Characterization of the fatty acid binding protein 3 (FABP3) promoter and its transcriptional regulation by cAMP response element binding protein 1 (CREB1) in goat mammary epithelial cells.

Fatty acid binding protein 3 (FABP3) is involved in signal transduction pathways, and in the uptake and utilization of long-chain fatty acids. However, the transcriptional regulation of FABP3 in goat is unclear. In this study, the FABP3 5' flanking region was amplified from goat (Capra hircus) genomic DNA. Luciferase reporter vectors containing promoter fragments of five different lengths were constructed and transfected into dairy goat mammary epithelial cells. The region of the promoter located between -1801 and -166 bp upstream of the transcription start site (TSS) exhibited the highest luciferase activity, and contained two cAMP response elements (CREs) located at -1632 bp and -189 bp. Interference with CREB1 significantly downregulated FABP3 promoter activity. In addition, FABP3 promoter activity was significantly reduced after mutation of the CRE1 (-1632 bp) and CRE2 (-189 bp) sites. Further analysis indicated that the CRE2 site was essential for the transcriptional activity induced by CREB1. These results demonstrated that CREB1 is involved in the transcriptional regulation of FABP3 expression in the goat mammary gland via a direct mechanism, thus revealing a novel signaling pathway involved in fatty acid metabolism in goat.

Diacylglycerol acyltransferase 2 promotes the adipogenesis of intramuscular preadipocytes in goat.

Diacylglycerol acyltransferase 2 (DGAT2) is the key enzyme that catalyzes the last step of triglyceride synthesis. However, its role in intramuscular fat (IMF) deposition in goat remains unclear. The purpose of this study was to explore the role of DGAT2 in regulating goat IMF deposition. In the present study, the expression of DGAT2 was highest in goat triceps brachii, and highest on the first day after oleic acid induction in goat intramuscular preadipocytes. The overexpression of DGAT2 promoted the accumulation of lipid droplets and triglyceride synthesis, accompanied by the expression upregulation of DGAT1, TIP47, ACC and ACOX1 significantly, and expression downregulation of AGPAT6, LPIN1, LPL, HSL, ATGL and ADRP significantly. In contrast, the silencing of DGAT2 decreased the accumulation of lipid droplets, inhibited the expression of DGAT1, GPAM, ADRP, AGPAT6, LPL, HSL, ATGL, ACC, FASN, ACOX1 significantly, and enhanced that of TIP47 significantly. Overall, these data underscore DGAT2 may play a potentially important role in lipid droplets formation and triglyceride accumulation, so as to maintain intramuscular fat deposition, beyond triglyceride synthesis in goat.

Identification and profiling of microRNAs involved in the regenerative involution of mammary gland.

Regenerative involution is important for the subsequent lactation, but molecular mechanism has not been revealed. The crucial miRNA in tissue development indicates that miRNAs might participate in regenerative involution. In the present study, the mammary tissues of the dairy goats (n = 3) were collected via biopsy at wk-8 (time to dry off), -6, -4, -1, and + 1 relative to lambing for the Hematoxylin and Eosin staining and miRNA sequencing. Alveolar structures collapsed during regenerative involution, but the structures remained intact and distended. Among the 50 miRNA expression trajectories categorized by short time-series expression miner, two significant patterns were clustered. The differentially expressed miRNAs in the two patterns were mainly related to the self-renewal of tissue and enriched in pathways containing vesical-mediated transport, tissue development, tube development, vasculature development and epithelial development. The identification of the miRNAs will help in elucidating their regulatory roles in mammary gland development.

Expression Variation of CPT1A Induces Lipid Reconstruction in Goat Intramuscular Precursor Adipocytes.

Intramuscular fat (IMF) deposition is one of the most important factors affecting meat quality and is closely associated with the expression of carnitine palmitoyl transferase 1A (CPT1A) which facilitates the transfer of long-chain fatty acids (LCFAs) into the mitochondria. However, the role of how CPT1A regulates the IMF formation remains unclear. Herein, we established the temporal expression profile of CPT1A during the differentiation of goat intramuscular precursor adipocytes. Functionally, the knockdown of CPT1A by siRNA treatment significantly increased the mRNA expression of adipogenic genes and promoted lipid deposition in goat intramuscular precursor adipocytes. Meanwhile, a CPT1A deficiency inhibited cell proliferation and promoted cell apoptosis significantly. CPT1A was then supported by the overexpression of CPT1A which significantly suppressed the cellular triglyceride deposition and promoted cell proliferation although the cell apoptosis also was increased. For RNA sequencing, a total of 167 differential expression genes (DEGs), including 125 upregulated DEGs and 42 downregulated DEGs, were observed after the RNA silencing of CPT1A compared to the control, and were predicted to enrich in the focal adhesion pathway, cell cycle, apoptosis and the MAPK signaling pathway by KEGG analysis. Specifically, blocking the MAPK signaling pathway by a specific inhibitor (PD169316) rescued the promotion of cell proliferation in CPT1A overexpression adipocytes. In conclusion, the expression variation of CPT1A may reconstruct the lipid distribution between cellular triglyceride deposition and cell proliferation in goat intramuscular precursor adipocyte. Furthermore, we demonstrate that CPT1A promotes the proliferation of goat adipocytes through the MAPK signaling pathway. This work widened the genetic regulator networks of IMF formation and delivered theoretical support for improving meat quality from the aspect of IMF deposition.

Bacillus amyloliquefaciens-9 Reduces Somatic Cell Count and Modifies Fecal Microbiota in Lactating Goats.

Subclinical mastitis is one of the major problems affecting dairy animals' productivity and is classified based on milk somatic cell counts (SCC). Previous data showed that marine-derived Bacillus amyloliquefaciens-9 (GB-9) improved the immunity and the nonspecific immune defense system of the body. In this study, the potential role of GB-9 in improving subclinical mastitis was assessed with Radix Tetrastigmae (RT) as a positive control in subclinical mastitis Saanen dairy goats. The current data showed that GB-9 and RT significantly reduced the SCC in dairy goats. After being fed with GB-9 or RT, the decreased concentrations of malondialdehyde, IgA, IgM, IL-2, IL-4, and IL-6 were observed. The amplicon sequencing analysis of fecal samples revealed that GB-9 significantly altered the bacterial community. Bacteroides and Phascolarctobacterium were the major genera that respond to GB-9 feeding. The correlation analysis using weighted gene co-expression network analysis showed a MePink module was most associated with the serum concentrations of immunoglobulin and interleukin. The MePink module contained 89 OTUs. The feeding of GB-9 in decreasing the SCC was associated with the altered abundance of Bacteroides, which was correlated with the concentrations of immunoglobulins and chemokines. Collectively, the current data suggested that marine-derived GB-9 could be a helpful probiotic to control subclinical mastitis.

Evaluation of the Antibacterial Material Production in the Fermentation of Bacillus amyloliquefaciens-9 from Whitespotted Bamboo Shark (Chiloscyllium plagiosum).

Bacillus amyloliquefaciens-9 (GBacillus-9), which is isolated from the intestinal tract of the white-spotted bamboo shark (Chiloscyllium plagiosum), can secrete potential antibacterial materials, such as ß-1,3-1,4-glucanase and some antimicrobial peptides. However, the low fermentation production has hindered the development of GBacillus-9 as biological additives. In this study, the Plackett-Burman design and response surface methodology were used to optimize the fermentation conditions in a shake flask to obtain a higher yield and antibacterial activity of GBacillus-9. On the basis of the data from medium screening, M9 medium was selected as the basic medium for fermentation. The data from the single-factor experiment showed that sucrose had the highest antibacterial activity among the 10 carbon sources. The Plackett-Burman design identified sucrose, NH4Cl, and MgSO4 as the major variables altering antibacterial activity. The optimal concentrations of these compounds to enhance antibacterial activity were assessed using the central composite design. Data showed that sucrose, NH4Cl, and MgSO4 had the highest antibacterial activities at concentrations of 64.8, 1.84, and 0.08 g L-1, respectively. The data also showed that the optimal fermentation conditions for the antibacterial material production of GBacillus-9 were as follows: Inoculum volume of 5%, initial pH of 7.0, temperature of 36 °C, rotating speed of 180 rpm, and fermentation time of 10 h. The optimal fermentation medium and conditions achieved to improve the yield of antibacterial materials for GBacillus-9 can enhance the process of developing biological additives derived from GBacillus-9.

A fragment activity assay reveals the key residues of TBC1D15 GTPase-activating protein (GAP) in Chiloscyllium plagiosum.

BACKGROUND: GTPase-activating proteins (GAPs) with a TBC (Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases. The related crystal structure has been studied and reported by other members of our research group in 2017 (Chen et al. in Protein Sci 26(4):834-846, 2017). The protein crystal structure and sequencing data accession numbers in Protein structure database (PDB) are 5TUB (Shark TBC1D15 GAP) and 5TUC (Sus TBC1D15 GAP), respectively. In this paper, we analyzed the Rab-GAP specificity of TBC1D15 in the evolution and influence of key amino acid residue mutations on Rab-GAP activity. RESULTS: Sequence alignment showed that five arginine residues of the TBC1D15-GAP domain are conserved among the species Sus/Mus/Homo but have been replaced by glycine or lysine in Shark. A fragment activity assay was conducted by altering the five residues of Shark TBC1D15-GAP to arginine, and the corresponding arginine in TBC1D15 GAP domains from Sus and Homo species were mutated to resemble Shark TBC1D15-GAP. Our data revealed that the residues of G28, K45, K119, K122 and K221 in the Shark TBC1D15-GAP domain had a key role in determining the specificity for Rab7 and Rab11. Mutation of the five residues significantly altered the Shark TBC1D15-GAP activity. CONCLUSIONS: These results revealed that the substrate specificity of TBC1D15 has had different mechanisms across the evolution of species from lower-cartilaginous fish to higher mammals. Collectively, the data support a different mechanism of Shark TBC1D15-GAP in substrate selection, which provides a new idea for the development of Marine drugs.

SCD1 Alters Long-Chain Fatty Acid (LCFA) Composition and Its Expression Is Directly Regulated by SREBP-1 and PPARγ 1 in Dairy Goat Mammary Cells.

Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme for the synthesis of the monounsaturated fatty acids (MUFA) palmitoleic acid and oleic acid. In non-ruminant species, SCD1 expression is known to be tightly regulated by a variety of transcription factors. Although the role of SCD1 and the transcriptional regulatory mechanism by SREBP-1 and PPARs in other species is clear, changes in lipid metabolism related to SCD1 and via the regulation of SREBP-1 or PPARG1 in ruminant mammary tissue remain largely unknown. Here, we demonstrated that SCD1 expression in goat mammary tissue is higher during lactation than the dry period. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation, whereas SCD1 silencing resulted in a significant decrease in oleic acid concentration and triacylglycerol (TAG) accumulation. The overexpression of SREBF1 in goat mammary epithelial cells (GMEC) enhanced SCD1 expression and its promoter activity, but that effect was abolished when SREBF1 was silenced. Furthermore, deletion of sterol regulatory element (SRE) and the nuclear factor (NF-Y)-binding sites within a -1713 to +65-base pair region of the SCD1 promoter completely abolished SREBP-1-induced SCD1 transcription. Otherwise, PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity directly via a PPAR response element (PPRE) in the SCD1 promoter. Together, these results indicate that SCD1 could markedly affect the fatty acid composition and rate of TAG synthesis through direct regulation via SREBP-1 and PPARG1, hence, underscoring an important role of the enzyme and this transcription regulator in controlling mammary gland lipid synthesis in the goat. J. Cell. Physiol. 232: 635-649, 2017. © 2016 Wiley Periodicals, Inc.

Genes regulating lipid and protein metabolism are highly expressed in mammary gland of lactating dairy goats.

Dairy goats serve as an important source of milk and also fulfill agricultural and economic roles in developing countries. Understanding the genetic background of goat mammary gland is important for research on the regulatory mechanisms controlling tissue function and the synthesis of milk components. We collected tissue at four different stages of goat mammary gland development and generated approximately 25 GB of data from Illumina de novo RNA sequencing. The combined reads were assembled into 51,361 unigenes, and approximately 60.07 % of the unigenes had homology to other proteins in the NCBI non-redundant protein database (NR). Functional classification through eukaryotic Ortholog Groups of Protein (KOG), gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the unigenes from goat mammary glands are involved in a wide range of biological processes and metabolic pathways, including lipid metabolism and lactose metabolism. The results of qPCR revealed that genes encoding FABP3, FASN, SCD, PLIN2, whey proteins (LALBA and BLG), and caseins (CSN1S1, CSN1S2, CSN2 and CSN3) at 100 and 310 days postpartum increased significantly compared with the non-lactating period. In addition to their role in lipid and protein synthesis, the higher expression at 310 days postpartum could contribute to mammary cell turnover during pregnancy. In conclusion, this is the first study to characterize the complete transcriptome of goat mammary glands and constitutes a comprehensive genomic resource available for further studies of ruminant lactation.

Crosstalk between innate immunity and rumen-fecal microbiota under the cold stress in goats.

The balance of the microbiome, which is sensitive to temperature changes, plays a crucial role in maintaining overall health and reducing the risk of diseases. However, the specific mechanisms by which immunity and microbiota interact to adapt to cold stress have yet to be addressed. In this study, Nanjiang Yellow goats were chosen as a model and sampled during the cold (winter, cold stress) and warm (spring) seasons, respectively. Analyses of serum immune factors, as well as the composition of rumen and fecal microbial communities, were conducted to explore the crosstalk between microbiota and innate immunity under cold stress. Significantly increased levels of IgA (P < 0.01) were observed in the cold season compared to the warm season. Conversely, the levels of IL-2 (P = 0.02) and IL-6 (P < 0.01) diminished under cold stress. However, no significant differences were observed in IgG (P = 0.89), IgM (P = 0.42), and IL-4 (P = 0.56). While there were no significant changes in the diversity of bacterial communities between the warm and cold seasons, positive correlations between serum IgA, IL-2, IL-6 concentrations and several genera were observed. Furthermore, the weighted gene co-expression network analysis indicated that the microbiota enriched in the MEbrown module positively correlated with IgA, while the microbiota enriched in the MEblue module positively correlated with IL-2 and IL-6. The strong correlation between certain probiotics, including Alistipes, Bacteroides, Blautia, and Prevotellaceae_UCG.004, and the concentration of IL-2, and IL-6 suggests their potential role in immunomodulatory properties. This study provides valuable insights into the crosstalk between microbial communities and immune responses under the challenge of cold stress. Further studies on the immunomodulatory properties of these probiotics would contribute to the development of strategies to enhance the stress resistance of animals for improved overall health and survival.

MCD Inhibits Lipid Deposition in Goat Intramuscular Preadipocytes.

Malonyl-CoA decarboxylase (MCD) is a major regulator of fatty acid oxidation catalyzing the decarboxylation of malonyl coenzyme A (malonyl-CoA). Although its involvement in human diseases has been well studied, its role in intramuscular fat (IMF) deposition remains unknown. In this present study, 1726 bp of MCD cDNA was cloned (OM937122) from goat liver, including 5'UTR of 27 bp, 3'UTR of 199 bp, and CDS of 1500 bp, encoding 499 amino acids. In this present study, although the overexpression of MCD increased the mRNA expression of FASN and DGAT2, the expression of ATGL and ACOX1 was also activated significantly and resulted in a decrease in cellular lipid deposition in goat intramuscular preadipocytes. Meanwhile, the silencing of MCD increased the cellular lipid deposition and was accompanied by the expression activation of DGAT2 and the expression suppression of ATGL and HSL, despite the expression suppression of genes related to fatty acid synthesis, including ACC and FASN. However, the expression of DGAT1 was not affected significantly (p > 0.05) by the expression alteration of MCD in this present study. Furthermore, 2025 bp of MCD promoter was obtained and predicted to be regulated by C/EBPα, SP1, SREBP1, and PPARG. In summary, although different pathways may respond to the expression alteration of MCD, the expression of MCD was negatively correlated with the cellular lipid deposition in goat intramuscular preadipocytes. These data may be beneficial for enhancing our understanding of the regulation of IMF deposition in goats.

Rumen and fecal microbiota profiles associated with immunity of young and adult goats.

Low immunity at birth increases risk of disease of young livestock, such as goat kids. Microbiomes change as animals mature, and a healthy microbiome is related to decreased risk of disease. The relationship between microbiota profiles and immunity at different developmental stages remains unclear. Young (female, n = 12, 30 d) and adult (female, n = 12, 2 yrs. old) Saanen dairy goats were used to investigate changes in rumen microbiomes, fecal microbiomes, and their correlations to circulating immune factors. Serum IgG (P = 0.02) and IgM (P < 0.01) were higher at 2 years than 30 d of age, but there were no differences in IgA (P = 0.34), IL-2 (P = 0.05), IL-4 (P = 0.37) and IL-6 (P = 0.73) between ages. Amplicon sequencing analysis revealed young goats had a higher diversity of bacterial communities in rumen and lower diversity in feces compared with adult goats. Ten genera in rumen and 14 genera in feces were positively correlated with serum IgM concentration across both ages. Olsenella, Methanosphaera, Quinella, Candidatus_Saccharimonas, and Methanobrevibacter in rumen and Ruminobacter, Treponema, Rikenelaceae_ RC9_ gut_ Group in feces were positively correlated with the concentration of IgG. The correlation analysis using weighted gene co-expression network analysis showed the MEblue module was positively associated with the IgG and IgM. These data provide novel insight into the association between rumen-feces microbiota and immune response. Further experiments are needed to investigate whether inoculating young livestock with immune-related bacteria identified can improve the immune status. Our data suggest a possible strategy to improve the immunity of the kids by alterative microbiota profiles.

The LXRB-SREBP1 network regulates lipogenic homeostasis by controlling the synthesis of polyunsaturated fatty acids in goat mammary epithelial cells.

BACKGROUND: In rodents, research has revealed a role of liver X receptors (LXR) in controlling lipid homeostasis and regulating the synthesis of polyunsaturated fatty acids (PUFA). Recent data suggest that LXRB is the predominant LXR subtype in ruminant mammary cells, but its role in lipid metabolism is unknown. It was hypothesized that LXRB plays a role in lipid homeostasis via altering the synthesis of PUFA in the ruminant mammary gland. We used overexpression and knockdown of LXRB in goat primary mammary epithelial cells (GMEC) to evaluate abundance of lipogenic enzymes, fatty acid profiles, content of lipid stores and activity of the stearoyl-CoA desaturase (SCD1) promoter. RESULTS: Overexpression of LXRB markedly upregulated the protein abundance of LXRB while incubation with siRNA targeting LXRB markedly decreased abundance of LXRB protein. Overexpression of LXRB plus T0901317 (T09, a ligand for LXR) dramatically upregulated SCD1 and elongation of very long chain fatty acid-like fatty acid elongases 5-7 (ELOVL 5-7), which are related to PUFA synthesis. Compared with the control, cells overexpressing LXRB and stimulated with T09 had greater concentrations of C16:0, 16:1, 18:1n7,18:1n9 and C18:2 as well as desaturation and elongation indices of C16:0. Furthermore, LXRB-overexpressing cells incubated with T09 had greater levels of triacylglycerol and cholesterol. Knockdown of LXRB in cells incubated with T09 led to downregulation of genes encoding elongases and desaturases. Knockdown of LXRB attenuated the increase in triacylglycerol and cholesterol that was induced by T09. In cells treated with dimethylsulfoxide, knockdown of LXRB increased the concentration of C16:0 at the expense of C18:0, while a significant decrease in C18:2 was observed in cells incubated with both siLXRB and T09. The abundance of sterol regulatory element binding transcription factor 1 precursor (pSREBP1) and its mature fragment (nSREBP1) was upregulated by T09, but not LXRB overexpression. In the cells cultured with T09, knockdown of LXRB downregulated the abundance for pSREBP1 and nSREBP1. Luciferase reporter assays revealed that the activities of wild type SCD1 promoter or fragment with SREBP1 response element (SRE) mutation were decreased markedly when LXRB was knocked down. Activity of the SCD1 promoter that was induced by T09 was blocked when the SRE mutation was introduced. CONCLUSION: The current study provides evidence of a physiological link between the LXRB and SREBP1 in the ruminant mammary cell. An important role was revealed for the LXRB-SREBP1 network in the synthesis of PUFA via the regulation of genes encoding elongases and desaturases. Thus, targeting this network might elicit broad effects on lipid homeostasis in ruminant mammary gland.

AMPK-ChREBP axis mediates de novo milk fatty acid synthesis promoted by glucose in the mammary gland of lactating goats.

To investigate the role of glucose in regulating milk fatty acid synthesis, 6 lactating Guanzhong dairy goats were infused with 0, 60, or 100 g/d glucose via the external pubic artery in a 3 × 3 repeated Latin square experiment. A concomitant in vitro experiment was conducted to investigate possible mechanisms whereby glucose regulates milk fatty acid synthesis. RNA sequencing was used for cellular transcriptome analysis. Drugs, MK-2206, rapamycin, and dorsomorphin were used to block cellular mammalian AMP-activated protein kinase (AMPK), AKT serine/threonine kinase 1, and mechanistic target of rapamycin kinase signaling pathways, respectively. Carbohydrate response element binding protein (ChREBP) was knockdown and overexpressed to investigate its role in regulating milk fatty acid synthesis in mammary epithelial cells. Glucose infusion linearly elevated the concentration of C8:0 (P = 0.039) and C10:0 (P = 0.041) in milk fat while it linearly decreased (P = 0.049) that of C16:0. This result was in agreement with the upregulation of genes related to de novo synthesis of fatty acids and lipid droplet formation, including adipose differentiation-related protein, butyrophilin subfamily 1 member A1, fatty acid synthase (FASN) and ChREBP. Their expression increased (P < 0.05) linearly in the lactating goat mammary gland. In vitro, glucose linearly stimulated the expression of genes related to de novo synthesis of fatty acids and cellular triacylglycerol in cultured mammary epithelial cells. RNA sequencing and inhibition studies revealed that glucose induced transcriptomic changes increasing lipogenic pathways, with AMPK responding to glucose by controlling ChREBP and FASN. Knockdown and overexpression of ChREBP highlighted its essential role in lipogenesis. The knockdown and overexpression of ChREBP protein also revealed an essential role in regulating the de novo synthesis of fatty acids. Collectively, our data highlight that glucose supplementation promotes de novo fatty acid synthesis via the AMPK-ChREBP axis, hence increasing milk fat yield in the goat mammary gland. Results from the current study provide possible strategies to manipulate the fatty acid composition as well as improve ruminant milk quality.

Bacillus Amyloliquefaciens-9 as an Alternative Approach to Cure Diarrhea in Saanen Kids.

Bacillus amyloliquefaciens-9 (GBacillus-9), derived from the intestinal tract of the white-spotted bamboo shark, secretes a variety of antimicrobial compounds that inhibit the growth of pathogenic bacteria. In this study, the role of GBacillus-9 in the prevention and treatment of Saanen kids with diarrhea was assessed. Six healthy kids (HL) and six kids with diarrhea (DL) were selected. All kids were fed with 0.3% (w/v) GBacillus-9 (spray power) in raw milk for two weeks. The proportion of kids with diarrhea decreased gradually as the trial progressed, and 100% DL kids were cured at day 15. GBacillus-9 increased the serum immunoglobulin (Ig) G, interleukin (IL)-4, and IL-6 concentration (p < 0.05). The amplicon sequencing analysis of the fecal bacterial community revealed that the fecal microbiota was remarkably different between the HL and the DL groups at day 0. After two weeks of feeding with GBacillus-9, no significant difference in fecal microbiota was observed between HL and DL groups at the phylum level. GBacillus-9 restored the intestinal microbial disorder associated with serum immunoglobulin and interleukin concentration. Correlation analysis showed that GBacillus-9 altered globulin and interleukin concentration and that immunoglobulin was associated with Firmicutes. Collectively, our results revealed that GBacillus-9 improved the gut health of kids by improving microbial homeostasis.

Comprehensive Transcriptome Profiling of Dairy Goat Mammary Gland Identifies Genes and Networks Crucial for Lactation and Fatty Acid Metabolism.

Milk fatty acids secreted by the mammary gland are one of the most important determinants of the nutritional value of goat milk. Unlike cow milk, limited data are available on the transcriptome-wide changes across stages of lactation in dairy goats. In this study, goat mammary gland tissue collected at peak lactation, cessation of milking, and involution were analyzed with digital gene expression (DGE) sequencing to generate longitudinal transcript profiles. A total of 51,299 unigenes were identified and further annotated to 12,763 genes, of which 9,131 were differentially expressed across various stages of lactation. Most abundant genes and differentially expressed genes (DEGs) were functionally classified through clusters of euKaryotic Orthologous Groups (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A total of 16 possible expression patterns were uncovered, and 13 genes were deemed novel candidates for regulation of lactation in the goat: POLG, SPTA1, KLC, GIT2, COPS3, PDP, CD31, USP16/29/37, TLL1, NCAPH, ABI2, DNAJC4, and MAPK8IP3. In addition, PLA2, CPT1, PLD, GGA, SRPRB, and AP4S1 are proposed as novel and promising candidates regulating mammary fatty acid metabolism. "Butirosin and neomycin biosynthesis" and "Glyoxylate and dicarboxylate metabolism" were the most impacted pathways, and revealed novel metabolic alterations in lipid metabolism as lactation progressed. Overall, the present study provides new insights into the synthesis and metabolism of fatty acids and lipid species in the mammary gland along with more detailed information on molecular regulation of lactogenesis. The major findings will benefit efforts to further improve milk quality in dairy goats.

Role of peroxisome proliferator-activated receptor-α on the synthesis of monounsaturated fatty acids in goat mammary epithelial cells.

A key member of the nuclear receptor superfamily is the peroxisome proliferator-activated receptor alpha (PPARA) isoform, which in nonruminants is closely associated with fatty acid oxidation. Whether PPARA plays a role in milk fatty acid synthesis in ruminants is unknown. The main objective of the present study was to use primary goat mammary epithelial cells (GMEC) to activate PPARA via the agonist WY-14643 (WY) or to silence it via transfection of small-interfering RNA (siRNA). Three copies of the peroxisome proliferator-activated receptor response element (PPRE) contained in a luciferase reporter vector were transfected into GMEC followed by incubation with WY at 0, 10, 20, 30, 50, or 100 µM. A dose of 50 µM WY was most effective at activating PPRE without influencing PPARA mRNA abundance. Transfecting siRNA targeting PPARA decreased its mRNA abundance to 20% and protein level to 50% of basal levels. Use of WY upregulated FASN, SCD1, ACSL1, DGAT1, FABP4, and CD36 (1.1-, 1.5-, 2-, 1.4-, 1.5-, and 5-fold, respectively), but downregulated DGAT2 and PGC1A (-20% and -40%, respectively) abundance. In contrast, triacylglycerol concentration decreased and the content and desaturation index of C16:1 and C18:1 increased. Thus, activation of PPARA via WY appeared to channel fatty acids away from esterification. Knockdown of PPARA via siRNA downregulated ACACA, SCD1, AGPAT6, CD36, HSL, and SREBF1 (-43%, -67%, -16%, -56%, -26%, and -29%, respectively), but upregulated ACSL1, DGAT2, FABP3, and PGC1A (2-, 1.4-, 1.3-, and 2.5-fold, respectively) mRNA abundance. A decrease in the content and desaturation index of C16:1 and C18:1 coupled with an increase in triacylglycerol content accompanied those effects at the mRNA level. Overall, data suggest that PPARA could promote the synthesis of MUFA in GMEC through its effects on mRNA abundance of genes related to fatty acid synthesis, oxidation, transport, and triacylglycerol synthesis.

Fatty Acid Elongase 7 (ELOVL7) Plays a Role in the Synthesis of Long-Chain Unsaturated Fatty Acids in Goat Mammary Epithelial Cells.

In humans, fatty acid elongase 7 (ELOVL7) plays a role in synthesis of long-chain saturated fatty acids. Whether ELOVL7 protein plays a role in ruminants is unclear. The transcript abundance of ELOVL7 in goat mammary tissue was assessed at three stages of lactation. Results showed that ELOVL7 had the highest expression in the dry period compared with peak and late lactation period. Results revealed that ELOVL7 overexpression was correlated with lower expression in diacylglycerol O-acyltransferase 2 (DGAT2) and stearoyl-CoA desaturase 1 (SCD1), and had no significant effect on triacylglycerol concentration. Overexpression of ELOVL7 significantly decreased the concentration of palmitoleic (C16:1n7) and oleic (C18:1n9) acid, and increased the concentration of vaccenic (C18:1n7) and linoleic (C18:2) acid. Overexpression of ELOVL7 significantly upregulated the elongation index of C16:1 in goat epithelial mammary cells (GMEC), but had a minor effect on that of palmitate (C16:0). Knockdown of ELOVL7 decreased mRNA expression of fatty acid binding protein 3 (FABP3) and fatty acid desaturase 2 (FADS2) and had a minor effect on triacylglycerol concentration; however, it increased concentration of C18:1n9 in GMEC. The elongation indices of C16:0 and C16:1 did not differ due to knockdown of ELOVL7. The minor change for the C16:0 and stearate (C18:0) was observed after activation of ELOVL7, suggesting the two fatty acids are not the preferential substrates of ELOVL7 in cultured GMEC. However, changes in C18:1n9 and C18:2 after overexpression or knockdown of ELOVL7 indicated a biological functional role of ELOVL7. Collectively, our data highlighted a role of ELOVL7 in long-chain unsaturated fatty acid elongation in goat mammary epithelial cells.

Effect of short-chain fatty acids on triacylglycerol accumulation, lipid droplet formation and lipogenic gene expression in goat mammary epithelial cells.

Short-chain fatty acids (SCFAs) are the major energy sources for ruminants and are known to regulate various physiological functions in other species. However, their roles in ruminant milk fat metabolism are still unclear. In this study, goat mammary gland epithelial cells (GMECs) were treated with 3 mmol/L acetate, propionate or butyrate for 24 h to assess their effects on lipogenesis. Data revealed that the content of triacylglycerol (TAG) and lipid droplet formation were significantly stimulated by propionate and butyrate. The expression of FABP3, SCD1, PPARG, SREBP1, DGAT1, AGPAT6 and ADRP were upregulated by propionate and butyrate treatment. In contrast, the messenger RNA (mRNA) expression of FASN and LXRα was not affected by propionate, but reduced by butyrate. Acetate had no obvious effect on the content of TAG and lipid droplets but increased the mRNA expression of SCD1 and FABP3 in GMECs. Additionally, it was observed that propionate significantly increased the relative content of mono-unsaturated fatty acids (C18:1 and C16:1) at the expense of decreased saturated fatty acids (C16:0 and C18:0). Butyrate and acetate had no significant effect on fatty acid composition. Overall, the results from this work help enhance our understanding of the regulatory role of SCFAs on goat mammary cell lipid metabolism.

Transcriptome-Wide Analysis Reveals the Role of PPARγ Controlling the Lipid Metabolism in Goat Mammary Epithelial Cells.

To explore the large-scale effect of peroxisome proliferator-activated receptor γ (PPARG) in goat mammary epithelial cells (GMEC), an oligonucleotide microarray platform was used for transcriptome profiling in cells overexpressing PPARG and incubated with or without rosiglitazone (ROSI, a PPARγ agonist). A total of 1143 differentially expressed genes (DEG) due to treatment were detected. The Dynamic Impact Approach (DIA) analysis uncovered the most impacted and induced pathways "fatty acid elongation in mitochondria," "glycosaminoglycan biosynthesis-keratan sulfate," and "pentose phosphate pathway." The data highlights the central role of PPARG in milk fatty acid metabolism via controlling fatty acid elongation, biosynthesis of unsaturated fatty acid, lipid formation, and lipid secretion; furthermore, its role related to carbohydrate metabolism promotes the production of intermediates required for milk fat synthesis. Analysis of upstream regulators indicated that PPARG participates in multiple physiological processes via controlling or cross talking with other key transcription factors such as PPARD and NR1H3 (also known as liver-X-receptor-α). This transcriptome-wide analysis represents the first attempt to better understand the biological relevance of PPARG expression in ruminant mammary cells. Overall, the data underscored the importance of PPARG in mammary lipid metabolism and transcription factor control.