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Department of Animal Science, Cornell University, Ithaca, NY 14853
4To whom correspondence should be addressed. E-mail: deb6{at}cornell.edu.
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ABSTRACT |
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50% reduction of 14C-acetate incorporation into total lipid and corresponding reductions in mRNA abundance for acetyl CoA carboxylase, fatty acid synthase, and stearoyl CoA desaturase, whereas cis-9, trans-11 CLA had no effect on these genes. There was no reduction in SREBP-1 mRNA or precursor protein, but the abundance of the activated nuclear fragment of the protein was significantly reduced by treatment with 75 µmol/L trans-10, cis-12 CLA. These results indicate that trans-10, cis-12 CLA reduces lipid synthesis in the bovine mammary gland through inhibition of the proteolytic activation of SREBP-1 and subsequent reduction in transcriptional activation of lipogenic genes.
KEY WORDS: • CLA • lipid synthesis • nutritional genomics • SREBP-1
The lipid content and composition in milk varies across species and can be altered by dietary manipulation. One example of this is the low-fat milk syndrome in dairy cows, also referred to as milk fat depression (MFD).5 In MFD, diets high in concentrates and low in effective fiber or supplemented with polyunsaturated oils can cause a dramatic reduction in milk fat content (1). This phenomenon was causally related to the formation and absorption of trans-10, cis-12 conjugated linoleic acid (CLA) and possibly other unique fatty acids formed by rumen biohydrogenation. The trans-10, cis-12 isomer of CLA was also shown to affect lipid metabolism in many other models of both mammary and adipose metabolism, and a number of possible mechanisms were proposed (2).
Milk fat is composed predominantly of triglycerides containing fatty acids that arise from 2 sources, de novo synthesis within the mammary gland and the uptake of long-chain fatty acids from circulation (1). Recent studies involving diet-induced MFD and MFD induced by abomasal infusion of trans-10, cis-12 CLA indicate that all pathways involved in fatty acid and triglyceride synthesis are coordinately suppressed. There is a reduction in the synthesis and secretion of all fatty acid chain lengths and reduced mRNA abundance for genes of key enzymes in the pathways of milk fat synthesis (3–8). These results support the hypothesis that a central regulator of lipid synthesis is involved in MFD and a role for the sterol response element-binding protein (SREBP) family of transcription factors was postulated (6,9). The role of SREBP in the regulation of lipid metabolism was elegantly characterized in rodents (10,11). Gene promoters for key enzymes in the pathways of cholesterol and fatty acid synthesis and metabolism contain sterol response elements (SRE), and SREBP contributes to the coordinated regulation of these genes in the rodent liver (11,12). However, SREBPs have not been investigated in any bovine tissues or in mammary tissue of any species.
The first objective of the present study was to validate an in vitro model for analysis of the effects of trans-10, cis-12 CLA on lipogenesis in the bovine mammary gland. For this, we chose the MAC-T bovine mammary epithelial cell line (13) and investigated effects on lipogenesis as well as mRNA abundance for lipogenic enzymes. The second objective was to assess the potential for involvement of SREBP-1 in bovine mammary cells. We accomplished this by identifying SREBP-1 mRNA and protein, and by locating SREBP-related regulatory elements in the promoter region of the bovine fatty acid synthase (FAS) and stearoyl CoA desaturase (SCD) genes, which are critical for SREBP-1 regulation of these genes in rodents (14–16). The third objective was to evaluate the possible involvement of SREBP-1 in the inhibition of lipogenesis elicited by trans-10, cis-12 CLA in bovine mammary cells by analyzing effects on the abundance of SREBP-1 mRNA and precursor protein, and on the proteolytic activation of this transcription factor.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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lactose). After 48 h in serum-containing medium, cells were changed to lipid-free, serum-free medium (basal medium plus 1 g/L bovine serum albumin; BSA) supplemented with prolactin (2.5 mg/L) to promote lactogenesis. Cells were cultured in the lipid-free lactogenic medium for 24 h before the initiation of treatments. In all experiments, cells were treated with BSA-complexed fatty acid for the last 48 h of the experiment (1:3 mol/L ratio of BSA:fatty acid). Fatty acids were bound to BSA by a modification of the method of Ip and co-workers (18). Briefly, pure fatty acid was weighed into borosilicate tubes and mixed with a slight molar excess of NaOH (5 mol/L). The resulting sodium salts were diluted in warm (40°C) Medium 199 containing 10 g/L BSA to a final concentration of 4.5 mmol/L fatty acid (3:1 ratio with BSA) and sonicated with a probe for six 10-s bursts. The resulting solution was added to the lactogenic medium to attain the desired final concentration. Lipogenesis assay. Lipogenesis was determined in the cell cultures by quantifying the incorporation of 14C-labeled acetate into total lipid over the last 4 h of the fatty acid treatment period. Cells were cultured in 6-well plates. After 44 h of fatty acid treatment, 14C-labeled acetate was added to the wells at a final concentration of 1 mCi/L. After 4 h, cells were harvested and total lipid was extracted by vortex treatment of the cells in hexane:isopropanol (3:1) and washing the resultant solvent layer with sodium sulfate (150 mg/L) to remove any traces of propanol. An aliquot of the final solvent layer was dried under nitrogen before redissolving in 5 mL scintillation cocktail for quantification of label incorporation using a Packard 2200CA liquid scintillation analyzer (Packard Instrument). All lipogenesis experiments were replicated 3 times.
Northern blotting. Total RNA was extracted using the RNeasy kit (Qiagen). RNA was separated in an agarose-formaldehyde gel and transferred to nylon membranes by upward capillary transfer. Northern blots were performed by hybridization with 32P-labeled cDNA probes for ovine acetyl CoA carboxylase (ACC) and ovine SCD (courtesy of M. T. Travers and M. C. Barber, Hannah Research Institute, UK), ovine fatty acid synthase (FAS; courtesy of C. Leroux, LGBC-INRA, France), human sterol response element binding protein 1 (SREBP-1; ATCC) and ß-actin (Superarray). The expression of all analyzed genes was quantified with a Fujix Bio-Imaging Analyzer BAS 1000 phosphoimager (Fuji Medical Systems) and was normalized to the expression of ß-actin mRNA.
Western blotting. Western blots were performed using a monoclonal antibody raised against the amino terminal fragment of SREBP-1 (2A4; Santa Cruz Biotechnology). All protein extraction buffers included protease inhibitor cocktail (final concentration of protease inhibitors: 2.8 mg/L aprotinin, 25 mg/L N-acetyl-leucyl-leucyl-norleucinal, 10 mg/L leupeptin, 5 mg/L pepstatin A, and 0.5 mmol/L Pefabloc). Whole-cell extracts were prepared by the method of Fu et al. (19). Nuclear and membrane protein extracts used in Western blots were obtained from cells by the method of Hannah et al. (20), except that the final centrifugation step in the preparation of the membrane fraction was 100,000 x g for 30 min. Protein concentration was determined using the bicinchoninic acid assay (Pierce).
Gene promoter analysis. Partial sequences of the bovine FAS and SCD gene promoters were obtained from GenBank (accession numbers AF285607 and AY241932, respectively). We performed searches within the bovine genomic sequence for regulatory elements matching those that are critical for normal regulation of the FAS and SCD1 genes in rodents (14–16). For FAS, these included a Sp1 site (5'-TGGGCGGCGC-3') and tandem sterol regulatory elements (SREs; 5'-TCAGCCCATGTGGCGTGT-3'). For SCD, these included a SRE (5'-AGCAGATTGCG-3') and nuclear factor-Y (NF-Y) binding sites (5'-CCAAT-3').
Statistical analysis. Statistical analysis of mRNA and protein abundance was performed using Fisher’s Least Significant Difference pairwise comparisons in the general linear models procedure of SAS (SAS Institute). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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125 and 68 kDa, representing the membrane-bound precursor and activated nuclear fragments of SREBP-1, respectively (Fig. 3b).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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SREBP are synthesized as 125-kDa precursors that are retained in the endoplasmic reticulum until proteolytically cleaved to release the amino terminal fragment of 68 kDa that migrates to the nucleus to activate gene transcription [see reviews (10,11)]. Two genes are responsible for the production of the 3 isoforms of SREBP; SREBP-1a and 1c are transcribed from a single gene through the use of alternative start sites and are associated with fatty acid metabolism, whereas SREBP-2 is transcribed from a distinct gene and controls primarily cholesterol metabolism (10). Most methods to detect SREBP-1a or 1c protein or mRNA do not distinguish between them, and for the purposes of this study, they are referred to collectively as SREBP-1. To date, there has been no published demonstration of SREBP-1 presence in mammary cells of any species, or in any bovine tissue. There has also been no identification of the SREBP genes in the bovine genome. However, this regulatory pathway was well characterized in rodents and was implicated in the regulation of the entire program of lipid synthesis in the rodent liver (11).
In the present study, we established that the trans-10, cis-12 isomer of CLA reduced lipid synthesis in a bovine mammary epithelial cell line (MAC-T), and this decline corresponded to a reduction of mRNA abundance for ACC, FAS, and SCD of a similar magnitude. Based on the reduction in lipogenesis, we expect that protein levels for these enzymes were reduced to a similar degree, although this was not explicitly investigated in the present study. We analyzed the proximal promoter of the bovine FAS and SCD genes and found that they contain regulatory elements identical to those identified as critical for the SREBP regulation of these genes in rodents (14–16,21). We also established that the lactating bovine mammary gland and the MAC-T bovine mammary epithelial cell line contain SREBP-1 mRNA, and we confirmed the presence of SREBP-1 protein in the MAC-T cell line. Incubation of MAC-T cells with 75 µmol/L trans-10, cis-12 CLA for 48 h reduced mRNA abundance for critical genes in the lipid synthesis pathway (ACC, FAS, SCD), whereas cis-9, trans-11 CLA treatment had no effect. The coordinated reductions in mRNA for these lipogenic genes are consistent with observations in studies conducted with dairy cows involving administration of trans-10, cis-12 CLA (4) or diet-induced MFD (6–8). Further, in the present study, neither CLA isomer affected SREBP-1 mRNA or precursor protein (membrane associated, 125 kDa), whereas the abundance of the mature fragment of SREBP-1 protein (nuclear, 68 kDa) was significantly reduced by the trans-10, cis-12 CLA and not the cis-9, trans-11 CLA treatment.
SREBP-1 activity can be regulated at multiple levels. Downregulation of SREBP-1 activation can be mediated through a reduction in the amount of SREBP-1 mRNA available for translation into the precursor protein. This can be achieved either through reduced transcription of the SREBP-1 gene, or through accelerated decay of SREBP-1 mRNA. Xu et al. (22) suggested that PUFA reduce SREBP-1 activity by accelerating SREBP-1 transcript decay. Hannah et al. (20) suggested that unsaturated fatty acids suppress SREBP-1 activity at the level of SREBP-1 mRNA as well as by inhibiting the proteolytic cleavage that would release the activated nuclear SREBP-1 fragment. Our results indicate that in the short term (48 h), treatment with trans-10, cis-12 CLA affects the SREBP-1 pathway at the level of proteolytic cleavage rather than by affecting the availability of the precursor protein. Functional assays such as the chromatin immunoprecipitation assay or the use of reporter gene constructs would be useful in providing further support for this model. Because SREBP-1 gene expression is regulated in part by SREBP-1 itself (23), it is reasonable to expect that longer-term treatment with trans-10, cis-12 CLA would lead to a reduced abundance of SREBP-1 mRNA as well as precursor protein; however, our results would indicate that this is a secondary effect.
In the present study, we showed that a bovine mammary epithelial cell line expresses SREBP-1 and that coincident with reductions in lipid synthesis, proteolytic activation of SREBP-1 is affected by incubation with trans-10, cis-12 CLA. These results support the hypothesis that the in vivo effects of trans-10, cis-12 CLA on milk fat synthesis in dairy cows involve alterations in the activation of this transcription factor.
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FOOTNOTES |
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2 Supported in part by the National Research Initiative Competitive Grants Program, Cooperative State Research, Education, and Extension Service, USDA (#2003–35206–12819), USDA IFAFS (#2001–52100–11211), Northeast Dairy Foods Research Center and Cornell Agricultural Experiment Station.
3 Present address: Animal Science Department, California Polytechnic State University, San Luis Obispo, CA 93407.
5 Abbreviations used: ACC, acetyl-CoA carboxylase; BSA, bovine serum albumin; CLA, conjugated linoleic acid; FAS, fatty acid synthase; MFD, milk fat depression; NF-Y, nuclear factor-Y; SCD, stearoyl-CoA desaturase; SRE, sterol response element; SREBP, sterol response element-binding protein.
Manuscript received 21 June 2004. Initial review completed 7 July 2004. Revision accepted 15 July 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Bauman, D. E. & Griinari, J. M. (2003) Nutritional regulation of milk fat synthesis. Annu. Rev. Nutr. 23:203-227.[Medline]
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15. Tabor, D. E., Kim, J. B., Spiegelman, B. M. & Edwards, P. A. (1999) Identification of conserved cis-elements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase 1 and 2. J. Biol. Chem. 274:20603-20610.
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19. Fu, Y., Sies, H. & Lei, X. G. (2001) Opposite roles of selenium dependent glutathione peroxidase-1 in superoxide generator diquat- and peroxinitrite-induced apoptosis and signaling. J. Biol. Chem. 276:43004-43009.
20. Hannah, V. C., Ou, J., Luong, A., Goldstein, J. L. & Brown, M. S. (2001) Unsaturated fatty acids down-regulate SREBP isoforms 1a and 1c by two mechanisms in HEK-293 cells. J. Biol. Chem. 276:4365-4372.
21. Bene, H., Lasky, D. & Ntambi, J. M. (2001) Cloning and characterization of the human stearoyl-CoA desaturase gene promoter: transcriptional activation by sterol response element binding protein and repression by polyunsaturated fatty acids and cholesterol. Biochem. Biophys. Res. Commun. 284:1194-1198.[Medline]
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