Determination of the effect of functional single-nucleotide polymorphisms associated with glycerolipid synthesis on intramuscular fat deposition in Korean cattle steer

Intramuscular fat deposition in the longissimus dorsi muscle (LM) of Korean cattle steer is regulated by several genes related to lipid metabolism. One of these genes encodes the enzyme bovine glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), which is located on the mitochondrial outer membrane and catalyzes the initial and committed step of glycerolipid synthesis in lipid metabolism of cattle. Previous studies have shown that the 3′-untranslated region (UTR) of the GPAM is quite extended and contains a polyadenylation signal site, erythroid 15-lipoxygenase differentiation control elements (15-LOX-DICEs), and cytoplasmic polyadenylation elements (CPEs) that affect the regulation of triacylglycerol synthesis. Therefore, the aim of this study was to identify single-nucleotide polymorphisms (SNPs) related to the regulation of glycerolipid synthesis in the 3′-UTR of GPAM and to verify the function of SNPs affecting the deposition of intramuscular fat in Korean cattle steer. In the present study, 11 SNPs were discovered in the 3′-UTR of GPAM. Among these SNPs, g.54853A>G, g.55441A>G, and g.55930C>T were significantly associated with marbling score in a Korean cattle steer population and were strongly correlated with each other within the GPAM gene. Furthermore, based on the results predicted by the RNAhybrid program, four putative microRNAs (miRNAs) were identified, and the above SNPs were found to present in the seed region of these miRNAs. These miRNAs have a differential binding affinity for each allele of SNPs g.54853A>G, g.55441A>G, and g.55930C>T. The in vivo evidence of intramuscular fat deposition in the LM tissue showed that these SNPs affected the regulation of intramuscular fat deposition in Korean cattle steer. Thus, the g.54853A>G, g.55441A>G, and g.55930C>T could be considered as causal mutations regulating intramuscular fat deposition in Korean cattle steer.

Messenger RNA levels of enzymes involved in glycerolipid synthesis in the brain of the mouse and its alterations in Agpat2-/- and db/db mice

Background and Aims: Expression of genes encoding enzymes involved in glycerolipid and monoacylglycerol pathways in specific brain regions is poorly known and their alterations in insulin resistance (IR) and type 2 diabetes (T2D) remain unreported. We determined the mRNA levels of enzymes involved in glycerolipid synthesis in specific regions of the mouse brain and their changes in two models of severe IR, the lipodystrophic Agpat2−/− and the obese Leprdb/db mice. Methods Cerebral cortex, hypothalamus, hippocampus and cerebellum were dissected from adult Agpat2−/− mice, Leprdb/db mice and their respective wild type littermates. Total RNA was isolated and the relative mRNA abundance of enzymes was determined by RT-qPCR. Results GPAT1, AGPAT1-4, LIPIN1/2, DGAT1/2 and MOGAT1 mRNAs were detected in all studied brain regions, whereas GPAT2, LIPIN3 and MOGAT2 were undetectable. Abundance of GPAT1, AGPAT1, AGPAT2, AGPAT4, LIPIN1, and MOGAT1, was higher in the hypothalamus. AGPAT3 and DGAT1 were higher in cortex and cerebellum, and LIPIN2 and DGAT2 were higher in cortex and hippocampus. In Agpat2−/− mice, LIPIN1 levels were increased in all the brain regions. By contrast, GPAT1 and AGPAT4 in hypothalamus, AGPAT3 in hippocampus and hypothalamus, and MOGAT1 in cortex, hypothalamus and cerebellum were lower in Agpat2−/− mice. Leprdb/db mice showed fewer and milder changes, with increased levels of GPAT1 and LIPIN1 in cerebellum, and AGPAT3 in hypothalamus. Conclusions Enzymes involved in glycerolipids synthesis are differentially expressed across regions of the mouse brain and IR and T2D determine altered gene expression of these enzymes in the mouse brain.

Plastid Glycerol-3-phosphate Acyltransferase Enhanced Plant Growth and Prokaryotic Glycerolipid Synthesis in Brassica napus

Plastid-localized glycerol-3-phosphate acyltransferase (ATS1) catalyzes the first-step reaction in glycerolipid assembly through transferring an acyl moiety to glycerol-3-phosphate (G3P) to generate lysophosphatidic acid (LPA), an intermediate in lipid metabolism. The effect of ATS1 overexpression on glycerolipid metabolism and growth remained to be elucidated in plants, particularly oil crop plants. Here, we found that overexpression of BnATS1 from Brassica napus enhanced plant growth and prokaryotic glycerolipid biosynthesis. BnATS1 is localized in chloroplasts and an in vitro assay showed that BnATS1 had acylation activity toward glycerol 3-phosphate to produce LPA. Lipid profiling showed that overexpression of BnATS1 led to increases in multiple glycerolipids including phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), phosphatidylcholine (PC), and phosphatidylinositol (PI), with increased polyunsaturated fatty acids. Moreover, increased MGDG was attributed to the elevation of 34:6- and 34:5-MGDG, which were derived from the prokaryotic pathway. These results suggest that BnATS1 promotes accumulation of polyunsaturated fatty acids in cellular membranes, thus enhances plant growth under low-temperature conditions in Brassica napus.

Macrophage-associated lipin-1 transcriptional co-regulatory activity is involved in atherosclerosis

During atherosclerosis, macrophages engulf and break down deposited modified low-density lipoproteins (modLDLs) into lipids and free fatty acids. The lipids and free fatty acids from these modLDLs either need to be stored during a process called glycerolipid synthesis or broken down during β-oxidation. In addition, free fatty acids can activate transcription factors to promote a pro-resolving macrophage phenotype. The protein lipin-1 is involved in both glycerolipid synthesis and β-oxidation. Lipin-1 enzymatic activity is a key step in the glycerolipid synthesis pathway; lipin-1 transcriptional co-regulatory activity either augments or represses various transcription factors that are activated via free fatty acids that promote β-oxidation and inhibit inflammation. Lipin-1 enzymatic activity increases pro-inflammatory macrophage phenotypes and is atherogenic. In contrast, we have also demonstrated that lipin-1 transcriptional co-regulatory activity promotes pro-resolving macrophage phenotypes leading us to the hypothesis that lipin-1 transcriptional co-regulatory activity is atheroprotective. Using a mouse model to delete lipin-1 in myeloid cells, we have demonstrated that loss of lipin-1 increases plaque size and pro-inflammatory gene expression. We have also shown mice lacking lipin-1 in myeloid cells have increased plaque collagen deposition and larger necrotic core formation. Combined, these data suggest that though lipin-1 enzymatic activity is atherogenic, lipin-1 transcriptional co-regulatory activity is atheroprotective. Overall, the results suggest that the dual activities of lipin-1 contribute to atherosclerosis progression in opposite ways.

Regulated lipid synthesis and LEM2/CHMP7 jointly control nuclear envelope closure

The nuclear permeability barrier depends on closure of nuclear envelope (NE) holes. Here, we investigate closure of the NE opening surrounding the meiotic spindle in C. elegans oocytes. ESCRT-III components accumulate at the opening but are not required for nuclear closure on their own. 3D analysis revealed cytoplasmic membranes directly adjacent to NE holes containing meiotic spindle microtubules. We demonstrate that the NE protein phosphatase, CNEP-1/CTDNEP1, controls de novo glycerolipid synthesis through lipin to prevent invasion of excess ER membranes into NE holes and a defective NE permeability barrier. Loss of NE adaptors for ESCRT-III exacerbates ER invasion and nuclear permeability defects in cnep-1 mutants, suggesting that ESCRTs restrict excess ER membranes during NE closure. Restoring glycerolipid synthesis in embryos deleted for CNEP-1 and ESCRT components rescued NE permeability defects. Thus, regulating the production and feeding of ER membranes into NE holes together with ESCRT-mediated remodeling is required for nuclear closure.

Messenger RNA levels of enzymes involved in glycerolipid synthesis in the brain of the mouse and its alterations in Agpat2-/- and db/db mice

AimsExpression of genes encoding enzymes involved in glycerolipid and monoacylglycerol pathways in specific brain regions is poorly known and its impact in insulin resistance (IR) and type 2 diabetes (T2D) in the brain remains unreported. We determined mRNA levels of enzymes involved in glycerolipid synthesis in different regions of the mouse brain and evaluated their changes in two models of IR and T2D, the Agpat2-/- and Leprdb/db mice.MethodsCerebral cortex, hypothalamus, hippocampus and cerebellum were dissected from adult Agpat2-/- mice, Leprdb/db mice and their respective wild type littermates. Total RNA was isolated and mRNA abundance was measured by RT-qPCR.Key findingsGPAT1, AGPAT1-4, LIPIN1/2, DGAT1/2 and MOGAT1 mRNAs were detected in all studied brain regions, whereas GPAT2, LIPIN3 and MOGAT2 were undetectable. Abundance of AGPATs, LIPIN1 and DGAT1, was higher in cerebellum and hypothalamus. LIPIN2 and MOGAT1 levels were higher in hypothalamus, and DGAT2 was higher in cortex and hypothalamus. In Agpat2-/- mice, LIPIN1 levels were increased in all the brain regions. By contrast, GPAT1 in cortex and hypothalamus, AGPAT3 in hippocampus and hypothalamus, AGPAT4 in hypothalamus, and MOGAT1 in cortex, hypothalamus and cerebellum were lower in Agpat2-/- mice. Leprdb/db mice showed fewer and milder changes, with increased levels of GPAT1 and LIPIN1 in cerebellum, and AGPAT3 in hypothalamus.Conclusions and SignificanceEnzymes of glycerolipids synthesis are differentially expressed across regions of the mouse brain. Two mouse models of IR and T2D have altered gene expression of glycerolipid enzymes in the brain.

Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages

Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3−/− and Gpat4−/− BMDM was impaired. Additionally, inhibiting fatty acid β-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4−/− BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.