Absence of monoacylglycerol pathway for triacylglycerol synthesis in goat mammary gland.
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ABSTRACT:
SUBMITTER: Hansen HO
PROVIDER: S-EPMC1147112 | biostudies-other | 1986 Aug
REPOSITORIES: biostudies-other
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Project description:The monoacylglycerol acyltransferase (MGAT) pathway has a well-established role in the small intestine where it facilitates the absorption of dietary fat. In enterocytes, MGAT participates in the resynthesis of triacylglycerol using substrates (monoacylglycerol and fatty acids) generated in the gut lumen from the breakdown of triacylglycerol consumed in the diet. MGAT activity is also present in the liver, but its role in triacylglycerol metabolism in this tissue remains unclear. The predominant MGAT isoforms present in human liver appear to be MGAT2 and MGAT3. The objective of this study was to use selective small molecule inhibitors of MGAT2 and MGAT3 to determine the contributions of these enzymes to triacylglycerol production in liver cells. We found that pharmacological inhibition of either enzyme had no effect on TG mass in HepG2 cells but did alter lipid droplet size and number. Inhibition of MGAT2 did result in decreased DG and TG synthesis and TG secretion. Interestingly, MGAT2 preferentially utilized 2-monoacylglycerol derived from free glycerol and not from exogenously added 2-monoacylglycerol. In contrast, inhibition of MGAT3 had very little effect on TG metabolism in HepG2 cells. Additionally, we demonstrated that the MGAT activity of DGAT1 only makes a minor contribution to TG synthesis in intact HepG2 cells. Our data demonstrated that the MGAT pathway has a role in hepatic lipid metabolism with MGAT2, more so than MGAT3, contributing to TG synthesis and secretion.
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Project description:ATP alone had no effect on incorporation of fatty acids synthesized de novo and membrane-bound diacylglycerol into triacylglycerol. Combined addition of ATP and Mg2+ totally inhibits incorporation of fatty acids synthesized de novo and stimulated incorporation of membrane-bound diacylglycerol. ATP, Mg2+ and glycerol 3-phosphate stimulate incorporation of fatty acids synthesized de novo into triacylglycerol, but inhibited the incorporation of membrane-bound diacylglycerol. Diacylglycerol generated in situ was shown to be superior to diacylglycerols preloaded on the membrane as substrate for the diacylglycerol acyltransferase. A model is proposed to explain the effect of absorbed exogenous fatty acid on fatty acid synthesis de novo in goat mammary gland.
| S-EPMC1153655 | biostudies-other
Project description:BACKGROUND: Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT). Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.
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Project description:CCAAT/enhancer binding protein α (C/EBPα) is the key transcription factor involved in lipid metabolism, however, the role of C/EBPα in milk fat synthesis of dairy goats remains unknown. The objective of the present research was to clarify the function of C/EBPα in goat mammary epithelial cells (GMECs) and its impact on peroxisome proliferator-activated receptor gamma (PPARG) promoter activity. In this study, C/EBPα overexpression increased its mRNA and protein levels by 42-fold and 6-fold, respectively. In contrast, transfecting siRNA targeting C/EBPα decreased its mRNA level to 20% and protein abundance to 80% of the basal level. The contents of lipid droplets, triacylglycerol (TAG), and cholesterol were increased (P < 0.05) in C/EBPα-overexpressing GMECs, and knockdown of C/EBPα led to the opposite results. Overexpression of C/EBPα significantly increased the expression levels of genes involved in TAG synthesis (AGPAT6, DGAT2, P < 0.01), lipid droplet formation (PLIN2, P < 0.01), and fatty acid synthesis (FADS2, P < 0.05; ELOVL6, P < 0.01). Knockdown of C/EBPα decreased (P < 0.05) the expression levels of AGPAT6, DGAT1, DGAT2, PLIN2, FADS2, and ELOVL6. C/EBPα upregulated the expression level of PPARG (P < 0.05), and four C/EBPα binding regions were identified in the PPARG promoter at -1,112 to -1,102 bp, -734 to -724 bp, -248 to -238 bp, and -119 to -109 bp. Knockdown of C/EBPα reduced (P < 0.05) the PPARG promoter activity when the C/EBPα binding regions were mutated at -1,112 to -1,102 bp, -734 to -724 bp, and -248 to -238 bp locations of the promoter. However, the promoter activity did not change when the mutation was located at -119 bp. In conclusion, our results suggest that C/EBPα can promote TAG synthesis in GMECs through its effects on mRNA abundance of genes related to lipid metabolism and regulation of the PPARG promoter activity via C/EBPα binding regions.
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Project description:Mesenchymal stem cells have been widely used in the treatment of various chronic diseases. The objective of this survey was to evaluate the therapeutic and regenerative potential of stem cells from adipose tissue (ASCs) in the milk production recovery repair of tissue injury in mastitis goats treated with antimicrobial agents prior to cell therapy. After the diagnosis of mastitis and treatment with gentamicin, eight lactating goats were selected for cellular and subsequent therapy, physical-chemical analysis of milk, ultrasonographic and histopathological examinations. The ASCs were taken from the subcutaneous fat of a young goat cultivated in vitro, marked with Qdots-655 and injected in the left mammary gland, being the right mammary gland used as the control. After 30 days the ultrasonographic and histopathological analyzes were repeated and, in the first lactation period, the physical-chemical analysis of the milk was reapeated. Before the cellular therapy, the physical-chemical quality of the milk was compromised and the ultrasonographic and histopathological analysis revealed a chronic inflammatory process and fibrous tissue. The marking of the ASCs with Qdots enabled the tracking, by fluorescence microscopy (BX41-OLYMPUS), in the mammary tissue. In the ASCs therapy, cultures showed high cellularity and characteristics favorable to preclinical studies; with the therapy the physical-chemical parameters of the milk, fat, protein, temperature and pH showed significant differences among the groups; five animals treated with ASCs reconstituted the functionality of the gland and the connective tissue reduced in quantity and inflammatory infiltrate cells. ASCs have potential for the possible regeneration of fibrous mastitis lesions in the mammary gland, however, it would be necessary to increase injection time for the histopathological analysis, since the reconstitution of the glandular acini within the assessed period was not finalized. ASCs can be used to reestablish milk production in goat with chronic mastitis repair mammary lesions, with potential to be a promising clinical alternative for animal rehabilitation for productivity.
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Project description:To explore the function of PPAR γ in the goat mammary gland, we cloned the whole cDNA of the PPAR γ gene. Homology alignments revealed that the goat PPAR γ gene is conserved among goat, bovine, mouse, and human. Luciferase assays revealed that rosiglitazone enhanced the activity of the PPAR γ response element (PPRE) in goat mammary epithelial cells (GMECs). After rosiglitazone (ROSI) treatment of GMECs, there was a significant (P < 0.05) increase in the expression of genes related to triacylglycerol synthesis and secretion: LPL, FASN, ACACA, PLIN3, FABP3, PLIN2, PNPLA2, NR1H3, SREBF1, and SCD. The decreases in expression observed after knockdown of PPAR γ relative to the control group (Ad-NC) averaged 65%, 52%, 67%, 55%, 65%, 58%, 85%, 43%, 50%, and 24% for SCD, DGAT1, AGPAT6, SREBF1, ACACA, FASN, FABP3, SCAP, ATGL, and PLIN3, respectively. These results provide direct evidence that PPAR γ plays a crucial role in regulating the triacylglycerol synthesis and secretion in goat mammary cells and underscore the functional importance of PPAR γ in mammary gland tissue during lactation.
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Project description:During lactation, large amounts of calcium are exported from the mammary gland into milk to ensure skeletal growth of the offspring. Recent studies revealed that serotonin (5-HT) is essential to stimulate skeletal calcium resorption for milk synthesis. Our objective was to explore the correlation between circulating 5-HT and serum calcium and parathyroid hormone-related protein (PTHrP) concentrations around parturition in dairy goats. We also investigated the effect of 5-HT on PTHrP expression in cultured primary goat mammary epithelial cells (GMEC). Blood samples of multiparous Guanzhong dairy goats were collected on day -5 to 3 postpartum for analysis of serum concentrations of calcium, 5-HT, and PTHrP. Results revealed that from day -3 to 0 postpartum serum calcium and 5-HT concentrations decreased progressively, but serum PTHrP concentration only had a sharp drop in the postpartum period sampled. Correlation analysis of circulating 5-HT and serum calcium and PTHrP concentrations on day 1 and 2 postpartum revealed that low serum 5-HT concentration was positively correlated with serum total calcium or PTHrP concentration. By knocking down tryptophan hydroxylase-1 (TPH1) or adding 5-hydroxytryptophan (5-HTP) to decrease or increase the levels of 5-HT in GMEC, we observed that 5-HTP increased PTHrP expression in a dose-dependent manner and siTPH1 decreased PTHrP protein expression. Furthermore, 5-HT increased mRNA abundance of calcium-sensing receptor (CaSR) in a dose-dependent manner and decreased the expression of plasma membrane Ca2+ ATPase-1 (PMCA1). Taken together, 5-HT seems to induce PTHrP expression in goat mammary cells during and after parturition. These findings suggest that increasing 5-HT biosynthesis could be a potential therapeutic target for prevention of hypocalcemia in dairy goats.
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Project description:cAMP response element binding protein 1 (CREB1) is a member of the leucine zipper transcription factor family of DNA binding proteins. Although studies in non-ruminants have demonstrated a crucial role of CREB1 in lipid synthesis in liver and adipose tissue, it is unknown if this transcription regulator exerts control of fatty acid synthesis in ruminant mammary cells. To address this question, we first defined the expression dynamics of CREB1 in mammary tissue during lactation. Analysis of CREB1 in mammary tissue revealed higher mRNA abundance in mammary tissue harvested at peak lactation. Overexpression of CREB1 markedly upregulated sterol regulatory element binding transcription factor 1 (SREBP1), fatty acid synthase (FASN), acetyl-coenzyme A carboxylase α (ACACA), elongase of very long chain fatty acids 6 (ELOVL6), lipoprotein lipase (LPL), fatty acid binding protein 3 (FABP3), lipin 1 (LPIN1) and diacylglycerol acyltransferase 1 (DGAT1), but had no effect on glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) or 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6). In addition, overexpressing CREB1 led to a significant increase in the concentration and desaturation index of C16:1 (palmitoleic acid) and C18:1 (oleic acid), along with increased concentration of triacylglycerol. Taken together, these results highlight an important role of CREB1 in regulating lipid synthesis in goat mammary epithelial cells. Thus, manipulation of CREB1 in vivo might be one approach to improve the quality of goat milk.
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Project description:Goat mammary-gland microsomal fraction by itself induces synthesis of medium-chain-length fatty acids by goat mammary fatty acid synthetase and incorporates short- and medium-chain fatty acids into triacylglycerol. Addition of ATP in the absence or presence of Mg2+ totally inhibits triacylglycerol synthesis from short- and medium-chain fatty acids, and severely inhibits synthesis de novo of medium-chain fatty acids. The inhibition by ATP of fatty acid synthesis and triacylglycerol synthesis de novo can be relieved by glycerol 3-phosphate. The effect of ATP could not be mimicked by the non-hydrolysable ATP analogue, adenosine 5'-[beta,gamma-methylene]triphosphate and could not be shown to be caused by inhibition of the diacylglycerol acyltransferase by a phosphorylation reaction. Possible explanations for the mechanism of the inhibition by ATP are discussed, and a hypothetical model for its action is outlined.
| S-EPMC1153654 | biostudies-other
Project description:BACKGROUND:Nutrition affects milk composition thus influencing its nutritional properties. Nutrition also modifies the expression of mammary genes, whose regulation is not fully understood. MicroRNAs (miRNA) are small non coding RNA which are important post-transcriptional regulators of gene expression by targeting messenger RNAs. Our goal was to characterize miRNA whose expression is regulated by nutrition in the lactating goat mammary gland, which may provide clues to deciphering regulations of the biosynthesis and secretion of milk components. METHODOLOGY/PRINCIPAL FINDINGS:Using high-throughput sequencing technology, miRNomes of the lactating mammary gland were established from lactating goats fed ad libitum or deprived of food for 48 h affecting milk production and composition. High throughput miRNA sequencing revealed 30 miRNA with an expression potentially modulated by food deprivation; 16 were down-regulated and 14 were up-regulated. Diana-microT predictive tools suggested a potential role for several nutriregulated miRNA in lipid metabolism. Among the putative targets, 19 were previously identified as differently expressed genes (DEG). The functions of these 19 DEG revealed, notably, their involvement in tissue remodelling. CONCLUSION/SIGNIFICANCE:In conclusion, this study offers the first evidence of nutriregulated miRNA in the ruminant mammary gland. Characterization of these 30 miRNA could contribute to a clearer understanding of gene regulation in the mammary gland in response to nutrition.
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