Acetyl-coenzyme A Carboxylase Alpha Research Articles

MiR-206 Suppresses Triacylglycerol Accumulation via Fatty Acid Elongase 6 in Dairy Cow Mammary Epithelial Cells.

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA-mRNA networks.

Tissue-Specific Expression of Circ_015343 and Its Inhibitory Effect on Mammary Epithelial Cells in Sheep.

Circular RNAs (circRNAs) are a kind of non-coding RNA that have an important molecular function in mammary gland development and lactation of mammals. In our previous study, circ_015343 was found to be highly expressed in the ovine mammary gland tissue at the peak-lactation period by using RNA sequencing (RNA-seq). In the present study, the authenticity of circ_015343 was confirmed by using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and Sanger sequencing. The circ_015343 was derived from the complete 10 exons of aminoadipic semialdehyde synthase (AASS), ranging from exon 2 to exon 11 and mainly located in cytoplasm of ovine mammary epithelial cells. The circRNA was found to be expressed in eight ovine tissues, with the highest expression level in the mammary gland and the least expression in Longissimus dorsi muscle. The circ_015343 had a lower level of expression in a sheep breed with higher milk yield and milk fat content. The disturbed circ_015343 increased the viability and proliferation of the ovine mammary epithelial cells. The inhibition of circ_015343 also increased the expression levels of three milk fat synthesis marker genes: acetyl-coenzyme A carboxylase alpha (ACACA), fatty acid-binding protein 4 (FABP4), and sterol regulatory element-binding protein 1 (SREBP1), as well as three proliferation-related genes: cyclin dependent kinase 2 (CDK2), cyclin dependent kinase 4 (CDK4) and proliferating cell nuclear antigen (PCNA), but decreased the expression level of its parent gene AASS. A circRNA-miRNA-mRNA interaction network showed that circ_015343 would bind some microRNAs (miRNAs) to regulate the expression of functional genes related to the development of mammary gland and lactation. This study contributes to a better understanding of the roles of circ_015343 in the mammary gland of sheep.

Relative gene expression of fatty acid synthesis genes at 60 days postpartum in bovine mammary epithelial cells of Surti and Jafarabadi buffaloes.

Aim:Aim of the study was to study the relative gene expression of genes associated with fatty acid synthesis at 60 days postpartum (pp) in bovine mammary epithelial cells (MECs) of Surti and Jafarabadi buffaloes.Materials and Methods:A total of 10 healthy Surti and Jafarabadi buffaloes of each breed were selected at random from Livestock Research Station, Navsari and Cattle Breeding Farm, Junagadh, Gujarat, respectively, for this study. Milk sample was collected from each selected buffalo at day 60 pp from these two breeds to study relative gene expression of major milk fat genes using non-invasive approach of obtaining primary bovine MECs (pBMEC) from milk samples.Results:In this study overall, the relative expression of the six major milk lipogenic genes butyrophilin subfamily 1 member A1 (BTN1A1), stearoyl-CoA desaturase (SCD), lipoprotein lipase (LPL), glycerol-3-phosphate acyltransferase mitochondrial (GPAM), acetyl-coenzyme A carboxylase alpha (ACACA), and lipin (LPIN) did not show changes in expression patterns at 60th day of lactation in both Surti and Jafarabadi buffaloes.Conclusion:The pBMEC can be successfully recovered from 1500 ml of milk of Surti and Jafarabadi buffaloes using antibody-mediated magnetic bead separation and can be further used for recovering RNA for down step quantification of major milk lipogenic gene expression. The relative expression of the six major milk lipogenic genes BTN1A1, SCD, LPL, GPAM, ACACA, and LPIN did not show changes in expression patterns in both Surti and Jafarabadi buffaloes, suggesting expression levels of lipogenic genes are maintained almost uniform till peak lactation without any significant difference.

Long form PRLR (lPRLR) regulates genes involved in the triacylglycerol synthesis in goat mammary gland epithelial cells

The prolactin receptor (PRLR) is a single-pass transmembrane receptor belonging to the class-1 cytokine receptor superfamily and known to play a crucial role in lactation and milk synthesis. However, very little information is available on the roles of PRLR in the regulation of lipid metabolism in the goat mammary gland. In this study, we cloned the whole cDNA of goat long form PRLR (lPRLR). The full-length cDNA of lPRLR was comprised of a 327-bp 5′-untranslated region, 1746-bp open reading frame and 183-bp 3′-UTR. The deduced amino acid sequence encoded 581 amino acids (aa) with signal peptide (24 aa) and single transmembrane domain (22 aa). Homology alignments revealed that the goat lPRLR was highly conserved among sheep, bovine, and buffalo. Real-time quantitative PCR suggested that goat lPRLR was predominantly expressed in the muscle and liver. Treatment of goat mammary epithelial cells (GMECs) with prolactin resulted in Protein Kinase B (AKT) phosphorylation, elevated Peroxisome Proliferator-Activated Receptor γ (PPARG), Sterol Regulatory Element-Binding Protein-1 (SREBP1), Fatty Acid Synthesis (FASN), and Acetyl-Coenzyme A Carboxylase Alpha (ACACA) mRNA levels, and significantly increased the triglyceride content (P<0.05). The decreases in expression observed after knockdown of PRLR relative to the control group averaged 76%, 55%, 52%, and 68% of PPARG, SREBP1, FASN and ACACA, respectively. These results possibly provide direct evidence that lPRLR is an extensive-distribution transmembrane protein, involved in regulating the triacylglycerol synthesis in GMECs during lactation.

Epistatic effect between ACACA and FABP2 gene on abdominal fat traits in broilers

Epistasis is generally defined as the interaction between two or more genes or their mRNA or protein products to influence a single trait. Experimental evidence suggested that epistasis could be important in the determination of the genetic architecture of complex traits in domestic animals. Acetyl-coenzyme A carboxylase alpha (ACACA) and fatty acid binding protein 2 (FABP2) are both key factors of lipogenesis and transport. They may play a crucial role in the weight variability of abdominal adipose tissue in the growing chicken. In this study, the polymorphisms of c.2292G>A in ACACA and c.-561A>C in FABP2 were detected among individuals from two broiler lines which were divergently selected for abdominal fat content. Epistasis between the two SNPs on abdominal fat weight (AFW) and abdominal fat percentage (AFP) was analyzed. The additive x additive epistatic components between these two SNPs were found significant or suggestively significant on both AFW and AFP in lean lines of the 9th and 10th generation; whereas, it was not significantly associated with either AFW or AFP in fat lines. At the same time, there were not any other significant epistatic components found in both generations or in both lines. Significant epistatic effects between these two SNPs found only in the lean lines could partly be due to the fact that the abdominal fat traits in these two experimental lines have been greatly modified by strong artificial selection. The results suggested that the epistasis mode may be different between the lean and fat chicken lines. Our results could be helpful in further understanding the genetic interaction between candidate genes contributing to phenotypic variation of abdominal fat content in broilers.

Effects of homocysteine on the expression of osteoblastic genes and on genes related to cholesterol and fat metabolism

Epistasis is generally defined as the interaction between two or more genes or their mRNA or protein products to influence a single trait. Experimental evidence suggested that epistasis could be important in the determination of the genetic architecture of complex traits in domestic animals. Acetyl-coenzyme A carboxylase alpha (ACACA) and fatty acid binding protein 2 (FABP2) are both key factors of lipogenesis and transport. They may play a crucial role in the weight variability of abdominal adipose tissue in the growing chicken. In this study, the polymorphisms of c.2292G>A in ACACA and c.-561A>C in FABP2 were detected among individuals from two broiler lines which were divergently selected for abdominal fat content. Epistasis between the two SNPs on abdominal fat weight (AFW) and abdominal fat percentage (AFP) was analyzed. The additive × additive epistatic components between these two SNPs were found significant or suggestively significant on both AFW and AFP in lean lines of the 9th and 10th generation; whereas, it was not significantly associated with either AFW or AFP in fat lines. At the same time, there were not any other significant epistatic components found in both generations or in both lines. Significant epistatic effects between these two SNPs found only in the lean lines could partly be due to the fact that the abdominal fat traits in these two experimental lines have been greatly modified by strong artificial selection. The results suggested that the epistasis mode may be different between the lean and fat chicken lines. Our results could be helpful in further understanding the genetic interaction between candidate genes contributing to phenotypic variation of abdominal fat content in broilers.

Hepatic Lipid Composition and Stearoyl-Coenzyme A Desaturase 1 mRNA Expression Can Be Estimated from Plasma VLDL Fatty Acid Ratios

Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the limiting step of monounsaturated fatty acid synthesis in humans and is an important player in triglyceride generation. SCD1 has been repeatedly implicated in the pathogenesis of metabolic and inflammatory diseases. Therefore it is of great importance to determine SCD1 activity in human samples. In this study we aimed to evaluate a hepatic SCD1 activity index derived from plasma VLDL triglyceride composition as a tool to estimate hepatic SCD1 expression in humans. Additionally, we further evaluated commonly used fatty acid ratios [elongase, de novo lipogenesis, and Delta5 and Delta6 desaturase] in plasma VLDL and hepatic lipid fractions. Liver biopsies and plasma samples were simultaneously collected from 15 individuals. Plasma VLDL was obtained by ultracentrifugation. Hepatic and plasma VLDL lipids were fractionated by thin-layer chromatography, and the fatty acid composition of each fraction was analyzed by gas chromatography. Hepatic SCD1 expression was determined by real-time PCR. Hepatic SCD1 mRNA expression was associated with the product/precursor ratios (16:1/16:0 and 18:1/18:0) of hepatic lipid fractions. The 16:1/16:0 ratio in hepatic and VLDL triglycerides as well as the 18:1/18:0 ratio in plasma VLDL were closely associated with hepatic SCD1 expression. The hepatic de novo lipogenesis index from triglycerides was associated with expression of lipogenic genes [fatty acid synthase (FASN), acetyl-Coenzyme A carboxylase alpha (ACACA), and sterol regulatory element binding transcription factor 1 (SREBP-1)] and is closely reflected by the de novo lipogenesis index in VLDL triglycerides. We demonstrated for the first time that hepatic SCD1 expression can be estimated noninvasively from routine blood samples by measuring the SCD1 activity index in fasting plasma VLDL.

Polymorphism of the pig acetyl‐coenzyme A carboxylase α gene is associated with fatty acid composition in a Duroc commercial line

Acetyl-coenzyme A carboxylase alpha (ACACA) catalyses the first committed step in the biosynthesis of long-chain fatty acids (FA) by converting acetyl-CoA into malonyl-CoA. In pigs, the ACACA gene maps to a chromosome 12 QTL with important effects on FA composition. In the present study, we have sequenced the coding region of the pig ACACA gene in 15 pigs, identifying 21 polymorphic sites that were either synonymous or non-coding. Ten of these SNPs segregated in a Duroc commercial population (n = 350) for which lipid metabolism and meat and carcass quality trait records were available. Significant associations were found between two linked single nucleotide polymorphisms (c.4899G>A and c.5196T>C) and percentages of carcass lean, intramuscular fat, monounsaturated, saturated (myristic, palmitic and stearic) and polyunsaturated (linoleic) FAs in the longissimus thoracis et lumborum muscle, along with serum HDL-cholesterol concentration. The most important allele substitution effects were observed for the polyunsaturated/saturated FA ratio (13-21% of the phenotypic mean) as well as for the percentages of omega-6 and polyunsaturated FAs, especially linoleic acid (7-16% of the phenotypic mean). These results suggest the existence of a causal mutation, mapping to the chromosomal region containing the pig ACACA gene, with marked effects on FA composition of meat.

The Kampo Medicines Orengedokuto, Bofutsushosan and Boiogito Have Different Activities to Regulate Gene Expressions in Differentiated Rat White Adipocytes: Comprehensive Analysis of Genetic Profiles

Three Kampo medicines, Boiogito (BOT), Bofutsushosan (BTS) and Orengedokuto (OGT), used for obese patients were investigated for their effects on adipogenesis in cultured rat white adipocytes. Administration of the three extracts suppressed adipogenesis in concentration-dependent manners (1-100 microg/ml) without any cytotoxicity. Changes in mRNA expression levels were analyzed using a Rat 230 2.0 Affymetrix GeneChip microarray system. DNA microarray analysis (total probe set: 31099) using cDNAs prepared from adipocytes revealed that BOT, BTS and OGT increased the expression of 133-150 genes and decreased the expression of 42-110 genes by > or =2-fold. We identified 329 downregulated genes and 189 upregulated genes among a total set of 514 probes (overlap: 4). Overall, genes related to cellular movement, cell death, cell growth/differentiation and immune responses were the most downregulated, while those related to lipid metabolism and cell signaling were the most upregulated. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted to confirm the microarray results. Analysis of the clustering profiles of the microarray results revealed that BOT and BTS changed the expression levels of similar genes mainly involved in small molecule biochemistry and cell differentiation, while OGT altered 10 genes related to lipid metabolism, in contrast to the effects of BOT and BTS. We also measured mRNA expression levels of seven selected genes highly contributing to the lipid metabolism by using semiquantitative RT-PCR assay, that were acetyl-Coenzyme A carboxylase alpha (ACACA), AE binding protein 1 (AEBP1), patatin-like phospholipase domain containing 8 (PNPLA8), secretoglobin (SCGB1A1), adrenergic (ADRB3), adiponectin (ADIPOQ), monoglyceride lipase (MGLL). Beta-actin (ACTB) gene was used as an endogenous internal standard. The present findings indicate that these three herbal extracts have the potential to prevent adipogenesis in rat white adipocytes through different mechanisms via modulation of gene expression levels.

QTL detection on porcine chromosome 12 for fatty‐acid composition and association analyses of thefatty acid synthase, gastric inhibitory polypeptideandacetyl‐coenzyme A carboxylase alphagenes

Refinement of previous QTL on porcine chromosome 12 for fatty-acid composition and a candidate gene association analysis were conducted using an Iberian x Landrace cross. The concentrations of ten fatty acids were assayed in backfat tissue from which four metabolic ratios were calculated for 403 F2 animals. Linkage analysis identified two significant QTL. The first QTL was associated with the average chain length ratio and the percentages of myristic, palmitic and gadoleic acids. The second QTL was associated with percentages of palmitoleic, stearic and vaccenic acids. Based upon its position on SSC12, fatty acid synthase was tested as a candidate gene for the first QTL and no significant effects were found. Similarly, gastric inhibitory polypeptide (GIP) and acetyl-coenzyme A carboxylase alpha (ACACA) were tested as candidate genes for the second QTL using three SNPs in GIP and 15 synonymous SNPs in ACACA cDNA sequences. Two missense SNPs in GIP showed significant effects with palmitoleic and stearic fatty-acid concentration. Highly significant associations were found for two SNPs in ACACA with stearic, palmitoleic and vaccenic fatty-acid concentrations. These associations could be due to linkage disequilibrium with the causal mutations.

BRCA1 and acetyl‐CoA carboxylase: The metabolic syndrome of breast cancer

Breast cancer-associated mutations affecting the highly-conserved C-terminal BRCT domains of the tumor suppressor gene breast cancer susceptibility gene 1 (BRCA1) fully disrupt the ability of BRCA1 to interact with acetyl coenzyme A carboxylase alpha (ACCA), the rate-limiting enzyme catalyzing de novo fatty acid biogenesis. Specifically, BRCA1 interacts solely with the phosphorylated (inactive) form of ACCA (P-ACCA), and the formation of the BRCA1/P-ACCA complex interferes with ACCA activity by preventing P-ACCA dephosphorylation. One of the hallmarks of aggressive cancer cells is a high rate of energy-consuming anabolic processes driving the synthesis of lipids, proteins, and DNA (all of which are regulated by the energy status of the cell). The ability of BRCA1 to stabilize the phosphorylated/inactive form of ACCA strongly suggests that the tumor suppressive function of BRCA1 closely depends on its ability to mimic a cellular-low-energy status, which is known to block tumor cell anabolism and suppress the malignant phenotype. Interestingly, physical exercise and lack of obesity in adolescence have been associated with significantly delayed breast cancer onset for Ashkenazi Jewish women carrying BRCA1 gene mutations. Further clinical work may explore a chemopreventative role of "low-energy-mimickers" deactivating the ACCA-driven "lipogenic phenotype" in women with inherited mutations in BRCA1. This goal might be obtained with current therapeutic approaches useful in treating the metabolic syndrome and associated disorders in humans (e.g., type 2 diabetes and obesity), including metformin, thiazolidinediones (TZDs), calorie deprivation, and exercise. Alternatively, new forthcoming ACCA inhibitors may be relevant in the management of BRCA1-dependent breast cancer susceptibility and development.

Metformin-Induced Stimulation of Adenosine 5′ Monophosphate-Activated Protein Kinase (PRKA) Impairs Progesterone Secretion in Rat Granulosa Cells1

Metformin is an anti-diabetic drug commonly used to treat cycle disorders and anovulation in women with polycystic ovary syndrome. However, the effects and molecular mechanism of metformin in the ovary are not entirely understood. We investigated the effects of this drug on steroidogenesis and proliferation in rat granulosa cells. Metformin (10 mM) treatment for 48 h reduced progesterone and estradiol (E2) production in both basal conditions and under FSH stimulation. It also decreased the levels of the HSD3B, CYP11A1, STAR, and CYP19A1 proteins in response to FSH (10(-8) M) and of HSD3B in the basal state only. Metformin treatment (10 mM, 24 h) also reduced cell proliferation and the levels of CCND2 and CCNE proteins without affecting cell viability, both in the basal state and in response to FSH. Furthermore, metformin treatment for 1 h simultaneously increased the Thr172 phosphorylation of PRKAA (adenosine 5' monophosphate-activated protein kinase alpha) and the Ser79 phosphorylation of ACACA (acetyl-Coenzyme A carboxylase alpha). The adenovirus-mediated production of dominant-negative PRKAA totally abolished the effects of metformin on progesterone secretion, HSD3B and STAR protein production, and MAPK3/1 phosphorylation. Conversely, total inhibition of PRKAA Thr172 phosphorylation with the dominant-negative PRKAA adenovirus did not restore the decrease in E2 production and cell proliferation induced by metformin. Our results therefore strongly suggest that metformin reduces progesterone production via a PRKAA-dependent mechanism, whereas PRKAA activation is not essential for the decrease in E2 production and cell growth induced by metformin in rat granulosa cells.

BRCA1 Affects Lipid Synthesis through Its Interaction with Acetyl-CoA Carboxylase

Germ line alterations in BRCA1 (breast cancer susceptibility gene 1) are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 acts as a scaffold protein implicated in multiple cellular functions, such as transcription, DNA repair, and ubiquitination. However, the molecular mechanisms responsible for tumorigenesis are not yet fully understood. We have recently demonstrated that BRCA1 interacts in vivo with acetyl coenzyme A carboxylase alpha (ACCA) through its tandem of BRCA1 C terminus (BRCT) domains. To understand the biological function of the BRCA1.ACCA complex, we sought to determine whether BRCA1 is a regulator of lipogenesis through its interaction with ACCA. We showed here that RNA inhibition-mediated down-regulation of BRCA1 expression induced a marked increase in the fatty acid synthesis. We then delineated the biochemical characteristics of the complex and found that BRCA1 interacts solely with the phosphorylated and inactive form of ACCA (P-ACCA). Finally, we demonstrated that BRCA1 affects lipid synthesis by preventing P-ACCA dephosphorylation. These results suggest that BRCA1 affects lipogenesis through binding to P-ACCA, providing a new mechanism by which BRCA1 may exert a tumor suppressor function.

A novel methodology for the identification of drugable enzymes as targets in cancer via meta analysis of gene expression microarrays

9602 Background: Many studies have used expression microarrays to identify cancer signatures for diagnostic and prognostic purposes. We hypothesized that identifying enzymes with known therapeutic inhibitors, which are highly over-expressed in cancer as compared to benign tissue, may uncover drugable targets shared by multiple cancers and those specific to a cancer type. To test the methodology, we analyzed arrays from untreated prostate and non-small cell lung cancer (NSCLC) patients for their variation in tumor biology and large patient samples. Methods: Utilizing ONCOMINE (www.oncomine.com), we meta-analyzed eight prostate & six lung cancer arrays published in literature. Prior well characterized drug targets such as DNA topoisomerase 1 & 2 were excluded. Differential expression analysis was done utilizing t-statistics and corrected for multiple comparisons using adjusted Qvalues (Q = p* n/I; where n = total number of genes and I=sorted rank of p value). Results: We identified 10 significant enzymes (Q ≤ 1.0e-2): 4 shared, 3 unique to prostate and 4 to NSCLC. Shared targets include two epigenetic enzymes, histone deacetylase1 (HDAC1) and poly (ADP-ribose)polymerase1 (PARP1), guanine monophosphate synthetase (GMPS), an enzyme in nucleoside catabolism involved in DNA synthesis and cellular metabolism, and cyclin-dependent kinase 4 (CDK4), a cell-cycle regulatory protein. Targets unique to prostate cancer are endothelin converting enzyme1 (ECE1) which forms endothelin-1 from its precursor, and two critical enzymes in fatty acid systhesis pathways implicated in prostate cancer pathogenesis: fatty acid synthase (FASN) and acetyl-Coenzyme A carboxylase alpha (ACACA). NSCLC enzymes are a protein that regulates p53 function, NADPH dehydrogenase (NQO1), macrophage migration inhibitory factor (MIF), minichromosome maintenance deficient 6 (MCM6) which is a DNA helicase and casein kinase 2 (CSNK2A1), a putative target of the active anti-oxidant of red wine, resveratrol. Conclusions: Meta-analysis of expression microarrays is a novel methodology which can quickly uncover targets over expressed in multiple cancers and help identify potential therapeutic inhibitors. No significant financial relationships to disclose.