Unveiling Novel Mechanism of CIDEB in Fatty Acid Synthesis Through ChIP-Seq and Functional Analysis in Dairy Goat.

Goat milk is abundant in nutrients, particularly in milk fats, which confer health benefits to humans. Exploring the regulatory mechanism of fatty acid synthesis is highly important to understand milk composition manipulation. In this study, we used chromatin immunoprecipitation sequencing (ChIP-seq) on goat mammary glands at different lactation stages which revealed a novel lactation regulatory factor: cell death-inducing DFFA-like effector B (CIDEB). RT-qPCR results revealed that CIDEB was significantly upregulated during lactation in dairy goats. CIDEB overexpression significantly increased the expression levels of genes involved in fatty acid synthesis (ACACA, SCD1, p < 0.05; ELOVL6, p < 0.01), lipid droplet formation (XDH, p < 0.05), and triacylglycerol (TAG) synthesis (DGAT1, p < 0.05; GPAM, p < 0.01) in goat mammary epithelial cells (GMECs). The contents of lipid droplets, TAG, and cholesterol were increased (p < 0.05) in CIDEB-overexpressing GMECs, and knockdown of CIDEB led to the opposite results. In addition, CIDEB knockdown significantly decreased the proportion of C16:0 and total C18:2. Luciferase reporter assays indicated that X-box binding protein 1 (XBP1) promoted CIDEB transcription via XBP1 binding sites located in the CIDEB promoter. Furthermore, CIDEB knockdown attenuated the stimulatory effect of XBP1 on lipid droplet accumulation. Collectively, these findings elucidate the critical regulatory roles of CIDEB in milk fat synthesis, thus providing new insights into improving the quality of goat milk.

Sirtuin 1 Inhibits Fatty Acid Synthesis through Forkhead Box Protein O1-Mediated Adipose Triglyceride Lipase Expression in Goat Mammary Epithelial Cells.

Sirtuin 1 (SIRT1) is a key upstream regulator of lipid metabolism; however, the molecular mechanisms by which SIRT1 regulates milk fat synthesis in dairy goats remain unclear. This study aimed to investigate the regulatory roles of SIRT1 in modulating lipid metabolism in goat mammary epithelial cells (GMECs) and its impact on the adipose triglyceride lipase (ATGL) promoter activity using RNA interference (RNAi) and gene overexpression techniques. The results showed that SIRT1 is significantly upregulated during lactation compared to the dry period. Additionally, SIRT1 knockdown notably increased the expressions of genes related to fatty acid synthesis (SREBP1, SCD1, FASN, ELOVL6), triacylglycerol (TAG) production (DGAT2, AGPAT6), and lipid droplet formation (PLIN2). Consistent with the transcriptional changes, SIRT1 knockdown significantly increased the intracellular contents of TAG and cholesterol and the lipid droplet abundance in the GMECs, while SIRT1 overexpression had the opposite effects. Furthermore, the co-overexpression of SIRT1 and Forkhead box protein O1 (FOXO1) led to a more pronounced increase in ATGL promoter activity, and the ability of SIRT1 to enhance ATGL promoter activity was nearly abolished when the FOXO1 binding sites (FKH1 and FKH2) were mutated, indicating that SIRT1 enhances the transcriptional activity of ATGL via the FKH element in the ATGL promoter. Collectively, our data reveal that SIRT1 enhances the transcriptional activity of ATGL through the FOXO1 binding sites located in the ATGL promoter, thereby regulating lipid metabolism. These findings provide novel insights into the role of SIRT1 in fatty acid metabolism in dairy goats.

Chromosome-level dairy goat genome reveals the regulatory landscape of lactation.

Goat milk is rich in various nutrients that are beneficial for human health. However, the genomic evolution and genetic basis underlying the nutritional value and unique flavor formation in dairy goats remain poorly understood. In the present study, we generate a chromosome-level genome assembly for dairy goats comprising 2.63 Gb with a contig N50 of 43 Mb and a scaffold N50 of 101 Mb. Genome quality comparisons revealed that the dairy goat genome has higher integrity and continuity than the published goat and sheep genomes. The identification of genes under positive selection in dairy goats highlights potential candidates to explain their high milk production. Comparative genomic analysis elucidates the adaptive evolutionary mechanisms of dairy goats such as strong disease resistance, broad adaptability, and unique milk flavor. Moreover, we demonstrate the conservation of the lactation gene network and identify new potential regulators associated with lipid metabolism. Additionally, we establish the regulatory landscape of lactation for the first time in dairy goats, revealing its unique gene regulatory characteristics. Hence, our study not only provides the first chromosome-level reference genome for dairy goat, but also offers potential research directions for dairy production and genetic improvement.

Integrated analysis of genomics and transcriptomics revealed the genetic basis for goaty flavor formation in goat milk.

Goat milk exhibits a robust and distinctive "goaty" flavor. However, the underlying genetic basis of goaty flavor remains elusive and requires further elucidation at the genomic level. Through comparative genomics analysis, we identified divergent signatures of certain proteins in goat, sheep, and cow. MMUT has undergone a goat-specific mutation in the B12 binding domain. We observed the goat FASN exhibits nonsynonymous mutations in the acyltransferase domain. Structural variations in these key proteins may enhance the capacity for synthesizing goaty flavor compounds in goat. Integrated omics analysis revealed the catabolism of branched-chain amino acids contributed to the goat milk flavor. Furthermore, we uncovered a regulatory mechanism in which the transcription factor ZNF281 suppresses the expression of the ECHDC1 gene may play a pivotal role in the accumulation of flavor substances in goat milk. These findings provide insights into the genetic basis underlying the formation of goaty flavor in goat milk. STATEMENT OF SIGNIFICANCE: Branched-chain fatty acids (BCFAs) play a crucial role in generating the distinctive "goaty" flavor of goat milk. Whether there is an underlying genetic basis associated with goaty flavor is unknown. To begin deciphering mechanisms of goat milk flavor development, we collected transcriptomic data from mammary tissue of goat, sheep, cow, and buffalo at peak lactation for cross-species transcriptome analysis and downloaded nine publicly available genomes for comparative genomic analysis. Our data indicate that the catabolic pathway of branched-chain amino acids (BCAAs) is under positive selection in the goat genome, and most genes involved in this pathway exhibit significantly higher expression levels in goat mammary tissue compared to other species, which contributes to the development of flavor in goat milk. Furthermore, we have elucidated the regulatory mechanism by which the transcription factor ZNF281 suppresses ECHDC1 gene expression, thereby exerting an important influence on the accumulation of flavor compounds in goat milk. These findings provide insights into the genetic mechanisms underlying flavor formation in goat milk and suggest further research to manipulate the flavor of animal products.

Knockdown of PROX1 promotes milk fatty acid synthesis by targeting PPARGC1A in dairy goat mammary gland.

Goat milk is rich in various fatty acids that are beneficial to human health. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA-seq analyses of goat mammary glands at different lactation stages revealed a novel lactation regulatory factor, Prospero homeobox 1 (PROX1). However, the mechanism whereby PROX1 regulates lipid metabolism in dairy goats remains unclear. We found that PROX1 exhibits the highest expression level during peak lactation period. PROX1 knockdown enhanced the expression of genes related to de novo fatty acid synthesis (e.g., SREBP1 and FASN) and triacylglycerol (TAG) synthesis (e.g., DGAT1 and GPAM) in goat mammary epithelial cells (GMECs). Consistently, intracellular TAG and lipid droplet contents were significantly increased in PROX1 knockdown cells and reduced in PROX1 overexpression cells, and we observed similar results in PROX1 knockout mice. Following PROX1 overexpression, RNA-seq showed a significant upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) expression. Further, PPARGC1A knockdown attenuated the inhibitory effects of PROX1 on TAG contents and lipid-droplet formation in GMECs. Moreover, we found that PROX1 promoted PPARGC1A transcription via the PROX1 binding sites (PBSs) located in the PPARGC1A promoter. These results suggest a novel target for manipulating the goat milk-fat composition and improving the quality of goat milk.

The FoxO1-ATGL axis alters milk lipolysis homeostasis through PI3K/AKT signaling pathway in dairy goat mammary epithelial cells.

Goat milk is enriched in fatty acids which are beneficial to human health. Previous research has revealed that 98% of milk fat is composed of triglycerides. However, the mechanisms regulating milk fat composition remain unclear. Forkhead box protein O1 (FoxO1) is a crucial regulatory factor involved in lipid metabolism across various cell types. Chromatin immunoprecipitation sequencing (ChIP)-seq data) and RNA sequencing (RNA-seq) data revealed that have indicated a close association between FoxO1 was closely related to lipid metabolism during lactation in dairy goats. The objective of this study was to investigate the mechanisms by which FoxO1 regulates lipid metabolism in goat mammary epithelial cells (GMECs). FoxO1 knockdown significantly downregulated the expression of adipose triglyceride lipase (ATGL) and suppressed the activity of the ATGL promoter. Consistently, the number of lipid droplets decreased significantly in FoxO1-overexpressing cells and increased in ATGL-knockdown cells. To further verify the effect of FoxO1 on ATGL promoter activity, cells were transfected with four promoter fragments of different lengths. We found that the core region of the ATGL promoter was located between -882 bp and -524 bp, encompassing two FoxO1 binding sites (FKH1 and FKH2). Mutations in the FoxO1 binding sites significantly downregulated ATGL promoter activity in GMECs. Luciferase reporter assays demonstrated that FoxO1 overexpression markedly enhanced ATGL promoter activity. Furthermore, site-directed mutation confirmed that FKH1 and FKH2 sites were simultaneously mutated significantly attenuated the stimulatory effect of FoxO1 on ATGL promoter activities simultaneous mutation of FKH1 and FKH2 sites significantly attenuated the stimulatory effect of FoxO1 on ATGL promoter activity. ChIP assays showed that FoxO1 directly binds to the FKH2 element located in the ATGL promoter in vivo. Finally, immunofluorescence staining revealed that insulin promotes the translocation of FoxO1 from the nucleus to the cytoplasm, thereby attenuating the FoxO1-induced activation of the ATGL promoter. Collectively, these findings uncover a novel pathway where by FoxO1 may regulate lipid metabolism in GMECs specifically by modulating the transcriptional activity of ATGL.

Forkhead box protein O1(FoxO1) is a key cellular regulatory factor that was involved in lipid metabolism in several cell types. This study was performed to explore the regulatory mechanism of FoxO1 in adipose triglyceride lipase (ATGL) promoter-driven transcription during lactation in dairy goats. Chromatin immunoprecipitation (ChIP)-seq and RNA sequencing (RNA-seq) data revealed that FoxO1 was closely related to lipid metabolism and inflammation during lactation in dairy goats. FoxO1 overexpression significantly decreased cellular triglyceride (TAG) content lipid droplet accumulation in goat mammary epithelial cells (GMECs), while ATGL knockdown attenuated this effect of FoxO1. Furthermore, the relative content of free fatty acid (FFAs) was markedly increased in FoxO1-overexpressed cells. Additionally, site-directed mutation and ChIP assays confirmed that FoxO1 promotes ATGL transcription through FoxO1 binding sites (FKH) located in the ATGL promoter. Moreover, insulin attenuated the FoxO1-induced activation of the ATGL promoter. Our data reveal that FoxO1 regulates the activity of ATGL in GMECs by binding to FKH elements located in the ATGL promoter.

Docosahexaenoic Acid Alters Lipid Metabolism Processes via H3K9ac Epigenetic Modification in Dairy Goat.

Goat milk is increasingly recognized by consumers due to its high nutritional value, richness in short- and medium-chain fatty acids, and richness in polyunsaturated fatty acids (PUFA). Exogenous supplementation of docosahexaenoic acid (DHA) is an important approach to increasing the content of PUFA in goat milk. Several studies have reported benefits of dietary DHA in terms of human health, including potential against chronic diseases and tumors. However, the mechanisms whereby an increased supply of DHA regulates mammary cell function is unknown. In this study, we investigated the effect of DHA on lipid metabolism processes in goat mammary epithelial cells (GMEC) and the function of H3K9ac epigenetic modifications in this process. Supplementation of DHA promoted lipid droplet accumulation increased the DHA content and altered fatty acid composition in GMEC. Lipid metabolism processes were altered by DHA supplementation through transcriptional programs in GMEC. ChIP-seq analysis revealed that DHA induced genome-wide H3K9ac epigenetic changes in GMEC. Multiomics analyses (H3K9ac genome-wide screening and RNA-seq) revealed that DHA-induced expression of lipid metabolism genes (FASN, SCD1, FADS1, FADS2, LPIN1, DGAT1, MBOAT2), which were closely related with changes in lipid metabolism processes and fatty acid profiles, were regulated by modification of H3K9ac. In particular, DHA increased the enrichment of H3K9ac in the promoter region of PDK4 and promoted its transcription, while PDK4 inhibited lipid synthesis and activated AMPK signaling in GMEC. The activation of the expression of fatty acid metabolism-related genes FASN, FADS2, and SCD1 and their upstream transcription factor SREBP1 by the AMPK inhibitor was attenuated in PDK4-overexpressing GMEC. In conclusion, DHA alters lipid metabolism processes via H3K9ac modifications and the PDK4-AMPK-SREBP1 signaling axis in goat mammary epithelial cells, providing new insights into the mechanism through which DHA affects mammary cell function and regulates milk fat metabolism.

Goat FADS2 controlling fatty acid metabolism is directly regulated by SREBP1 in mammary epithelial cells.

Goat milk provides benefits to human health due to its richness in bioactive components, such as polyunsaturated fatty acids (PUFAs). The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in PUFAs biosynthesis. However, its role and transcriptional regulation mechanisms in fatty acid metabolism in dairy goat remain unclear. Here, our study revealed that the FADS2 gene was highly expressed during the peak lactation compared with the dry period, early lactation, and late lactation. The content of triacylglycerol (TAG) was enhanced with the increasing mRNA expression of TAG synthesis genes (diacylglycerol acyltransferase 1/2, DGAT1/2) in FADS2-overexpressed goat mammary epithelial cells (GMECs). Overexpression of FADS2 was positively correlated with the elevated concentrations of dihomo-gamma-linolenic acid (DGLA) and docosahexaenoic acid (DHA) in GMECs. BODIPY staining showed that FADS2 promoted lipid droplet accumulation in GMECs. To clarify the transcriptional regulatory mechanisms of FADS2, 2,226 bp length of FADS2 promoter was obtained. Deletion mutation assays revealed that the core region of FADS2 promoter was located between the -375 and -26 region, which contained SRE1 (-361/-351) and SRE2 (-191/-181) cis-acting elements of transcription factor sterol regulatory element-binding protein 1 (SREBP1). Overexpression of SREBP1 enhanced relative luciferase activity of the single mutant of SRE1 or SRE2, vice versa, and failed to alter the relative luciferase activity of the joint mutant of SRE1 and SRE2. Chromatin immunoprecipitation (ChIP) and site-directed mutation assays further demonstrated that SREBP1 regulated the transcription of the FADS2 gene by binding to SRE sites in vivo and in vitro. In addition, the mRNA levels of FADS2 were significantly decreased by targeting SRE1 and SRE2 sites in the genome via the CRISPR interference (CRISPRi) system. These findings establish a direct role for FADS2 regulating TAG and fatty acid synthesis by SREBP1 transcriptional regulation in dairy goat, providing new insights into fatty acid metabolism in mammary gland of ruminants.

The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in polyunsaturated fatty acids (PUFAs) biosynthesis in mammals. This study aimed to investigate the function and transcriptional regulation mechanism of FADS2 in goat mammary epithelial cells (GMECs). The content of triacylglycerol (TAG) was enhanced with lipid droplet accumulation in FADS2-overexpressed GMECs. Overexpression of FADS2 was positively correlated with elevated concentrations of docosahexaenoic acid (DHA) in GMECs. Furthermore, site-directed mutation and chromatin immunoprecipitation (ChIP) assays simultaneously demonstrated that FADS2 was directly regulated by SREBP1 transcriptional factor binding to sterol regulatory element (SRE) in vitro and in vivo. In addition, genetic ablation of SRE1 and SRE2 in the genome resulted in a significant reduction in the mRNA levels of FADS2 via the CRISPR interference (CRISPRi) system. Altogether, this study discovered that the SREBP1 exerts control on FADS2 to regulate milk fatty acids, and provides a theoretical approach for improving milk quality via genetic approaches.

Functional study and epigenetic targets analyses of SIRT1 in intramuscular preadipocytes via ChIP-seq and mRNA-seq.

The SIRT1 epigenetic regulator is involved in hepatic lipid homoeostasis. However, the role of SIRT1 in regulating intramuscular fat deposition as well as the pathways and potential epigenetic targets involved remain unknown. Herein, we investigate SIRT1 function, its genome-wide epigenetic target profile, and transcriptomic changes under SIRT1 overexpression during yak intramuscular preadipocytes differentiation. To this end, we analysed the relationship between SIRT1 and intramuscular fat content as well as lipid metabolism-related genes in longissimus dorsi tissue. We found that SIRT1 expression negatively correlates with intramuscular fat content as well as with the expression of genes related to lipid synthesis, while positively correlating with that of fatty acid oxidation-involved genes. SIRT1 overexpression in intramuscular preadipocytes significantly reduced adipose differentiation marker expression, intracellular triacylglycerol content, and lipid deposition. Chromatin immunoprecipitation coupled with high-throughput sequencing of H3K4ac (a known direct target of SIRT1) and high-throughput mRNA sequencing results revealed that SIRT1 may regulate intramuscular fat deposition via three potential new transcription factors (NRF1, NKX3.1, and EGR1) and four genes (MAPK1, RXRA, AGPAT1, and HADH) implicated in protein processing within the endoplasmic reticulum pathway and the MAPK signalling pathway in yaks. Our study provides novel insights into the role of SIRT1 in regulating yak intramuscular fat deposition and may help clarify the mechanistic determinants of yak meat characteristics.

Genome-wide analysis of the acyl-coenzyme A synthetase family and their association with the formation of goat milk flavour.

Goat milk is rich in fat and protein, thus, has high nutritional values and benefits human health. However, goaty flavour is a major concern that interferes with consumer acceptability of goat milk and the 4-alkyl-branched-chain fatty acids (vBCFAs) are the major substances relevant to the goaty flavour in goat milk. Previous research reported that the acyl-coenzyme A synthetases (ACSs) play a key role in the activation of fatty acids, which is a prerequisite for fatty acids entering anabolic and catabolic processes and highly involved in the regulation of vBCFAs metabolism. Although ACS genes have been identified in humans and mice, they have not been systematically characterized in goats. In this research, we performed genome-wide characterization of the ACS genes in goats, identifying that a total of 25 ACS genes (without ACSM2A) were obtained in the Capra hircus and each ACS protein contained the conserved AMP-binding domain. Phylogenetic analysis showed that out of the 25 genes, 21 belonged to the ACSS, ACSM, ACSL, ACSVL, and ACSBG subfamilies. However, AACS, AASDH, ACSF, and ACSF3 genes were not classified in the common evolutionary branch and belonged to the ACS superfamily. The genes in the same clade had similar conserved structures, motifs and protein domains. The expression analysis showed that the majority of ACS genes were expressed in multi tissues. The comparative analysis of expression patterns in non-lactation and lactation mammary glands of goat, sheep and cow indicated that ACSS2 and ACSF3 genes may participate in the formation mechanisms of goaty flavour in goat milk. In conclusion, current research provides important genomic resources and expression information for ACSs in goats, which will support further research on investigating the formation mechanisms of the goaty flavour in goat milk.

Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells.

αS1-Casein (encoded by the CSN1S1 gene) is associated with food allergy more than other milk protein components. Milk allergy caused by αS1-casein is derived from cow milk, goat milk and other ruminant milk. However, little is known about the transcription regulation of αS1-casein synthesis in dairy goats. This study aimed to investigate the regulatory roles of signal transducer and activator of transcription 5 (STAT5) on αS1-casein in goat mammary epithelial cells (GMEC). Deletion analysis showed that the core promoter region of CSN1S1 was located at -110 to -18 bp upstream of transcription start site, which contained two putative STAT5 binding sites (gamma-interferon activation site, GAS). Overexpression of STAT5a gene upregulated the mRNA level and the promoter activity of the CSN1S1 gene, and STAT5 inhibitor decreased phosphorylated STAT5 in the nucleus and CSN1S1 transcription activity. Further, GAS site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays revealed that GAS1 and GAS2 sites in the CSN1S1 promoter core region were binding sites of STAT5. Taken together, STAT5 directly regulates CSN1S1 transcription by GAS1 and GAS2 sites in GMEC, and the mutation of STAT5 binding sites could downregulate CSN1S1 expression and decrease αS1-casein synthesis, which provide the novel strategy for reducing the allergic potential of goat milk and improving milk quality in ruminants.

miR-204-5p and miR-211 Synergistically Downregulate the αS1-Casein Content and Contribute to the Lower Allergy of Goat Milk.

αS1-Casein (encoded by the CSN1S1 gene) is associated with higher rates of allergy than other milk protein components for humans. microRNAs (miRNAs) as small noncoding RNA molecules regulate gene expression and influence diverse biological processes. However, little is known about the regulation of milk protein synthesis by miRNAs in ruminants. In this study, we aim to investigate the regulatory roles of miR-204 family members (miR-204-5p and miR-211) on αS1-casein in goat mammary epithelial cells (GMEC). Here, we observed that the CSN1S1 mRNA level is upregulated, while miR-204-5p and miR-211 (miR-204-5p/-211) abundance is downregulated during peak lactation compared with middle lactation of dairy goats. We found that miR-204-5p/-211 synergistically inhibit αS1-casein expression via directly binding to the 3'-untranslated region (3'UTR) of CSN1S1 in GMEC. miR-204-5p/-211 increase ß-casein mRNA (CSN2) and protein abundance, as well as the signal transducer and activator of transcription 5a (STAT5a) activity. Further, miR-204-5p/-211 enhance ß-casein expression via the CSN1S1-STAT5a signaling axis and promote ß-casein transcription by activating the STAT5 response element located in the CSN2 promoter. In conclusion, miR-204-5p/-211 regulate αS1-casein and ß-casein synthesis via targeting CSN1S1 in GMEC, which provide the strategy for manipulating miR-204 family members to reduce milk allergy potential and improve ruminant milk quality for human consumption.

ELOVL6 promoter binding sites directly targeted by sterol regulatory element binding protein 1 in fatty acid synthesis of goat mammary epithelial cells.

The elongation of long-chain fatty acid family member 6 (ELOVL6) gene plays an important role in the synthesis of long-chain saturated and monounsaturated fatty acids. Although some studies have revealed that ELOVL6 is the target of sterol regulatory element binding protein 1 (SREBP1; gene name SREBF1) in rodents, the mechanism underlying ELOVL6 regulation during lactation in dairy goats remains unknown. The present study aimed to investigate the transcriptional regulation mechanism of ELOVL6 in goat mammary epithelial cells (GMEC). We used PCR to clone and sequenced a 2,370 bp fragment of the ELOVL6 5' flanking region from goat genomic DNA. Deletion analysis revealed a core promoter region located -105 to -40 bp upstream of the transcriptional start site. Mutant sterol regulatory elements (SRE) 1 and 3 significantly reduced the ELOVL6 promoter activities in GMEC. Both SRE1 and SRE3 binding sites were required for the basal transcriptional activity of ELOVL6. Luciferase reporter assays showed that SREBF1 knockdown decreased ELOVL6 promoter activities in GMEC. Furthermore, SRE1 and SRE3 sites were simultaneously mutated completely abolished the stimulatory effect of SREBF1 and the repressive effect of linoleic acid on ELOVL6 gene promoter activities. Furthermore, chromatin immunoprecipitation assays confirmed that SREBP1 directly bound to SRE sites in the ELOVL6 promoter. In conclusion, these results indicate that SREBP1 regulates ELOVL6 transcription via the SRE elements located in the ELOVL6 promoter in goat mammary gland.

FoxO1 Knockdown Promotes Fatty Acid Synthesis via Modulating SREBP1 Activities in the Dairy Goat Mammary Epithelial Cells.

FoxO1 is a crucial transcription factor involved in lipid metabolism in mouse liver through repressing a key regulator of lipogenesis, sterol regulatory element binding protein 1 (SREBP1). However, it remains elusive whether FoxO1 plays roles in the regulation of fatty acid metabolism during lactation in dairy goats. In this study, we aim to investigate the function of FoxO1 in goat mammary epithelial cells (GMECs). We found that the expression of FoxO1 is significantly upregulated during lactation compared with the dry period. FoxO1 knockdown enhanced the expression of genes related to de novo fatty acid synthesis (e.g., FASN, ELOVL6 and SCD1) and triacylglycerol (TAG) synthesis (e.g., DGAT2 and GPAM). Consistently, intracellular TAG was significantly increased in FoxO1 knockdown cells and reduced in FoxO1 overexpression cells. Immunofluorescence staining revealed that insulin suppresses FoxO1 transcription by promoting its nuclear export. Further, we found that FoxO1 inhibits insulin-induced SREBP1 promoter activities in GMECs. Moreover, FoxO1 suppresses SREBP1 transcription via the LXR response element (LXRE) and SREBP response element (SRE) located in the SREBP1 promoter. Our data reveal that FoxO1 plays critical roles in regulating the synthesis of the fatty acid and triacylglycerol (TAG) in GMECs.

Identification and characterization of putative ovarian lincRNAs in dairy goats treated for repeated estrous synchronization.

The goal of this study was to identify and characterize effects of repeated estrous synchronization (ES) treatments on the regulation of ovarian intergenic long non-coding RNAs (lincRNAs) in dairy goats. Six does were randomly assigned to a group administered three ES treatment regimens separated by 2 weeks or to a group administered only one ES treatment regimen (control) at the same time as the third ES treatment in the does administered the three hormonal regimens for ES. The paired-end RNA Sequencing procedures were used to evaluate lincRNAs of ovarian tissues. A total of 134 lincRNAs were differentially abundant between the two treatment groups. Several target genes were annotated and were related to hormone activity, cellular response to hormone stimulus, response to hormone, female pregnancy, as well as regulation of hormone secretion. These genes were noticeably enriched in MAPK, Hippo, estrogen signaling pathways, oocyte meiosis, progesterone-mediated oocyte maturation, ovarian steroidogenesis as well as GnRH signaling pathways. According to the enriched GO terms and KEGG pathways of regulated genes, 13 differentially abundant lincRNAs could be promising candidates for regulating reproductive functions of female goats. Current results indicate that repeated treatments with gonadotropins affected hormone sensitivity, estrogen synthesis, and ovarian function. The results also indicated that when there was imposing of the three hormonal treatment regimens for ES, there were several lincRNAs that could contribute to dysregulation of several genes that are important for reproduction in dairy goats. Findings provide novel insights for further investigation of lncRNAs biological functions in goats.

Insulin-induced gene 1 and 2 isoforms synergistically regulate triacylglycerol accumulation, lipid droplet formation, and lipogenic gene expression in goat mammary epithelial cells.

The insulin-induced genes INSIG1 and INSIG2 (INSIG) are known to regulate adipogenesis in nonruminants. Although data in bovine mammary tissue underscore a role for INSIG1 during lactation, regulatory mechanisms of INSIG action in ruminant mammary lipid metabolism are not well known. In the present study, INSIG1 and INSIG2 were overexpressed or silenced through adenoviral transfection to evaluate their role in lipid metabolism in goat mammary epithelial cells (GMEC). The INSIG were overexpressed using an adenovirus system with recombinant green fluorescent protein as the control. Downregulation of INSIG was performed via small interfering RNA targeting INSIG with a scrambled small interfering RNA as a negative control. The GMEC were treated with these constructs for 48 h before analyses. Responses to overexpressing INSIG1 or INSIG2 included downregulation of SREBF1, ACACA, FASN, SCD1, GPAM, DGAT2, ATGL, and HSL coupled with a decrease in content of triacylglycerol (TAG), total cholesterol (TC), and lipid droplet accumulation. The marked decrease in content of TAG and TC in response to overexpression of INSIG2, along with a modest decrease in content of TAG when INSIG1 was overexpressed, suggested that TAG synthesis is mainly regulated by INSIG2, whereas TC synthesis is equally regulated by INSIG2 and INSIG1. The lack of difference in mRNA expression of genes related to lipid metabolism, content of TAG, and accumulation of lipids in response to interference alone of INSIG1 or INSIG2 indicated that INSIG proteins play a biological role in the maintenance of lipid homeostasis. However, in response to simultaneous interference of INSIG1 and INSIG2, the marked increase in content of TAG and TC and accumulation of lipids along with significant upregulation of SREBF1, ACACA, SCD1, AGPAT6, and DGAT2 suggested that INSIG1 and INSIG2 synergistically regulate milk fat synthesis in GMEC. These results highlight an essential role of INSIG in regulating lipid synthesis in dairy goat mammary cells and underscore the complexity of mammary lipid synthesis in ruminants.

Association between the expression of miR-26 and goat milk fatty acids.

microRNA (miRNA) are small noncoding RNA that regulate protein abundance and are involved in diverse aspects of cellular function including aspects of lipid metabolism in mammary gland of ruminants. Although our previous studies showed that the miR-26 family and its host genes control components of the cellular fatty acid metabolic machinery in goat mammary epithelial cells, a direct relationship between the miR-26 family and milk fatty acids remains unknown. Bioinformatics analysis in this study indicated that the miR-26 family targets belong to the PI3K-Akt signalling pathway, MAPK signalling pathway, and fatty acid biosynthesis pathway. Studies on the relationship of miR-26 family and their host genes with milk composition during mid-lactation revealed that the expression of the miR-26 family and their host genes were associated with total fat yield and short-chain, medium-chain and long-chain fatty acid content, but not lactose or milk protein content. In addition, a significant positive correlation was detected for the expression of the miR-26 family with C16:1 and C18:3 in milk fat. Taken together, our findings demonstrate that the expression of miR-26 is directly related to milk fatty acid composition and underscores the significance of miRNAs in milk fat synthesis regulation.

miR-26b promoter analysis reveals regulatory mechanisms by lipid-related transcription factors in goat mammary epithelial cells.

MicroRNA (miRNA) regulate protein abundance and control diverse aspects of cellular processes and biological functions associated with lipid metabolism. MiR-26b and its host gene CTDSP1 regulate triacylglycerol synthesis by synergistically suppressing the insulin-induced gene 1 (INSIG1); however, the direct regulators of miR-26b expression remain unknown. In the present study, we characterized the activity of a novel putative promoter region in miR-26b. Results revealed that promoter activity and miR-26b expression are dynamically regulated by different transcription factors including peroxisome proliferator-activated receptor gamma (PPARG), sterol regulatory element binding transcription factor 1 (SREBF1), and liver X receptor α (LXRα). Two binding sites for the SREBF1 (SRE1 and SRE3) and the PPARG (peroxisome proliferator response element 1 and 2; PPRE1 and PPRE2), respectively, were identified in the miR-26b promoter, which demonstrated that those binding sites are responsible for the activation by PPARG and SREBF1. In silico analysis and site-directed mutagenesis of LXRα binding elements (LXRE) and SREBF1 binding elements (SRE) revealed that the effects of Ad-LXRα + T0901317 requires the presence of SRE, whereas potential LXRE had no effects on miR-26b expression. This suggested that regulation of miR-26b by LXRα is indirectly via an SRE, and miR-26b is regulated by transcription factors dually through DNA methylation and directly through binding to its promoter, all of which implies that regulation of miR-26b in ruminant mammary epithelial cells results from various mechanisms. In conclusion, we demonstrate a novel dual-regulatory mechanism whereby transcription factors regulate the expression of miR-26b. Overall, these findings contribute to our understanding of the interactions between specific promoter elements and the control of transcription and translation of miRNA.

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.