1.2 and 58.2±1.3, mean ± SEM percent of total fatty acids for Fat-1 and wt animals respectively). Lipoprotein percent n-3 PUFA was also similar between groups. However, phospholipids and triglycerides extracted from mammary and liver tissues demonstrated significantly higher n-3 PUFA and a corresponding decrease in the ratio n-6/n-3 PUFA in Fat-1 compared to wt mice. This was accompanied by higher SDC-1 in mammary glands and livers of Fat-1 mice, thus demonstrating that endogenously synthesized n-3 PUFA may upregulate SDC-1 in the presence of high dietary n-6 PUFA.

Show MeSH
Related in: MedlinePlus