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Genomics, Proteomics, and Metabolomics

ACSL1, AGPAT6, FABP3, LPIN1, and SLC27A6 Are the Most Abundant Isoforms in Bovine Mammary Tissue and Their Expression Is Affected by Stage of Lactation^1–3,

Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801

^* To whom correspondence should be addressed. E-mail: jloor{at}uiuc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The lactating bovine mammary gland is a formidable triacylglycerol-synthesizing machine and, as such, represents an ideal model for studying putative functions of distinct isoforms of solute carrier family 27 transporters [(SLC27A) 1, 2, 3, 5, 6], long chain acyl-CoA synthetases [(ACSL) 1, 3, 4, 5, 6], fatty acid binding proteins [(FABP) 1, 3, 4, 5, 6], 1-acylglycerol-3-phosphate O-acyltransferases [(AGPAT) 1, 2, 3, 4, 5, 6, 7, 8], and lipins [(LPIN) 1, 2, 3]. The relative percentage of mRNA abundance and fold-changes in the expression of isoforms in mammary tissue from 6 cows each at –15, 15, 60, and 240 d relative to parturition were analyzed using quantitative PCR. Transcripts of FABP isoforms were most abundant, accounting for 78% of the 28 genes measured, and SLC27A isoforms were least abundant (<0.5% of genes measured). mRNA of AGPAT, ACSL, and LPIN accounted for 12, 7, or 2%, respectively, of all genes measured. The mRNA abundance at 60 d postpartum for FABP3, ACSL1, AGPAT6, and LPIN1 was 80-, 7-, 15-, and 20-fold greater relative to –15 d. Transcripts of these isoforms constituted the most abundant within each specific gene family. SLC27A2, SLC27A5, and SLC27A6 had peak expression at 240, 240, or 15 d relative to parturition, respectively. Results suggest that SLC27A6, ACSL1, FABP3, AGPAT6, and LPIN1 coordinately regulate the channeling of fatty acids toward copious milk fat synthesis in bovine mammary.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Among the key proteins involved in TG synthesis, several FABP isoforms were identified in the early 1980s (3). Other isoform families, such as ACSL, have received increased attention only recently (4). There are many definitions of "isoform", but from a transcriptional standpoint (i.e., mRNA), an isoform refers to a protein variant produced either as a result of different genes or from differential processing of a single gene transcript (i.e., splice variant). Even if isoform proteins are highly similar and have a conserved functional domain, both the specific cellular localization as well as the differences in affinity for substrates can play an important role in determining the fate of fatty acids (FA), e.g., esterification vs. β-oxidation. This has been demonstrated for ACSL isoforms (5). Divergent tissue expression patterns also are suggestive of functional specificity, as has been highlighted for FABP and SLC27A isoforms (6,7). Isoforms of LPIN and AGPAT have been characterized recently and, for both, diverse biological functions of the different isoforms have been suggested (8,9).

To our knowledge, expression patterns of isoforms associated with pathways of TG synthesis in bovine mammary have not been studied. The formidable capacity of bovine mammary to synthesize fat during lactation (i.e., 25–30% total milk solids) makes it an optimal model to study putative functions of isoforms. Regulation of the above-mentioned gene families occurs almost exclusively at the gene expression level (4,6–9). Furthermore, ACSL (4), FABP (6), and AGPAT (8) isoform expression is under the control of PPAR in several tissues. In addition, PPAR coactivator 1 regulates the expression of LPIN1 (10). The expression of SLC27A1 appears to be under the control of resistin and leptin in the mouse heart (7). Thus the measurement of isoform mRNA expression in mammary tissue will provide valid information to aid in generating hypotheses for more detailed functional studies. Our objective was to characterize longitudinal expression profiles and relative mRNA abundance among isoforms of ACSL, AGPAT, FABP, LPIN, and SLC27A in bovine mammary tissue throughout the lactation cycle.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Animals, sampling, and diet. Details of the experimental design were reported previously (11). Briefly, Holstein dairy cows (n = 6, 2nd or greater lactation) averaged 9434 ± 1328 kg milk/lactation, 3.7 ± 0.4% fat in milk/lactation, and 343 ± 57 kg fat/lactation. Percutaneous biopsies from each of the 6 cows were obtained from the right or left rear quarter of the mammary gland at –15 (–13 ± 3), 15, 60, and 240 d relative to parturition. During lactation, cows were fed a typical diet composed of (g/kg dry matter) 281 corn silage, 201 alfalfa silage, 97 cottonseed, and 421 concentrate [composed of 618 corn, 143 mechanically extracted soybean meal, 122 soybean meal, 34 soy hulls, 33 limestone, 23 NaHCO₃, 12 CaHPO₄, 3 MgO, 10 other salts, and 1 vitamin E (containing 3.03 g/kg dl--tocopherol)]. Diet composition was kept constant throughout the entire lactation and adjusted weekly based on dry matter content. All procedures were conducted under protocols approved by the University of Illinois Institutional Animal Care and Use Committee.

    RNA extraction, PCR, and primer design and evaluation. Specific details of these procedures are presented in the Supplemental Materials and Methods, Supplemental Table 1 (primer sequence and features), Supplemental Table 2 (edited amplicon sequencing for each primer pair), and Supplemental Table 3 (best BLAST hit for amplicon sequencing).

    Data processing and statistical analysis. Normalized quantitative real-time RT-PCR (qPCR) data are presented as fold-change relative to –15 d. To estimate standard errors at –15 d and prevent biases in statistical analysis, normalized data were transformed to obtain a perfect mean of 1.0 at –15 d, leaving the proportional difference between the biological replicate. The same proportional change was calculated at all other time points to obtain a fold-change relative to –15 d. This final dataset was analyzed using a mixed model with repeated measures in SAS (release 8.0; SAS Inst.) to evaluate the effect of time relative to parturition on gene expression. Compound symmetry was the most appropriate covariate structure used for repeated measures analysis. The model included the fixed effect of time (–15, 15, 60, and 240 d relative to parturition) and the random effect of cow. The PDIFF statement (pairwise difference) with Tukey adjustment was used as a post hoc test. Temporal fold-change data (Fig. 1) are reported as means with pooled SEM. Significance was declared at P < 0.05.

View larger version (13K): [in this window] [in a new window]   FIGURE 1  Patterns of SLC27A (or FATP), ACSL, FABP, LPIN, and AGPAT mRNA isoform expression in bovine mammary tissue throughout the lactation cycle. Values are means, n = 6, and are expressed as fold of d –15 (late pregnancy), which was set to 1.0. Pooled SEM are SLC27A1, 0.12; SLC27A2, 4.73; SLC27A3, 0.09; SLC27A5, 0.76; SLC27A6, 1.25; ACSL1, 0.61; ACSL3, 0.14; ACSL4, 0.10; ACSL5, 0.16; ACSL6, 1.94; FABP1, 0.82; FABP3, 6.18; FABP4, 0.75; FABP5, 0.37; FABP6, 0.19; LPIN1, 3.9; LPIN2, 0.09; LPIN3, 0.12; AGPAT1, 0.82; AGPAT2, 0.27; AGPAT3, 0.28; AGPAT4, 0.11; AGPAT5, 0.11; AGPAT6, 1.24; AGPAT7, 0.17; AGPAT8, 0.23. * Different from d –15, P < 0.05.

Ct

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Milk production and fat yield. Milk production was 32.1, 37.6, and 29.7 kg/d (SEM = 2.6), respectively, on 15, 60, and 240 d postpartum. In addition, milk fat yield was 1.42 (4.4% of milk), 1.61 (4.3%), and 1.24 (4.2%) kg of milk fat/d (SEM = 0.15), respectively, on 15, 60, and 240 d postpartum.

    Protein and nucleotide similarity among bovine isoforms. Bovine nucleotide and protein isoform alignment results are reported in Supplemental Table 5 (description of each isoform), Supplemental Table 6 (accession number and length of nucleotide and protein), and Supplemental Table 7 (percentage nucleotide/amino acid identity).

    SLC27A mRNA abundance and expression pattern. Overall, SLC27A isoform mRNA abundance represented 1% of genes measured (Table 1). Very little information exists regarding SLC27A isoform function, activity, or expression in bovine. Except for SLC27A1, which has been characterized (13), annotations of other bovine SLC27A mRNA sequences in this study were predicted (Supplemental Table 6). During lactation, mRNA for SLC27A6 was predominant, accounting for >80% of all SLC27A isoforms (Table 1), followed by mRNA of SLC27A1, which ranged from 13 to 50% of all SLC27A isoforms. SLC27A5 mRNA was detected in the lowest abundance (3 to 5%). Other isoforms were present in trace amounts and isoform 4 was undetectable (Table 1). Despite these differences, mRNA expression of SLC27A 2 and 5 increased significantly throughout lactation to peak values at 240 d (Fig. 1). SLC27A6 mRNA peaked at 15 d (P < 0.05 vs. –15 d) and then declined gradually to prepartum values by 240 d.

    FABP mRNA abundance and expression pattern. Mammary tissue had a high mRNA abundance of FABP3, FABP4, and FABP5. The remaining isoforms were barely detectable and mRNA of FABP2 was undetectable (Table 1). Among isoforms, mRNA of FABP3 was predominant (>75%) during lactation (Table 1). FABP3 expression was tremendously upregulated during the transition from the nonlactating period to lactation (>40-fold at 15 d; Fig. 1) and, along with FABP4, was the most abundant mRNA of those under investigation. Expression of FABP4 and 5 (Fig. 1) was upregulated significantly during lactation.

    AGPAT isoform mRNA abundance and expression pattern. AGPAT6 was the most abundant isoform, accounting for 60% of all AGPAT mRNA, followed by AGPAT1 and AGPAT3 (18 and 10%, respectively, of AGPAT isoforms; Table 1). Other AGPAT isoforms had lower mRNA abundance but all were detectable. AGPAT6 expression (Fig. 1) was the most affected by stage of lactation (>14-fold at 60 vs. –15 d), followed by AGPAT1 (>4-fold), AGPAT8 (>1-fold), AGPAT7 (>0.5-fold), and AGPAT3 (0.3-fold), all statistically significant.

    LPIN mRNA abundance and expression pattern. A total of 2 LPIN1 variants (a and b) with distinct subcellular localization, tissue expression, and function have been discovered in mouse (14). However, to our knowledge, at the present time there are no bovine sequences that distinguish between these isoforms. Thus present data should be regarded as the combination of the 2 LPIN1 variants. Mammary tissue expressed all 3 LPIN isoforms, with mRNA of LPIN2 predominating during the end of pregnancy (70% of all LPIN) and mRNA of both LPIN2 (30 to 47%) and LPIN1 (33 to 63%) predominating during lactation (Table 1). Expression of LPIN1 and LPIN2 was affected by lactation (P < 0.05) in this study, but mRNA of LPIN1 had a larger and significant increase (>19-fold at 60 vs. –15 d) compared with mRNA of LPIN2 (0.3-fold) (Fig. 1). LPIN3 expression increased numerically during late lactation (0.4-fold at 240 vs. –15 d).

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Role of SLC27A isoforms in FA uptake by mammary cells. The predominant route of long chain FA (LCFA) transport into cells is via a saturable, protein-mediated mechanism (15). FATP (or soluble carrier protein 27A) is a family of 6 related proteins encoded by 6 different genes (SLC27A1–6) (7). SLC27A family members translocate LCFA across the plasma membrane and also have acyl-CoA activity (7). The mechanism of LCFA translocation remains unclear (7); however, evidence of LCFA uptake capacity has been demonstrated for SLC27A isoforms 1, 2, 4, and 6 (7).

Isoform 6, the predominant isoform in this gene family, was the only SLC27A with a dramatic increase (5-fold) in mRNA during the transition from late prepartum (–15 d) through lactation (15 d), followed by a gradual decrease thereafter (Fig. 1). In humans, SLC27A6 is expressed primarily in the heart, is localized in the sarcolemma juxtaposed to blood vessels, and has a high affinity for palmitic acid and oleic acid (7) (Supplemental Table 8). Mammary tissue SLC27A6 expression has not been previously evaluated. Other isoforms increased during lactation to a peak at 240 d postpartum (i.e., late lactation), with expression of SLC27A2 having a >20-fold response vs. –15 d (Fig. 1). Unlike bovine, murine mammary tissue had a 2-fold upregulation of SLC27A3 mRNA at 9 d of lactation (1), suggesting species differences in expression and function during mammary TG synthesis (Table 2).

    ACSL isoforms and FA channeling toward milk TG synthesis. ACSL activates FA through an ATP-requiring process prior to entry into different intracellular metabolic pathways (17). ACSL proteins have been well characterized in mouse, rat, and human (5,18–20) but no data are available for cow. Among bovine ACSL isoforms, only isoform 5 and 6 have been completely annotated (21). Others with a full mRNA sequence have been annotated based on similarity with the human, rat, and mouse orthologs [(21,22); Supplemental Table 6].

Our data showed mRNA of ACSL1 as predominant among acyl-CoA synthetase isoforms in bovine mammary tissue, with medium-to-high overall mRNA abundance compared with other genes examined (Table 1). Rat ACSL1 prefers palmitic acid > lauric > myristic > oleic acid (Supplemental Table 8). Palmitic acid and oleic acid constitute 2 abundant FA in bovine blood plasma (23) and both are major constituents of TG in bovine milk. Palmitic acid also is the main product of FA synthase in all species (24). Newly synthesized FA require activation to acyl-CoA before they can be metabolized or inserted into lipid droplets. In rat primary hepatocytes (25), murine liver (26) and adipose tissue (5), overexpression of ACSL1 resulted in the channeling of LCFA toward TG synthesis. The channeling of LCFA toward TG synthesis also is supported by specific localization of ACSL1 in the plasma membrane, endoplasmic reticulum, and the mitochondria-associated membrane (4) (Table 2).

    FABP isoforms and mammary intracellular LCFA trafficking. FABP are among the main proteins allowing for rapid diffusion and selective targeting of LCFA toward specific organelles for metabolism (27). They also bind LCFA CoAs, as demonstrated for the liver-specific FABP (28). Our data confirm the predominance of FABP3 mRNA in cow mammary as found in mouse (1) and other species (6). Expression of FABP3, FABP4, and FABP5, the most abundant among FABP isoforms, was upregulated by the onset of lactation and had large increases (1- to 78-fold) relative to prepartum levels (Fig. 1).

Based partly on mRNA abundance, previous authors have proposed that FABP isoforms channel FA toward specific metabolic pathways (6). Our data clearly point to a major role of FABP3, and a minor role of FABP4 and FABP5, in bovine mammary lipid synthesis. We suggest that FABP3, in cooperation with ACSL1 and SLC27A6, channel LCFA toward esterification into milk TG. This proposal is supported by the physical association and coexpression of FABP3 with CD36 (29), as well as the large binding affinity of FABP3 for palmitic acid, oleic acid, and stearic acid (Supplemental Table 8), which are 3 of the most abundant LCFA in bovine milk (23).

    AGPAT isoforms and monounsaturated FA esterification into TG. Acylation of lysophosphatidic acid at the sn-2 position during TG synthesis occurs via 1-acyl-sn-glycerol-3-phosphate O-acyltransferase (AGPAT) and results in the formation of phosphatidic acid (PA). Recently, Beigneux et al. (30) characterized AGPAT6 in the mouse and established an essential role for this isoform in milk biosynthesis. In support of this key role, different genetic lines of mice had greater AGPAT6 expression in mammary tissue during lactation compared to pregnancy (1,30). A previous study reported a reduction of monounsaturated FA in TG, diacylglycerol (DAG), and phospholipids in AGPAT6-knockout mice (31). Oleic acid is the most abundant monounsaturated FA and the 2nd most predominant FA esterified to the sn-2 position in bovine milk fat (23,32). We suggest that expression of AGPAT6 over the lactation cycle in bovine is at least in part associated with the need for oleic acid incorporation into the sn-2 position of TG. AGPAT1 also appears to play a role in mammary lipid synthesis in both murine (1) and bovine mammary tissue. Results indicate that AGPAT6, and to a lesser extent AGPAT1, are the most important AGPAT isoforms in bovine mammary and might play an important role in milk fat synthesis.

    LPIN1 and provision of DAG for milk TG synthesis. LPIN are PA phosphatases (9) that convert PA to DAG during TG synthesis (33). Genes of the murine LPIN family have been recently isolated and characterized (9). mRNA of LPIN1 and LPIN2 are expressed in murine mammary (1), and to our knowledge, we show for the first time their expression in bovine mammary. In both species, expression of LPIN1 was upregulated >3-fold during lactation. These findings, and its downregulation in response to trans10,cis12–18:2 incubation in MacT cells (A. K. Kadegowda, M. Bionaz, L. S. Piperova, R. A. Erdman, and J. J. Loor, unpublished results), offer support for a role of LPIN1 in milk fat synthesis. Additional support is provided by data showing that LPIN1 plays an essential role in adipogenesis in 3T3-L1 cells, and its overexpression in adipose increased accumulation of TG (34). A role of LPIN1 in transcription regulation of other genes involved in milk fat synthesis cannot be excluded as shown by Finck et al. (10), who demonstrated that LPIN1 is essential for PPAR activation. mRNA of LPIN1 is the most abundant among LPIN isoforms in bovine mammary tissue. It remains to be determined which, if any, variant (a or b) plays a more important role in bovine milk TG synthesis.

The potential importance of enzyme systems and their various isoforms in the overall process of milk TG synthesis was highlighted (33). Transcriptional repression of 1 or more of these isoforms likely could result in deficient milk TG synthesis, or low-fat milk, as shown in murine knockout models (e.g., AGPAT6). Although measurement of mRNA expression (n-fold change) together with relative abundance (percentage of transcript abundance) of isoforms is useful for studying putative function, the driving force ultimately is the amount of protein produced. Several regulatory factors control protein abundance and thus the correlation between mRNA and protein is not always high [e.g., (13)]. Despite this limitation, our results uncovered dramatic increases in mRNA (>3-fold) of SLC27A6, ACSL1, FABP3, FABP4, AGPAT6, and LPIN1 as lactation progressed, allowing us to conclude that these gene isoforms coordinately participate in the channeling of FA toward copious TG synthesis in bovine mammary.

	ACKNOWLEDGMENTS

 
We thank Dr. James K. Drackley for his suggestions during the development of this manuscript.

	FOOTNOTES

 
¹ Supported in part, for the gene expression work, by the Cooperative State Research, Education, and Extension Service, USDA, under project ILLU-538-307.

² Author disclosures: M. Bionaz and J. J. Loor, no conflicts of interest.

³ Supplemental Materials and Methods, Results, and References, and Supplemental Tables 1–8 are available with the online posting of this paper at jn.nutrition.org.

⁴ Abbreviations used: ACSL, long chain acyl-CoA synthetase; AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase; DAG, diacylglycerol; FA, fatty acid; FABP, fatty acid binding protein; FATP, fatty acid transport protein; LCFA, long chain fatty acid; LPIN, lipin; PA, phosphatidic acid; qPCR, quantitative real time RT-PCR; SLC27A, solute carrier family 27 (fatty acid transporter); TG, triacylglycerol.

Manuscript received 14 January 2008. Initial review completed 14 February 2008. Revision accepted 5 March 2008.

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TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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