HuR promotes triglyceride synthesis and intestinal fat absorption.

Triacylglyceride (TAG) synthesis in the small intestine determines the absorption of dietary fat, but the underlying mechanisms remain to be further studied. Here, we report that the RNA-binding protein HuR (ELAVL1) promotes TAG synthesis in the small intestine. HuR associates with the 3' UTR of Dgat2 mRNA and intron 1 of Mgat2 pre-mRNA. Association of HuR with Dgat2 3' UTR stabilizes Dgat2 mRNA, while association of HuR with intron 1 of Mgat2 pre-mRNA promotes the processing of Mgat2 pre-mRNA. Intestinal epithelium-specific HuR knockout reduces the expression of DGAT2 and MGAT2, thereby reducing the dietary fat absorption through TAG synthesis and mitigating high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and obesity. Our findings highlight a critical role of HuR in promoting dietary fat absorption.

Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta.

BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. RESULT: In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). CONCLUSIONS: This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Multiple transcription factors of Arabidopsis thaliana that are activated by LEAFY COTYLEDON 2 regulate triacylglycerol biosynthesis.

Plant triacylglycerols (TAG) are used in food and various industrial feedstocks. LEAFY COTYLEDON 2 (LEC2), a master positive regulator of TAG biosynthesis, regulates a complex network of transcription factors (TFs) during seed development. Aside from WRINKLED1 (WRI1), the TFs regulated by LEC2 related to TAG biosynthesis have not yet been identified. Previously, we identified 25 seed-expressing TFs that were upregulated in Arabidopsis leaves that overexpressed senescence-induced LEC2. In this study, each of the 25 TFs was transiently expressed in the leaves of Nicotiana benthamiana to identify unknown TFs that regulate TAG biosynthesis. The TAG content of the transformed leaves was analyzed using thin layer chromatography and gas chromatography. We observed that five TFs, ARABIDOPSIS RESPONSIVE REGULATOR 21 (ARR21), AINTEGUMENTA-LIKE 6 (AIL6), APETALA2/ETHYLENE RESPONSIVE FACTOR 55 (ERF55), WRKY DNA-BINDING PROTEIN 8 (WRKY8), and ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 38 (ANAC038) increased TAG synthesis in the leaves. Among these, the promoters of AIL6, ERF55, WRKY8, and ANAC038 contain RY motifs, which are LEC2-binding sites activated by LEC2. AIL6 overexpression in Arabidopsis increased the total fatty acid (FA) content in seeds and altered the FA composition, with increases in 16:0, 18:1, and 18:2 and decreases in 18:0, 18:3, and 20:1 compared with those in the wild type (WT). AIL6 overexpression activates several FA and TAG biosynthesis genes. Therefore, our study successfully identified several new TFs regulated by LEC2 in TAG biosynthesis and showed that AIL6 increased the TAG content in seeds.

LPIN1 promotes triglycerides synthesis and is transcriptionally regulated by PPARG in buffalo mammary epithelial cells.

Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase1 (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism. However, the functional role and regulatory mechanism of LPIN1 gene in milk fat synthesis of buffalo are still unknown. In this study, overexpression of buffalo LPIN1 gene transfected with recombinant fusion expression vector significantly increased the expression of AGPAT6, DGAT1, DGAT2, GPAM and BTN1A1 genes involved in triglyceride (TAG) synthesis and secretion, as well as PPARG and SREBF1 genes regulating fatty acid metabolism in the buffalo mammary epithelial cells (BMECs), while the lentivirus-mediated knockdown of buffalo LPIN1 dramatically decreased the relative mRNA abundance of these genes. Correspondingly, total cellular TAG content in the BMECs increased significantly after LPIN1 overexpression, but decreased significantly after LPIN1 knockdown. In addition, the overexpression or knockdown of PPARG also enhanced or reduced the expression of LPIN1 and the transcriptional activity of its promoter. The core region of buffalo LPIN1 promoter spans from - 666 bp to + 42 bp, and two PPAR response elements (PPREs: PPRE1 and PPRE2) were identified in this region. Site mutagenesis analysis showed that PPARG directly regulated the transcription of buffalo LPIN1 by binding to the PPRE1 and PPRE2 on its core promoter. The results here reveal that the LPIN1 gene is involved in the milk fat synthesis of BMECs, and one of the important pathways is to participate in this process through direct transcriptional regulation of PPARG, which in turn significantly affects the content of TAG in BMECs.

Prostaglandin E2 and F2α Alter Expression of Select Cholesteryl Esters and Triacylglycerols Produced by Human Meibomian Gland Epithelial Cells.

PURPOSE: PGF2α analogs are commonly used to treat glaucoma and are associated with higher rates of meibomian gland dysfunction (MGD). The purpose of this study was to evaluate the physiological effects of PGF2α and PGE2 on immortalized human meibomian gland epithelial cells (HMGECs). METHODS: HMGECs were immunostained for the 4 PGE2 receptors (EP1, EP2, EP3, and EP4) and 1 PGF2α receptor (FP) and imaged. Rosiglitazone-differentiated HMGECs were exposed to PGF2α and PGE2 (10-9 to 10-6 M) for 3 hours. Cell viability was assessed by an adenosine triphosphate-based luminescent assay, and lipid extracts were analyzed for cholesteryl esters (CEs), wax esters (WEs), and triacylglycerols (TAGs) by ESI-MSMSALL in positive ion mode by a Triple TOF 5600 Mass Spectrometer using SCIEX LipidView 1.3. RESULTS: HMGECs expressed 3 PGE2 receptors (EP1, EP2, and EP4) and the 1 PGF2α receptor (FP). Neither PGE2 nor PGF2α showed signs of cytotoxicity at any of the concentrations tested. WEs were not detected from any of the samples, but both CEs and TAGs exhibited a diverse and dynamic profile. PGE2 suppressed select CEs (CE 22:1, CE 26:0, CE 28:1, and CE 30:1). PGF2α dose dependently increased several CEs (CE 20:2, CE 20:1, CE 22:1, and CE 24:0) yet decreased others. Both prostaglandins led to nonspecific TAG remodeling. CONCLUSIONS: PGE2 and PGF2α showed minimal effect on HMGEC viability. PGF2α influences lipid expression greater than PGE2 and may do so by interfering with meibocyte differentiation. This work may provide insight into the mechanism of MGD development in patients with glaucoma treated with PGF2α analogs.

Role of miR-181c in Diet-induced obesity through regulation of lipid synthesis in liver.

We recently identified a nuclear-encoded miRNA (miR-181c) in cardiomyocytes that can translocate into mitochondria to regulate mitochondrial gene mt-COX1 and influence obesity-induced cardiac dysfunction through the mitochondrial pathway. Because liver plays a pivotal role during obesity, we hypothesized that miR-181c might contribute to the pathophysiological complications associated with obesity. Therefore, we used miR-181c/d-/- mice to study the role of miR-181c in hepatocyte lipogenesis during diet-induced obesity. The mice were fed a high-fat (HF) diet for 26 weeks, during which indirect calorimetric measurements were made. Quantitative PCR (qPCR) was used to examine the expression of genes involved in lipid synthesis. We found that miR-181c/d-/- mice were not protected against all metabolic consequences of HF exposure. After 26 weeks, the miR-181c/d-/- mice had a significantly higher body fat percentage than did wild-type (WT) mice. Glucose tolerance tests showed hyperinsulinemia and hyperglycemia, indicative of insulin insensitivity in the miR-181c/d-/- mice. miR-181c/d-/- mice fed the HF diet had higher serum and liver triglyceride levels than did WT mice fed the same diet. qPCR data showed that several genes regulated by isocitrate dehydrogenase 1 (IDH1) were more upregulated in miR-181c/d-/- liver than in WT liver. Furthermore, miR-181c delivered in vivo via adeno-associated virus attenuated the lipogenesis by downregulating these same lipid synthesis genes in the liver. In hepatocytes, miR-181c regulates lipid biosynthesis by targeting IDH1. Taken together, the data indicate that overexpression of miR-181c can be beneficial for various lipid metabolism disorders.

Inhibition of Granuloma Triglyceride Synthesis Imparts Control of Mycobacterium tuberculosis Through Curtailed Inflammatory Responses.

Lipid metabolism plays a complex and dynamic role in host-pathogen interaction during Mycobacterium tuberculosis infection. While bacterial lipid metabolism is key to the success of the pathogen, the host also offers a lipid rich environment in the form of necrotic caseous granulomas, making this association beneficial for the pathogen. Accumulation of the neutral lipid triglyceride, as lipid droplets within the cellular cuff of necrotic granulomas, is a peculiar feature of pulmonary tuberculosis. The role of triglyceride synthesis in the TB granuloma and its impact on the disease outcome has not been studied in detail. Here, we identified diacylglycerol O-acyltransferase 1 (DGAT1) to be essential for accumulation of triglyceride in necrotic TB granulomas using the C3HeB/FeJ murine model of infection. Treatment of infected mice with a pharmacological inhibitor of DGAT1 (T863) led to reduction in granuloma triglyceride levels and bacterial burden. A decrease in bacterial burden was associated with reduced neutrophil infiltration and degranulation, and a reduction in several pro-inflammatory cytokines including IL1ß, TNFα, IL6, and IFNß. Triglyceride lowering impacted eicosanoid production through both metabolic re-routing and via transcriptional control. Our data suggests that manipulation of lipid droplet homeostasis may offer a means for host directed therapy in Tuberculosis.

CD36 and DGAT2 facilitate the lipid-lowering effect of chitooligosaccharides via fatty acid intake and triglyceride synthesis signaling.

This study examined the impact of chitobiose (GlcN)2 and chitotriose (GlcN)3 on lipid accumulation modification and their inhibitory functionalities. (GlcN)2 and (GlcN)3 significantly inhibited the total cholesterol (TC), triglyceride (TG), and low-density lipid cholesterol (LDL-c) levels in the liver of the ob/ob-/- mice fed a non-high-fat diet. This phenomenon was associated with a reduction in the mRNA and protein expression of TG synthesis and fatty acid uptake-related signaling, significantly affecting the cluster of differentiation 36 (CD36) and diacylglycerol acyltransferase 2 (DGAT2). Furthermore, the CD36 and DGAT2 genes were overexpressed by constructing a plasmid and transfecting it into HepG2 cells, after which the phenotypic traits of lipid accumulation were assessed in vitro. Consequently, it was evident that (GlcN)2 and (GlcN)3 reduced the overexpression of these proteins and relieved cellular lipid accumulation. In conclusion, these results indicated that (GlcN)2 and (GlcN)3 acted positively against NAFLD while regulating steatosis in the non-high-fat diet NAFLD model. The potential NAFLD treatment strategies, such as targeting CD36 and DGAT2 signaling, could provide scientific insight into further applying food-derived ingredients to reduce the risk of high-fat metabolism.

TRIM15 promotes the invasion and metastasis of pancreatic cancer cells by mediating APOA1 ubiquitination and degradation.

Most pancreatic ductal adenocarcinomas (PDACs) are diagnosed at an advanced or metastatic stage. Metastasis is the one of the major obstacles to prolonging the survival time of patients with pancreatic cancer. The tripartite motif (TRIM) family member TRIM15 has been implicated in cancer development. Our bioinformatics analysis indicated that TRIM15 might be involved in the regulation of pancreatic cancer metastasis. However, the role of TRIM15 in PDAC remains unclear. Metabolic reprogramming involving dysregulated lipid synthesis is common in patients with PDAC. Targeting lipid anabolism has been proposed as a strategy to treat PDAC. In this study, we demonstrated that TRIM15 expression was elevated in PDAC tissues, and this elevated expression was associated with a poor prognosis. TRIM15 silencing suppressed the invasion and migration of pancreatic cancer cells. Importantly, the mass spectrometry analysis suggested that Apolipoprotein A1 (APOA1), the main component of high-density lipoprotein (HDL) that is involved in lipid transport and metabolism, might be one of the binding partners of TRIM15. Further experiment indicated that TRIM15 interacted with APOA1 through its PRY/SPRY domain and promoted APOA1 polyubiquitination via its RING domain. APOA1 degradation enhanced lipid anabolism and promoted lipid droplet accumulation in pancreatic cancer cells. Furthermore, we showed that TRIM15 might promote PDAC metastasis by regulating lipid metabolism via the APOA1-LDLR axis. Consequently, targeting the TRIM15-APOA1-LDLR axis may be a strategy to inhibit PDAC metastasis by blocking triglyceride synthesis.

ß2-Adrenergic receptor agonist induced hepatic steatosis in mice: modeling nonalcoholic fatty liver disease in hyperadrenergic states.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from hepatic steatosis [excessive accumulation of triglycerides (TG)] to nonalcoholic steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The molecular pathogenesis of steatosis and progression to more severe NAFLD remains unclear. Obesity and aging, two principal risk factors for NAFLD, are associated with a hyperadrenergic state. ß-Adrenergic responsiveness in liver increases in animal models of obesity and aging, and in both is linked to increased hepatic expression of ß2-adrenergic receptors (ß2-ARs). We previously showed that in aging rodents intracellular signaling from elevated hepatic levels of ß2-ARs may contribute to liver steatosis. In this study we demonstrate that injection of formoterol, a highly selective ß2-AR agonist, to mice acutely results in hepatic TG accumulation. Further, we have sought to define the intrahepatic mechanisms underlying ß2-AR mediated steatosis by investigating changes in hepatic expression and cellular localization of enzymes, transcription factors, and coactivators involved in processes of lipid accrual and disposition-and also functional aspects thereof-in livers of formoterol-treated animals. Our results suggest that ß2-AR activation by formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, increased but incomplete ß-oxidation of fatty acids with accumulation of potentially toxic long-chain acylcarnitine intermediates, and reduced TG secretion-all previously invoked as contributors to fatty liver disease. Experiments are ongoing to determine whether sustained activation of hepatic ß2-AR signaling by formoterol might be utilized to model fatty liver changes occurring in hyperadrenergic states of obesity and aging, and thereby identify novel molecular targets for the prevention or treatment of NAFLD.NEW & NOTEWORTHY Results of our study suggest that ß2-adrenergic receptor (ß2-AR) activation by agonist formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, incomplete ß-oxidation of fatty acids with accumulation of long-chain acylcarnitine intermediates, and reduced TG secretion. These findings may, for the first time, implicate a role for ß2-AR responsive dysregulation of hepatic lipid metabolism in the pathogenetic processes underlying NAFLD in hyperadrenergic states such as obesity and aging.

Inhibition of Estrogen-Related Receptor α Blocks Liver Steatosis and Steatohepatitis and Attenuates Triglyceride Biosynthesis.

The estrogen-related receptor (ERR) family of orphan nuclear receptors are transcriptional activators for genes involved in mitochondrial bioenergetics and metabolism. The goal of this study was to explore the role of ERRα in lipid metabolism and the potential effect of inhibiting ERRα on the development of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). In the current study, three experimental mouse models: high-fat diet, high-carbohydrate diet, and a genetic model of hepatic insulin resistance where the liver hyperinsulinemia signal is mimicked via hepatic deletion of Pten (phosphatase and tensin homolog deleted on chromosome 10), the negative regulator of the insulin/phosphatidylinositol 3-kinase signaling pathway, were used. A recently developed small-molecule inhibitor for ERRα was used to demonstrate that inhibiting ERRα blocked NAFLD development induced by either high-carbohydrate diet or high-fat diet feeding. ERRα inhibition also diminished lipid accumulation and attenuated NASH development in the Pten null mice. Glycerolipid synthesis was discovered as an additional mechanism for ERRα-regulated NAFLD/NASH development and glycerophosphate acyltransferase 4 was identified as a novel transcriptional target of ERRα. In summary, these results establish ERRα as a major transcriptional regulator of lipid biosynthesis in addition to its characterized primary function as a regulator for mitochondrial function. This study recognizes ERRα as a potential target for NAFLD/NASH treatment and elucidates novel signaling pathways regulated by ERRα.

Mycobacterium leprae promotes triacylglycerol de novo synthesis through induction of GPAT3 expression in human premonocytic THP-1 cells.

Mycobacterium leprae (M. leprae) is the etiological agent of leprosy, and the skin lesions of lepromatous leprosy are filled with numerous foamy or xanthomatous histiocytes that are parasitized by M. leprae. Lipids are an important nutrient for the intracellular survival of M. leprae. In this study, we attempted to determine the intracellular lipid composition and underlying mechanisms for changes in host cell lipid metabolism induced by M. leprae infection. Using high-performance thin-layer chromatography (HPTLC), we demonstrated specific induction of triacylglycerol (TAG) production in human macrophage THP-1 cells following M. leprae infection. We then used [14C] stearic acid tracing to show incorporation of this newly synthesized host cell TAG into M. leprae. In parallel with TAG accumulation, expression of host glycerol-3-phosphate acyltransferase 3 (GPAT3), a key enzyme in de novo TAG synthesis, was significantly increased in M. leprae-infected cells. CRISPR/Cas9 genome editing of GPAT3 in THP-1 cells (GPAT3 KO) dramatically reduced accumulation of TAG following M. leprae infection, intracellular mycobacterial load, and bacteria viability. These results together suggest that M. leprae induces host GPAT3 expression to facilitate TAG accumulation within macrophages to maintain a suitable environment that is crucial for intracellular survival of these bacilli.

Anti-hyperlipidemic potential of natural product based labdane-pyrroles via inhibition of cholesterol and triglycerides synthesis.

Hyperlipidemia is the clinical condition where blood has an increased level of lipids, such as cholesterol and triglycerides. Therefore controlling hyperlipidemia is considered to be a protective strategy to treat many associated diseases. Thus, a novel natural product derived pyrrole, and pyrazole-(E)-Labda-8(17),12-diene-15,16-dial conjugates with cholesterol and triglycerides synthesis inhibition potential was designed through scaffold hopping approach and synthesized via one-pot selective cycloaddition. Amongst the tested hybrids, 3i exhibited excellent activity against triglyceride and cholesterol synthesis with the percentage inhibition of 71.73 ± 0.78 and 68.61 ± 1.19, which is comparable to the positive controls fenofibrate and atorvastatin, respectively. Compounds 3j and 3k also exhibited the considerable potential of promising leads. The HMG CoA reductase inhibitory activity of the compounds was consistent with that of inhibitory activity of cholesterol synthesis. Compound 3i showed the highest inhibitory potential (78.61 ± 2.80) percentage of suppression, which was comparable to that of the positive control pravastatin (78.05 ± 5.4). Favourably, none of the compounds showed cytotoxicity (HepG2) in the concentration ranging from 0.5 to 100 µM.

OGR1 negatively regulates ß-casein and triglyceride synthesis and cell proliferation via the PI3K/AKT/mTOR signaling pathway in goat mammary epithelial cells.

Goat milk in some cases is less allergenic than cow milk, therefore, more people drink goat milk in the world, so it is necessary for us to improve the yield and quality of goat milk. Previous studies have shown that some genes are closely related to lactation. Ovarian cancer G protein-coupled 1 (OGR1) is a G protein-coupled receptor discovered recently. OGR1 is widely found in various tissues of organisms and is involved in cell skeleton reorganization, carcinogenesis, cell proliferation, and apoptosis by regulating multiple signaling pathways in cells. However, the modulating effect of OGR1 in lactation is still unknown. Therefore, the objective of this study is to investigate the function of OGR1 in goat mammary epithelial cells (GMECs). Flow cytometry, CCK8, EDU, enzyme-linked immunosorbent assay, and triglyceride test kit assays were performed and we found that OGR1 regulated Bcl-2/Bax ratio, Fas protein expression as well as the phosphorylation of AKT and mammalian target of rapamycin (mTOR). si-OGR1 could enhance the proliferation of GMECs by promoting G1/S phase progression and the synthesis of ß-casein and triglyceride. By contrast, OGR1 repressed GMECs proliferation and down-regulated the synthesis of ß-casein and triglyceride by blocking the PI3K/AKT/mTOR signaling pathway in GMECs.

Foam Cell Macrophages in Tuberculosis.

Mycobacterium tuberculosis infects primarily macrophages in the lungs. Infected macrophages are surrounded by other immune cells in well organised structures called granulomata. As part of the response to TB, a type of macrophage loaded with lipid droplets arises which we call Foam cell macrophages. They are macrophages filled with lipid laden droplets, which are synthesised in response to increased uptake of extracellular lipids, metabolic changes and infection itself. They share the appearance with atherosclerosis foam cells, but their lipid contents and roles are different. In fact, lipid droplets are immune and metabolic organelles with emerging roles in Tuberculosis. Here we discuss lipid droplet and foam cell formation, evidence regarding the inflammatory and immune properties of foam cells in TB, and address gaps in our knowledge to guide further research.

Lipopolysaccharide inhibits triglyceride synthesis in dairy cow mammary epithelial cells by upregulating miR-27a-3p, which targets the PPARG gene.

The fat content of milk determines the quality of milk, and triglycerides are the major components of milk fat. Milk fat synthesis is regulated by many factors. Lipopolysaccharide (LPS) has been shown to inhibit milk fat synthesis in bovine mammary epithelial cells, but research on the underlying mechanisms has been limited. MicroRNA (miRNA) are involved in many physiological processes, but there have been few studies on their regulation in milk fat synthesis. In this study, we aimed to investigate whether LPS upregulates miR-27a-3p, which targets PPARG, thereby inhibiting the synthesis of triglycerides in a dairy cow mammary epithelial cell line (MAC-T). After LPS stimulation of MAC-T cells, PPARG gene expression and milk fat synthesis were inhibited. TargetScan software was used to predict miRNA targeting PPARG, and miR-27a-3p was selected as a candidate. A dual luciferase reporter assay further confirmed the targeting connection between miR-27a-3p and the PPARG gene. To investigate the functions of miR-27a-3p, miR-27a-3p mimic and inhibitors were transfected into MAC-T cells. The mRNA and protein levels of PPAR-γ were negatively correlated with the expression of miR-27a-3p. Lipid droplet accumulation and triglyceride synthesis were also negatively correlated with miR-27a-3p expression. Inhibition of miR-27a-3p partially reversed the LPS-induced decreases in PPARG expression and milk fat synthesis. In summary, our results reveal that LPS can inhibit MAC-T cell milk fat synthesis by upregulating miR-27a-3p, which targets the PPARG gene.

Enhanced triacylglycerol production in oleaginous microalga Neochloris oleoabundans by co-overexpression of lipogenic genes: Plastidial LPAAT1 and ER-located DGAT2.

Microalgae accumulate lipid triacylglycerol (TAG), a promising feedstock for production of natural edible oils and biofuels. To make products derived from microalgal TAG economically viable, increasing TAG content and productivity are of high importance. To increase TAG content, two endogenous key enzymes of TAG biosynthesis: plastidial lysophosphatidic acid acyltransferase (NeoLPAAT1) and endoplasmic reticulum-located diacylglycerol acyltransferase 2 (NeoDGAT2) were co-overexpressed in oleaginous microalga Neochloris oleoabundans. The neutral lipid content in NeoLPAAT1-NeoDGAT2 co-overexpressing transformant detected by Nile red staining increased 2-fold without compromising cell growth. The transcriptional levels of NeoLPAAT1 and NeoDGAT2 levels were 1.9-fold higher in the transformant than wild type. Considerably higher lipid accumulation was found in the transformant than wild type: total lipid content (73.72 ± 4.17 % DCW) increased 1.6-fold, TAG content (50.63 ± 6.15 % DCW) increased 2.1-fold, total lipid productivity (16.84 ± 0.66 mg/L/day) increased 1.9-fold, and TAG productivity (11.68 ± 0.90 mg/L/day) increased 2.1-fold. Fatty acid composition was slightly altered in the transformant compared to wild type; saturated fatty acid C16:0 increased to 26% from 20%, whereas C18:0 was reduced to 7% from 14%. Long-term stability of NeoLPAAT1-NeoDGAT2 co-overexpression was observed in the transformant continuously maintained on solid medium in a period of 4 years. The results suggested that targeted engineering of genes in pathway located at different organelles should be possible in microalgal lipid metabolism.

Liver X receptor α participates in LPS-induced reduction of triglyceride synthesis in bovine mammary epithelial cells.

Lipopolysaccharides (LPS) could induce milk fat depression via regulating the body and blood fat metabolism. However, it is not completely clear how LPS might regulate triglyceride synthesis in dairy cow mammary epithelial cells (DCMECs). DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS. The level of triglyceride synthesis, the expression and activity of the liver X receptor α (LXRα), enzymes related to de novo fatty acid synthesis, and the expression of the fatty acid transporters were investigated. We found that LPS decreased the level of triglyceride synthesis via a down-regulation of the transcription, translation, and nuclear translocation level of the LXRα. The results also indicated that the transcription level of the LXRα target genes, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthetase (FAS), acetyl-CoA carboxylase-1 (ACC1), were significantly down-regulated in DCMECs after LPS treatment. Our data may provide new insight into the mechanisms of milk fat depression caused by LPS.

miR-497 regulates fatty acid synthesis via LATS2 in bovine mammary epithelial cells.

Both mRNA and miRNA play an important role in the regulation of mammary fatty acid metabolism and milk fat synthesis. Although studies have shown a strong transcriptional control of fatty acid metabolism, less is known about the regulatory mechanisms of milk fat synthesis as a function of miRNA-mRNA interactions. In this study, we carried out transcriptome sequencing using mammary tissues from the early lactation period, peak lactation, mid-lactation and late lactation in dairy cows and identified key genes regulating milk fatty acid metabolism. A total of 32 differentially co-expressed gene were screened out. Large tumor suppressor kinase 2 (LATS2) was chosen for further study using luciferase reporter assays, qRT-PCR and western blotting. The aim was to demonstrate that miR-497 is an upstream regulator of LATS2, i.e. miR-497 and LATS2 are a potential miRNA/mRNA regulatory pair. The results indicated that miR-497 could inhibit the production of triglycerides (TAG) and unsaturated fatty acids in bovine mammary epithelial cells (BMECs). In contrast, LATS2 can promote the production of TAG and unsaturated fatty acids. "Rescue" experiments further verified the miR-497/LATS2 regulatory network. Overall, data underscored that the miR-497/LATS2 pathway exerts control on milk fat metabolism and provides a theoretical approach for improving milk quality via genetic means.

Characterization of glycerol-3-phosphate acyltransferase 9 (AhGPAT9) genes, their allelic polymorphism and association with oil content in peanut (Arachis hypogaea L.).

GPAT, the rate-limiting enzyme in triacylglycerol (TAG) synthesis, plays an important role in seed oil accumulation. In this study, two AhGPAT9 genes were individually cloned from the A- and B- genomes of peanut, which shared a similarity of 95.65%, with 165 site differences. The overexpression of AhGPAT9 or the knock-down of its gene expression increased or decreased the seed oil content, respectively. Allelic polymorphism analysis was conducted in 171 peanut germplasm, and 118 polymorphic sites in AhGPAT9A formed 64 haplotypes (a1 to a64), while 94 polymorphic sites in AhGPAT9B formed 75 haplotypes (b1 to b75). The haplotype analysis showed that a5, b57, b30 and b35 were elite haplotypes related to high oil content, whereas a7, a14, a48, b51 and b54 were low oil content types. Additionally, haplotype combinations a62/b10, a38/b31 and a43/b36 were associated with high oil content, but a9/b42 was a low oil content haplotype combination. The results will provide valuable clues for breeding new lines with higher seed oil content using hybrid polymerization of high-oil alleles of AhGPAT9A and AhGPAT9B genes.