Glycerophosphate Acyltransferase Research Articles

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.

CTRP12 inhibits triglyceride synthesis and export in hepatocytes by suppressing HNF-4α and DGAT2 expression.

C1q/TNF-related protein 12 (CTRP12) is an antidiabetic adipokine whose circulating levels are reduced in obesity and diabetes. Although partial and complete loss-of-function mouse models suggest a role for CTRP12 in modulating lipid metabolism and adiposity, its effect on cellular lipid metabolism remains poorly defined. Here, we demonstrate a direct action of CTRP12 in regulating lipid synthesis and secretion. In hepatoma cells and primary mouse hepatocytes, CTRP12 treatment inhibits triglyceride synthesis by suppressing glycerophosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT) expression. CTRP12 treatment also downregulates the expression of hepatocyte nuclear factor-4α (HNF-4α) and its target gene microsomal triglyceride transfer protein (MTTP), leading to reduced very-low-density lipoprotein (VLDL)-triglyceride export from hepatocytes. Consistent with the invitro findings, overexpressing CTRP12 lowers fasting and postprandial serum triglyceride levels in mice. These results underscore the important function of CTRP12 in lipid metabolism in hepatocytes.

Evolution, dynamic expression changes and regulatory characteristics of gene families involved in the glycerophosphate pathway of triglyceride synthesis in chicken (Gallus gallus)

It is well documented that four gene families, including the glycerophosphate acyltransferases (GPATs), acylglycerophosphate acyltransferases (AGPATs), lipid phosphate phosphohydrolases (LPINs) and diacylglycerol acyltransferases (DGATs), are involved in the glycerophosphate pathway of de novo triglyceride (TG) biosynthesis in mammals. However, no systematic analysis has been conducted to characterize the gene families in poultry. In this study, the sequences of gene family members in the glycerophosphate pathway were obtained by screening the public databases. The phylogenetic tree, gene structures and conserved motifs of the corresponding proteins were evaluated. Dynamic expression changes of the genes at different developmental stages were analyzed by qRT-PCR. The regulatory characteristics of the genes were analyzed by in vivo experiments. The results showed that the GPAT, AGPAT and LPIN gene families have 2, 7 and 2 members, respectively, and they were classified into 2, 4 and 2 cluster respectively based on phylogenetic analysis. All of the genes except AGPAT1 were extensively expressed in various tissues. Estrogen induction upregulated the expression of GPAM and AGPAT2, downregulated the expression of AGPAT3, AGPAT9, LPIN1 and LPIN2, and had no effect on the expression of the other genes. These findings provide a valuable resource for further investigation of lipid metabolism in liver of chicken.

Re‐Purposing FDA‐approved Raloxifene to Target Apoptotic Proteins in KRAS‐mutant Pancreatic Cancer

Pancreatic cancer (PCa) has a very low 5‐year survival rate and is challenging to treat due to substantial chemoresistance. Changes in apoptotic protein regulation may account for PCa's chemoresistance. While 90% of PCa cases occur due to mutations in the human homolog of the Kirsten rat sarcoma (KRAS) viral oncogene, how this effects regulation of apoptotic proteins in PCa is unknown. We screened drugs approved by the Food and Drug Authority for their ability to induce apoptosis in both KRAS‐mutant and Wild‐type (Wt) human PCa cell lines. From this screen, we identified the selective estrogen receptor modulator raloxifene, which is approved for the treatment of osteoporosis and breast cancer in postmenopausal women. Our data show that raloxifene caused apoptosis in PCa cells. We hypothesized that raloxifene treatment compromised mitochondrial integrity either due to altered regulation of apoptotic proteins or due to changes in core mitochondrial membrane components like the phospholipid cardiolipin (CL). To investigate the effect of raloxifene treatment on mitochondrial CL production, we tested its effect on the expression of the CL‐synthesizing enzymes tafazzin and cardiolipin synthase 1. Our data show a raloxifene‐mediated dose‐dependent suppression of both enzymes. Interestingly, raloxifene does not affect the expression of other phospholipid synthesizing enzymes like glycerophosphate acyltransferase, which perhaps explains the lack of changes in the relative amount of phosphatidylcholine and lysophosphatidylcholine when KRAS‐mutant PCa cells were treated with increasing doses of raloxifene. However, suppression of tafazzin correlated with an inhibition of autophagy demonstrated by an increase in the autophagy marker proteins LCII and p62, suggesting that raloxifene prevents the autophagy‐based removal of damaged mitochondria in KRAS‐mutant PCa. To confirm that tafazzin suppression mediated the observed changes in autophagy regulating proteins, using CRISPR technology, we ablated the tafazzin gene. Western blot data show a 75% decrease in tafazzin expression over three days following CRISPR treatment. Interestingly, CRISPR‐based suppression of tafazzin also inhibited autophagy as suggested by changes in the autophagy markers, suggesting a central role for tafazzin in regulating mitochondrial viability in PCa. Additionally, raloxifene increased the expression of the pro‐apoptotic Bim, while having no effect on the expression of the anti‐apoptotic Bcl‐2 and the pro‐apoptotic Bax proteins. Further, there was a dose‐dependent decrease in the activation of caspases 3 and 7 over 72 hours. Western blot data confirmed a lack of the cleavage of procaspases 3 and 9, suggesting activation of caspase‐independent cell death mechanisms in KRAS‐mutant PCa. Raloxifene treatment increased MAP‐Kinase activation as an early event as evidenced by a sustained increase in phosphorylated ERK and p‐38 proteins within an hour of treatment. The impact of our research is to establish raloxifene as a drug that specifically targets the mitochondria in PCa which constitutes a novel strategy to combat this deadly disease by identifying mitochondrial proteins as synthetic lethal targets in the context of KRAS mutations.Support or Funding InformationElsa U. Pardee Foundation GrantResearch Incentive Grant, Pacific University School of Pharmacy

Transcriptional regulation of mitochondrial glycerophosphate acyltransferase is mediated by distal promoter via ChREBP and SREBP-1

We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor.

Distal and proximal promoters of rat mitochondrial glycerophosphate acyltransferase gene direct the tissue specific expression of their transcripts

We have previously shown that rat mtGPAT has two promoters : a regulatory distal promoter which is ~ 30kb upstream of the first translational codon and a constitutive proximal promoter which is 63 bp upstream of the second translational codon. These promoters are responsible for the production of two transcripts with different 5′‐ends. The tissue distribution of the two transcripts and the amount and specific activity of mtGPAT protein were investigated. 5'RACE PCR performed on total RNA isolated from different tissues of rat suggested the presence of alternatively spliced variants of the distal and proximal promoter products. There was presence of three spliced variants of mtGPAT mRNA in liver, kidney and adipose tissues while there was presence of only two variants in skeletal muscle, heart, lung and brain. Western blotting and GPAT specific activity performed on intact mitochondria isolated from the various tissues suggested that both the mtGPAT protein levels and their specific activities were relatively higher in liver, adipose tissues, heart, kidney and were much lower in the other tissues. The results also provide evidence that there is a constant level of the proximal transcript in all the tissues while the amount of the distal transcript varied in a tissue specific manner. Supported by NIH grant GM‐57643.

The distal, not the proximal promoter upregulates mitochondrial glycerophosphate acyltransferase in starved and refed rat liver cells

The effect of starvation and refeeding of liver cells on the activity of mitochondrial glycerophosphate acyltransferase (mtGPAT) was investigated. Rat liver cells were cultured in media lacking glucose, pyruvate, and fetal bovine serum for different periods of time, then placed in media, which contained those compounds, for additional time. After 24, 48 and 72 hrs of starvation and 24 hrs of refeeding of the cells, mtGPAT activity increased up to 5 fold. The viability of the cells was between 85% and 90%. Western blot results confirmed the presence of a larger amount of mtGPAT protein in the group of cells that was starved for 24, 48, and 72 hrs and refed for 24 hrs. The results of RT‐PCR showed enhancement of mRNA levels in the cells after starvation and refeeding. These results suggest that refeeding of liver cells induce an increase in mtGPAT. Additional experiments were done with liver cells transfected with pGL3‐ basic vector containing distal or proximal promoter of mtGPAT and luciferase as the reporter gene. Starvation and refeeding caused 2 to 2.5 fold increase in the luciferase activity in cells transfected with the distal promoter over control cells. No change in the luciferase activity was observed in the cells transfected with the proximal promoter. The results are in keeping with the idea that the increase in mtGPAT is regulated via the distal and not the proximal promoter. Supported by NIH grant GM‐57643.

Role of the distal and proximal promoters in transcriptional regulation of mitochondrial glycerophosphate acyltransferase

We have recently identified the presence of two promoter regions, distal and proximal, for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). The role of these two promoters in the regulation of mtGPAT transcription caused by some hormones and epidermal growth factor (EGF) was investigated. When rat liver cells were treated with insulin or EGF, the mtGPAT protein level and specific activity increased compared to that of the control cells. When cells were treated with glucagon or leptin, mtGPAT protein level and specific activity decreased compared to that of the control cells. 5′RACE PCR showed the presence of two transcripts synthesized presumably from the respective promoters. Analysis of distal transcript obtained from 5′RACE PCR showed similar increase or decrease in cells treated with the above agents in comparison to control cells while no such changes were observed in the proximal transcript. Quantitative analysis of the amounts of the distal transcript showed that cells treated with 1μM insulin or EGF was only 200% and 300% of those obtained from control cells, respectively. On the other hand, mRNA obtained from cells treated with 1μM glucagon or leptin was only 58% and 50% of those obtained from control cells, respectively. The combined results suggest that the regulation of mtGPAT might work through the distal, but not the proximal promoter. Supported by NIH grant GM‐57643.

Further characterization of mitochondrial glycerophosphate acyltransferase distal and proximal promoters

Further characterization of mitochondrial glycerophosphate acyltransferase distal and proximal promoters

First direct evidence of kinase mediated phosphorylation of mitochondrial Glycerol‐3‐phosphate acyltransferase

Mitochondrial glycerophosphate acyltransferase (mtGPAT) esterifies glycerol-3-phosphate at the sn-1 position and is responsible for the asymmetric distribution of fatty acids in glycerophospholipids. We previously presented indirect evidence for the regulation of this enzyme by PKC via phosphorylation. Here, we wished to determine if PKC could interact with and directly phosphorylate immunoprecipitated mtGPAT. Isolated rat liver mitochondria were solubilized and mtGPAT immunoprecipitated using an affinity purified polyclonal anti-GPAT antibody (IM-1). An on-bead kinase reaction was carried out by the addition of either PKC-α or PKC-α and γ 32P-ATP. SDS-PAGE followed by electroblotting and autoradiography of the PVDF revealed a band at 85 kDa, the molecular weight of mtGPAT in the PKC-α treated lane the molecular weight of mtGPAT. This is the first direct evidence of a kinase mediated phosphorylation of mtGPAT. Modified Far-Western blotting showed interaction between PKC and immobilized mtGPAT. PMA (cell permeable activator of PKC) stimulated COS-1 mtGPAT by 50%. Using mouse monoclonal anti-phosphsoserine/anti-phosphothreonine antibodies we showed that PMA treated mtGPAT was serine/threonine phosphorylated. Co-immunoprecipitation showed that PKC co-localized with mtGPAT. It is concluded that PKC stimulates mtGPAT both in vitro and in COS-1 cells. (Supported by NIH grant GM-57643 and a grant from Estee Lauder)

Expression and regulation of 1-acyl-sn-glycerol- 3-phosphate acyltransferases in the epidermis

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue

It has been observed previously that hormone-sensitive lipase-deficient (HSL-ko) mice have reduced white adipose tissue (WAT) stores compared to control mice. These findings contradict the expectation that the decreased lipolytic activity in WAT of HSL-ko mice would cause accumulation of triglycerides (TGs) in that tissue. Here we demonstrate that the cellular TG synthesis in HSL-deficient WAT is markedly reduced due to downregulation of the enzymatic activities of glycerophosphate acyltransferase, dihydroxyacetonphosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase. Fatty acid de novo synthesis is also decreased due to reduced cellular glucose uptake, reduced glucose incorporation into adipose tissue lipids, and reduced activities of acetyl:CoA carboxylase and fatty acid synthase. Finally, the activities of phosphoenolpyruvate carboxykinase (PEPCK), acyl:CoA synthetase (ACS), and glucose 6-phosphate dehydrogenase, the enzymes that provide glycerol-3-phosphate, acyl-CoA, and NADPH for TG synthesis, respectively, are decreased in HSL-ko mice. The reduced expression of the peroxisome proliferator-activated receptor gamma (PPAR gamma) target genes PEPCK, ACS, and aP2, as well as reduced mRNA levels of PPAR gamma itself, suggest the involvement of this transcription factor in the downregulation of lipogenesis. Taken together, these results establish that in the absence of HSL, the reduced NEFA production is counteracted by a drastic reduction of NEFA reesterification that provides sufficient quantities of NEFA for release into the circulation. These metabolic adaptations result in decreased fat mass in HSL-ko mice.

Stimulation of rat liver mitochondrial sn-glycerol-3-phosphate acyltransferase by polymyxin B via enhanced extraction of lysophosphatidic acid.

The purpose of this investigation was to determine how polymyxin B stimulates the activity of mitochondrial glycerophosphate acyltransferase. Polymyxin B did not change the integrity of the mitochondrial outer membrane as judged by testing the latency (>80%) of cytochrome oxidase activity. The stimulation totally disappeared when polymyxin B-treated mitochondria were washed. The FA side chain in polymyxin B was unnecessary for stimulation, as the nonapeptide was as effective as the whole antibiotic. The stimulation by polymyxin B or the nonapeptide was observed only in the presence of BSA. Cytochrome c, when added to the incubation medium instead of albumin, did not stimulate the mitochondrial enzyme, but did produce a stimulatory effect of polymyxin B on the mitochondrial acyltransferase. As reported earlier for the bacterial and microsomal acyltransferase, other polycationic compounds such as spermine and spermidine stimulated mitochondrial glycerophosphate acyltransferase. The stimulation of the mitochondrial acyltransferase by spermine and spermidine also occurred only in the presence of BSA. The analysis of the products of esterification demonstrated the presence of more lysophosphatidic acid (LPA) in the polymyxin B- and polyamine-stimulated assays in comparison to their respective control. Furthermore, in comparison to the albumin-treated control, there was 60% more LPA present in the assay supernatant fractions of polymyxin B-treated samples. Our results suggest that polymyxin B stimulates the mitochondrial glycerophosphate acyltransferase activity by enhancing the extraction of more LPA from the mitochondria to the supernatant fraction.

2P-0550 Possible mechanisms for the antiobesity effects of a novel compound OT-13540

Treatment of normolipidemic rats by alkylthiopropionic acid (CETTD), resulted in a dose- and time-dependent increase in total dihydroxyacetone phosphate acyltransferase (DHAPAT) activity, in extent comparable to that of 3-thiadicarboxylic acid (BCMTD) and alkylthioacetic acid (CMTTD). Thus, in CETTD- and CMTTD-treated rats, the specific DHAPAT activity increased in the microsomal, peroxisomal and mitochondrial fractions. In contrast, repeated administration of the peroxisome proliferator, BCMTD, decreased the specific DHAPAT activity both in the peroxisomal fraction and in purified peroxisomes. A three-fold increase in specific activity was, however, revealed in the mitochondrial fraction. Whether the variation of the DHAPAT activity in the mitochondrial and microsomal fractions among the feeding groups can be explained by increased number of enlarged and small peroxisomes sedimenting in the fractions, are to be considered. Subcellular fractionation studies confirmed previous findings that rat liver glycerophosphate acyltransferase (GPAT) was located both in mitochondria and the microsomal fraction. BCMTD was considerably more potent than CMTTD in stimulating the microsomal and mitochondrial GPAT activities. Administration of CETTD marginally affected the isoenzymes of GPAT. Diacylglycerol acyltransferase (DGAT) activity was increased by 35% in BCMTD and CMTTD treated rats, but by administration of CETTD the enzyme activity was decreased by more than 80%. The acyl-CoA cholesterol acyltransferase (ACAT) activity was marginally affected in animals treated with BCMTD, CMTTD and CETTD. Thus, the results indicate that the initial steps in the synthesis of triacylglycerols and ether glycerolipids as well as the last step in triacylglycerol synthesis could not be identified as mediating the fat accumulation or the lowering of triacylglycerol content in liver of CETTD, or BCMTD and CMTTD treated rats. On the other hand, CMTTD increased the palmitoyl-CoA oxidation in mitochondria, and CETTD considerably inhibited the activity. Therefore, it is conceivable that the development of fatty liver with CETTD is mostly due to inhibition of mitochondrial β-oxidation.

Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity

Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-λ treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [γ- 32P]ATP resulted in a 32P-labeled protein of 85 kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed.

Identification of Two Transmembrane Regions and a Cytosolic Domain of Rat Mitochondrial Glycerophosphate Acyltransferase

The topography of rat glycerophosphate acyltransferase (GAT) in the transverse plane of the mitochondrial outer membrane (MOM) was investigated. Computer analysis of the amino acid (aa) sequence derived from rat mitochondrial GAT cDNA (GenBanktrade mark accession nos. and ) predicts the presence of two possible transmembrane domains (aa 473-493 and 574-594) separated by an 80-aa stretch (aa 494-573). To determine the actual orientation of the native protein, we prepared anti-peptide antibodies to three regions: one in between (aa 543-559) and the other two (aa 420-435 and 726-740) flanking the two putative transmembrane regions. Both immunoreaction and immunoprecipitation experiments employing intact and solubilized mitochondria indicate that regions on the N- and C-terminal sides of the transmembrane regions are sequestered on the inner surface of the MOM, while the region between the transmembrane domains is present on the cytosolic face of the MOM. Additionally, two green fluorescent protein (GFP) fusion proteins consisting of full-length GAT fused to GFP at either the C terminus or inserted 115 amino acids from the N terminus were also constructed to determine the orientation of the N and C termini. COS-1 cells expressing these fusion proteins were fractionated to obtain mitochondria. Protease digestion of intact and solubilized COS-1 cell mitochondria revealed that the GFP domains of these fusion proteins are sequestered on the inner side of the MOM. The present findings indicate that GAT is a dual-spanning, transmembrane protein adopting an inverted "U" conformation in the transverse plane of the MOM, where the N and C termini are sequestered on the inner surface of the MOM, while aa 494-573 are exposed on the cytosolic surface of the MOM.

Induction of the Peroxisomal Glycerolipid-synthesizing Enzymes during Differentiation of 3T3-L1 Adipocytes: ROLE IN TRIACYLGLYCEROL SYNTHESIS

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D-[3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.

Phosphatidic Acid Synthesis in Mitochondria: TOPOGRAPHY OF FORMATION AND TRANSMEMBRANE MIGRATION

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80-100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A(2). The amount of phospholipase A(2)-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.

The inhibitory guanine nucleotide-binding protein Gi2alpha induces and potentiates adipocyte differentiation.

The present study further elucidates the involvement of the alpha-subunit of the GTP-binding protein Gi2 in the differentiation of murine 3T3-L1 cells. Control and vector-transfected cells attained a fully differentiated adipocyte phenotype showing ample lipid droplets. Cells expressing wild type (WT)-Gi2alpha or the constitutively active R179E-Gi2alpha, however, became enlarged, less confluent, and produced large amounts of lipids. Differentiation consistently increased the triglyceride (TAG) content in control cells. In both WT-Gi2alpha and R179E-Gi2alpha clones, a marked increase in TAG could be detected even prior to insulin/dexamethasone/isobutyl methylxanthine exposure. The activity of palmitoyl-CoA synthetase (PCS) and glycerophosphate acyltransferase (GPAT) also increased upon differentiation. WT-Gi2alpha and R179E-Gi2alpha overexpression also enhanced PCS and GPAT activities even before differentiation medium was added. The total amount of phospholipids (PL) generally increased upon differentiation; however, pre- and postdifferentiation values were insignificantly different in cells expressing WT-Gi2alpha and R179E-Gi2alpha. Differentiation altered the PL profile with a relative shift from phosphatidylcholine and phosphatidylethanolamine to phosphatidylinositol (PI) in differentiated cells. Finally, differentiation yielded a general increase in the activity of basal PI-phospholipase-C activity. Again, cells expressing WT-Gi2alpha and R179E-Gi2alpha demonstrated elevated enzyme activity and enhanced second messenger accumulation subsequent to differentiation. In summary, cells with the R179E-mutants of Gi2alpha exhibited stimulated lipid turnover and accumulation in both undifferentiated and differentiated cells.

Enhanced hepatic fatty acid oxidation and upregulated carnitine palmitoyltransferase II gene expression by methyl 3-thiaoctadeca-6,9,12,15-tetraenoate in rats

This study reports the effects of a novel polyunsaturated 3-thia fatty acid, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on serum lipids and key enzymes in hepatic fatty acid metabolism compared to a saturated 3-thia fatty acid, tetradecylthioacetic acid. Palmitic acid treated rats served as controls. Fatty acids were administered by gavage in daily doses of 150 mg/kg body weight for 10 days. The aim of the present study was: (a) To investigate the effect of a polyunsaturated 3-thia fatty acid ester, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on plasma lipids in normolipidemic rats; (b) to verify whether the lipid-lowering effect could be consistent with enhanced fatty acid oxidation; and (c) to study whether decreased activity of esterifying enzymes and diversion to phospholipid synthesis is a concerted mechanism in limiting the availability of free fatty acid as a substrate for hepatic triglyceride formation. Repeated administration of the polyunsaturated 3-thia fatty acid ester for 10 days resulted in a reduction of plasma triglycerides (40%), cholesterol (33%) and phospholipids (20%) compared to controls. Administration of polyunsaturated and saturated 3-thia fatty acids (daily doses of 150 mg/kg body weight) reduced levels of lipids to a similar extent and followed about the same time-course. Both mitochondrial and peroxisomal fatty acid oxidation increased (1.4-fold- and 4.2-fold, respectively) and significantly increased activities of carnitine palmitoyltransferase (CPT) (1.6-fold), 2,4-dienoyl-CoA reductase (1.2-fold) and fatty acyl-CoA oxidase (3.0-fold) were observed in polyunsaturated 3-thia fatty acid treated animals. This was accompanied by increased CPT-II mRNA (1.7-fold), 2,4-dienoyl-CoA reductase mRNA (2.9-fold) and fatty acyl-CoA oxidase mRNA (1.7-fold). Compared to controls, the hepatic triglyceride biosynthesis was retarded as indicated by a decrease in liver triglyceride content (40%). The activities of glycerophosphate acyltransferase, acyl-CoA: 1,2-diacylglycerol acyltransferase and CTP:phosphocholine cytidylyltransferase were increased. The cholesterol lowering effect was accompanied by a reduction in HMG-CoA reductase activity (80%) and acyl-CoA:cholesterol acyltransferase activity (33%). In hepatocytes treated with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate, fatty acid oxidation was increased 1.8-fold compared to controls. The results suggest that treatment with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate reduces plasma triglycerides by a decrease in the availability of fatty acid substrate for triglyceride biosynthesis via enhanced fatty acid oxidation, most likely attributed to the mitochondrial fatty acid oxidation. It is hypothesized that decreased phosphatidate phosphohydrolase activity may be an additive mechanism which contribute whereby 3-thia fatty acids reduce triglyceride formation in the liver. The cholesterol-lowering effect of the polyunsaturated 3-thia fatty acid ester may be due to changes in cholesterol/cholesterol ester synthesis as 60% of this acid was observed in the hepatic cholesterol ester fraction.