HMGA2 as a functional antagonist of PARP1 inhibitors in tumor cells.

Poly(ADP-ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage-induced PARP1 activity. We used human triple-negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage-induced PARP1 activity, which was dependent on functional DNA-binding AT-hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells.

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Glucose Uptake and Triacylglycerol Synthesis Are Increased in Barth Syndrome Lymphoblasts.

Barth syndrome (BTHS) is an X-linked genetic disease resulting in loss of cardiolipin (Ptd2Gro). Patients may be predisposed to hypoglycemia and exhibit increases in whole-body glucose disposal rates and a higher fat mass percentage. We examined the reasons for this in BTHS lymphoblasts. BTHS lymphoblasts exhibited a 60% increase (p < 0.004) in 2-[1,2-3H(N)]deoxy-D-glucose uptake, a 40% increase (p < 0.01) in glucose transporter-3 protein expression, an increase in phosphorylated-adenosine monophosphate kinase (AMPK) and a 58% increase (p < 0.001) in the phosphorylated-AMPK/AMPK ratio compared to controls. In addition, BTHS lymphoblasts exhibited a 90% (p < 0.001) increase in D-[U-14C]glucose incorporated into 1,2,3-triacyl-sn-glycerol (TAG) and a 29% increase (p < 0.025) in 1,2-diacyl-sn-glycerol acyltransferase-2 activity compared to controls. Thus, BTHS lymphoblasts exhibit increased glucose transport and increased glucose utilization for TAG synthesis. These results may, in part, explain why BTHS patients exhibit an increase in whole-body glucose disposal rates, may be predisposed to hypoglycemia and exhibit a higher fat mass percentage.

Dietary linoleate preserves cardiolipin and attenuates mitochondrial dysfunction in the failing rat heart.

AIMS: Cardiolipin (CL) is a tetra-acyl phospholipid that provides structural and functional support to several proteins in the inner mitochondrial membrane. The majority of CL in the healthy mammalian heart contains four linoleic acid acyl chains (L(4)CL). A selective loss of L(4)CL is associated with mitochondrial dysfunction and heart failure in humans and animal models. We examined whether supplementing the diet with linoleic acid would preserve cardiac L(4)CL and attenuate mitochondrial dysfunction and contractile failure in rats with hypertensive heart failure. METHODS AND RESULTS: Male spontaneously hypertensive heart failure rats (21 months of age) were administered diets supplemented with high-linoleate safflower oil (HLSO) or lard (10% w/w; 28% kilocalorie fat) or without supplemental fat (control) for 4 weeks. HLSO preserved L(4)CL and total CL to 90% of non-failing levels (vs. 61-75% in control and lard groups), and attenuated 17-22% decreases in state 3 mitochondrial respiration observed in the control and lard groups (P < 0.05). Left ventricular fractional shortening was significantly higher in HLSO vs. control (33 ± 2 vs. 29 ± 2%, P < 0.05), while plasma insulin levels were lower (5.4 ± 1.1 vs. 9.1 ± 2.3 ng/mL; P < 0.05), with no significant effect of lard supplementation. HLSO also increased serum concentrations of several eicosanoid species compared with control and lard diets, but had no effect on plasma glucose or blood pressure. CONCLUSION: Moderate consumption of HLSO preserves CL and mitochondrial function in the failing heart and may be a useful adjuvant therapy for this condition.

Mifepristone treatment results in differential regulation of glycerolipid biosynthesis in baby hamster kidney cells expressing a mifepristone-inducible ABCA1.

ATP binding cassette A1 (ABCA1) transports cholesterol, phospholipids and lipophilic molecules to and across cellular membranes. We examined if ABCA1 expression altered cellular de novo glycerolipid biosynthesis in growing Baby hamster kidney (BHK) cells. Mock BHK cells or cells expressing a mifepristone-inducible ABCA1 (ABCA1) were incubated plus or minus mifepristone and then with [(3)H]serine or [(3)H]inositol or [(3)H]ethanolamine or [methyl-(3)H]choline or [(3)H]glycerol or [(14)C]oleate and radioactivity incorporated into glycerolipids determined. Mifepristone did not affect [1,3-(3)H]glycerol or [(14)C]oleate or [(3)H]ethanolamine or [methyl-(3)H]choline uptake in BHK cells. In contrast, [(3)H]glycerol and [(14)C]oleate incorporated into phosphatidylserine (PtdSer) were elevated 2.4-fold (p < 0.05) and 54% (p < 0.05), respectively, upon ABCA1 induction confirming increased PtdSer biosynthesis from these precursors. However, mifepristone inhibited [(3)H]serine uptake and incorporation into PtdSer indicating that PtdSer synthesis from serine in BHK cells is dependent on serine uptake. Mifepristone stimulated [(3)H]inositol uptake in mock and ABCA1 cells but not its incorporation into phosphatidylinositol indicating that its synthesis from inositol is independent of inositol uptake in BHK cells. [(3)H]glycerol and [(14)C]oleate incorporated into triacylglycerol were reduced and into diacylglycerol elevated only in mifepristone-induced ABCA1 expressing cells due to a decrease in diacylglycerol acyltransferase-1 (DGAT-1) activity. The presence of trichostatin A, a class I and II histone deacetylase inhibitor, reversed the ABCA1-mediated reduction in DGAT-1 activity but did not affect DGAT-1 mRNA expression. Thus, mifepristone has diverse effects on de novo glycerolipid synthesis. We suggest that caution should be exercised when using mifepristone-inducible systems for studies of glycerolipid metabolism in cells expressing glucocorticoid responsive receptors.

The mevalonate cascade as a target to suppress extracellular matrix synthesis by human airway smooth muscle.

Smooth muscle cells promote fibroproliferative airway remodeling in asthma, and transforming growth factor ß1 (TGFß1) is a key inductive signal. Statins are widely used to treat hyperlipidemia. Growing evidence indicates they also exert a positive impact on lung health, but the underlying mechanisms are unclear. We assessed the effects of 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase inhibition with simvastatin on the fibrotic function of primary cultured human airway smooth muscle cells. Simvastatin blocked de novo cholesterol synthesis, but total myocyte cholesterol content was unaffected. Simvastatin also abrogated TGFß1-induced collagen I and fibronectin expression, and prevented collagen I secretion. The depletion of mevalonate cascade intermediates downstream from HMG-CoA underpinned the effects of simvastatin, because co-incubation with mevalonate, geranylgeranylpyrophosphate, or farnesylpyrophosphate prevented the inhibition of matrix protein expression. We also showed that human airway myocytes express both geranylgeranyl transferase 1 (GGT1) and farnesyltransferase (FT), and the inhibition of GGT1 (GGTI inhibitor-286, 10 µM), but not FT (FTI inhibitor-277, 10 µM), mirrored the suppressive effects of simvastatin on collagen I and fibronectin expression and collagen I secretion. Moreover, simvastatin and GGTI-286 both prevented TGFß1-induced membrane association of RhoA, a downstream target of GGT1. Our findings suggest that simvastatin and GGTI-286 inhibit synthesis and secretion of extracellular matrix proteins by human airway smooth muscle cells by suppressing GGT1-mediated posttranslational modification of signaling molecules such as RhoA. These findings reveal mechanisms related to evidence for the positive impact of statins on pulmonary health.

Cardiolipin synthase-1 mRNA expression does not correlate with endogenous cardiolipin synthase enzyme activity in vitro and in vivo in mammalian lipopolysaccharide models of inflammation.

We examined if lipopolysaccharide (LPS) treatment of mice affected cardiolipin (CL) synthesis. Mice were injected i.p. with LPS, the liver harvested, and CL synthase (CLS) enzyme activity and its mRNA expression examined. Treatment of mice with LPS resulted in a 55% decrease (p < 0.01) in mRNA expression of murine CLS compared to controls, but CLS enzyme activity was unaltered. The pool size of liver CL and other phospholipids were unaltered by LPS treatment. A similar effect was observed in murine epidermal fat pad and in vitro in RAW mouse macrophages and in human HepG2 cells. LPS treatment of HepG2 cells transiently expressing a histidine-tagged human cardiolipin synthase-1 (hCLS1) reduced hCLS1 mRNA and newly synthesized CLS activity indicating that LPS inhibits production of newly synthesized hCLS1 via reduction in hCLS1 mRNA. The results clearly indicate that CLS mRNA levels cannot be correlated with CLS enzyme activity nor CL content in the LPS model of inflammation.

The dynamics of cardiolipin synthesis post-mitochondrial fusion.

Alteration in mitochondrial fusion may regulate mitochondrial metabolism. Since the phospholipid cardiolipin (CL) is required for function of the mitochondrial respiratory chain, we examined the dynamics of CL synthesis in growing Hela cells immediately after and 12h post-fusion. Cells were transiently transfected with Mfn-2, to promote fusion, or Mfn-2 expressing an inactive GTPase for 24h and de novo CL biosynthesis was examined immediately after or 12h post-fusion. Western blot analysis confirmed elevated Mfn-2 expression and electron microscopic analysis revealed that Hela cell mitochondrial structure was normal immediately after and 12h post-fusion. Cells expressing Mfn-2 exhibited reduced CL de novo biosynthesis from [1,3-(3)H]glycerol immediately after fusion and this was due to a decrease in phosphatidylglycerol phosphate synthase (PGPS) activity and its mRNA expression. In contrast, 12h post-mitochondrial fusion cells expressing Mfn-2 exhibited increased CL de novo biosynthesis from [1,3-(3)H]glycerol and this was due to an increase in PGPS activity and its mRNA expression. Cells expressing Mfn-2 with an inactive GTPase activity did not exhibit alterations in CL de novo biosynthesis immediately after or 12h post-fusion. The Mfn-2 mediated alterations in CL de novo biosynthesis were not accompanied by alterations in CL or monolysoCL mass. [1-(14)C]Oleate incorporation into CL was elevated at 12h post-fusion indicating increased CL resynthesis. The reason for the increased CL resynthesis was an increased mRNA expression of tafazzin, a mitochondrial CL resynthesis enzyme. Ceramide-induced expression of PGPS in Hela cells or in CHO cells did not alter expression of Mfn-2 indicating that Mfn-2 expression is independent of altered CL synthesis mediated by elevated PGPS. In addition, Mfn-2 expression was not altered in Hela cells expressing phospholipid scramblase-3 or a disrupted scramblase indicating that proper CL localization within mitochondria is not essential for Mfn-2 expression. The results suggest that immediately post-mitochondrial fusion CL de novo biosynthesis is "slowed down" and then 12h post-fusion it is "upregulated". The implications of this are discussed.

StARD13(Dlc-2) RhoGap mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells.

To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C(2)-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1-2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C(2)-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C(2)-ceramide or tumor necrosis factor alpha, but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C(2)-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.

The phospholipidosis-lnducing potential of the chemopotentiating drug, N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE, tesmilifene) correlates with its stimulation of phosphatidylserine synthesis and exposure on the plasma membrane in MCF-7 breast cancer cells.

N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE, tesmilifene), a potent chemopotentiating drug currently in Phase III clinical trials of metastatic breast cancer, increases cytotoxicity of anthracyclines and taxanes in a variety of multi-drug resistance expressing (MDR+) tumor cell lines in vitro; inhibits binding of histamine to CYP3A4, a lipid/prostanoid-regulatory P450; and modulates serum levels of HDL/LDL cholesterol and phospholipids in vivo. Since increased exposure of phosphatidylserine (PS) on the outer cell membrane leaflet is associated with apoptosis, increased clearance of dead cells by phagocytes and inhibition of the P-glycoprotein pump, the effect of DPPE on PS synthesis was assessed in vitro in a human breast cancer cell line. MCF-7 cells were incubated with 5 microM DPPE for 24 hr or 5 days, followed by addition of [1-(14)C]arachidonic acid for 4 hr; or [3H]serine for 8 hr. Compared to untreated cells, a 27-42% (p < 0.05) increase in [1-(14)C]arachidonic acid incorporated into all phospholipids, including a 1.9-fold increase (p < 0.05) in PS was observed in DPPE-treated cells. [3H]Serine incorporation into PS was elevated 37%, while the pool size of PS was elevated 23% (p < 0.05) in DPPE-treated cells, indicating elevated de novo PS biosynthesis. Annexin-5 binding studies indicated an elevation in exposure of PS on the surface of the plasma membrane in DPPE-treated cells. DPPE-treatment also resulted in N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine accumulation indicative of phospholipidosis-inducing potential. Thus, the chemopotentiating effect of DPPE may be due to its phospholipidosis-inducing potential and stimulation of PS synthesis leading to an increased exposure of PS on the cell surface which could potentially enhance cancer cell clearance by phagocytes.

Cloning and characterization of a cDNA encoding human cardiolipin synthase (hCLS1).

Cardiolipin (CL) is a phospholipid localized to the mitochondria, and its biosynthesis is essential for mitochondrial structure and function. We report here the identification and characterization of a cDNA encoding the first mammalian cardiolipin synthase (CLS1) in humans and mice. This cDNA exhibits sequence homology with members of a CLS gene family that share similar domain structure and chemical properties. Expression of the human CLS (hCLS1) cDNA in reticulocyte lysates or insect cells led to a marked increase in CLS activity. The enzyme is specific for CL synthesis, because no significant increase in phosphatidylglycerol phosphate synthase activity was observed. In addition, CL pool size was increased in hCLS1-overexpressing cells compared with controls. Furthermore, the hCLS1 gene was highly expressed in tissues such as heart, skeletal muscle, and liver, which have been shown to have high CLS activities. These results demonstrate that hCLS1 encodes an enzyme that synthesizes CL.

Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.

AGPAT (1-acyl-sn-glycerol 3-phosphate acyltransferase) exists in at least five isoforms in humans, termed as AGPAT1, AGPAT2, AGPAT3, AGPAT4 and AGPAT5. Although they catalyse the same biochemical reaction, their relative function, tissue expression and regulation are poorly understood. Linkage studies in humans have revealed that AGPAT2 contributes to glycerolipid synthesis and plays an important role in regulating lipid metabolism. We report the molecular cloning, tissue distribution, and enzyme characterization of mAGPATs (murine AGPATs) and regulation of cardiac mAGPATs by PPARalpha (peroxisome-proliferator-activated receptor alpha). mAGPATs demonstrated differential tissue expression profiles: mAGPAT1 and mAGPAT3 were ubiquitously expressed in most tissues, whereas mAGPAT2, mAGPAT4 and mAGPAT5 were expressed in a tissue-specific manner. mAGPAT2 expressed in in vitro transcription and translation reactions and in transfected COS-1 cells exhibited specificity for 1-acyl-sn-glycerol 3-phosphate. When amino acid sequences of five mAGPATs were compared, three highly conserved motifs were identified, including one novel motif/pattern KX2LX6GX12R. Cardiac mAGPAT activities were 25% lower (P<0.05) in PPARalpha null mice compared with wild-type. In addition, cardiac mAGPAT activities were 50% lower (P<0.05) in PPARalpha null mice fed clofibrate compared with clofibrate fed wild-type animals. This modulation of AGPAT activity was accompanied by significant enhancement/reduction of the mRNA levels of mAGPAT3/mAGPAT2 respectively. Finally, mRNA expression of cardiac mAGPAT3 appeared to be regulated by PPARalpha activation. We conclude that cardiac mAGPAT activity may be regulated by both the composition of mAGPAT isoforms and the levels of each isoform.

On the mechanism of the increase in cardiolipin biosynthesis and resynthesis in hepatocytes during rat liver regeneration.

CL (cardiolipin) is a major mitochondrial membrane phospholipid important for the regulation of mitochondrial function. We examined CL de novo biosynthesis and its resynthesis in isolated rat liver hepatocytes prepared 48 h subsequent to two-thirds PHx (partial hepatectomy). The pool size of CL and its de novo biosynthesis from [1,3-(3)H]glycerol were increased 3.3-fold (P<0.05) and 3.1-fold (P<0.05) respectively in hepatocytes prepared from PHx rats compared with sham-operated controls. The reason for the increased CL biosynthesis was a 65% increase (P<0.05) in enzymic activity in PGP-S (phosphatidylglycerolphosphate synthase), a key enzyme in de novo CL biosynthesis. The increase in PGP-S activity was due to a 3-fold increase (P<0.05) of hepatic PGP-S mRNA expression. The increase in de novo CL biosynthesis and pool size corresponded to a 2.3-fold increase (P<0.05) in the amount of [1-14C]linoleic acid incorporated into CL of hepatocytes prepared from PHx rats compared with sham-operated controls, indicating an increase in CL resynthesis. The activity of MLCL-AT (monolysocardiolipin acyltransferase), a rate-limiting enzyme of CL resynthesis, was increased by 43% (P<0.05) in hepatocytes prepared from PHx rats compared with sham-operated controls; this result would explain the increase in [1-14C]linoleic acid incorporation into CL. The increase in MLCL-AT activity was due to an increase in hepatic MLCL-AT protein expression. The results show that CL de novo biosynthesis and its resynthesis are increased during liver regeneration.

Stimulation of cardiac cardiolipin biosynthesis by PPARalpha activation.

The role of peroxisome proliferator-activated receptor alpha (PPARalpha)-stimulated phospholipase A2 (PLA2) in cardiac mitochondrial cardiolipin (CL) biosynthesis was examined in both in vivo and in vitro models. Treatment of rat heart H9c2 cells with clofibrate increased the expression and activity of 14 kDa PLA2 but did not affect the pool size of CL. Clofibrate treatment stimulated de novo CL biosynthesis via an increase in phosphatidylglycerolphosphate (PGP) synthase activity, accounting for the unaltered CL content. Cardiac PLA2, PGP synthase, and CDP-1,2-diacyl-sn-glycerol synthase (CDS-2) activities and CDS-2 mRNA levels were elevated in mice fed clofibrate for 14 days compared with controls. In PPARalpha-null mice, clofibrate feeding did not alter cardiac PLA2, PGP synthase activities, or CDS-2 activity and mRNA level, confirming that these enzymes are regulated by PPARalpha activation. In contrast to mouse heart, clofibrate treatment did not affect the activity or mRNA levels of CDS-2 in H9c2 cells, indicating that CDS-2 is regulated differently in rat heart H9c2 cells in vitro and in mouse heart in vivo. These results clearly indicate that cardiac CL de novo biosynthesis is stimulated by PPARalpha activation in responsive rodent models and that CDS-2 is an example of an enzyme that exhibits alternative regulation in vivo and in cultured cell lines. This study is the first to demonstrate that CL de novo biosynthesis is regulated by PPARalpha activation.

Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells.

We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.

FATP1 channels exogenous FA into 1,2,3-triacyl-sn-glycerol and down-regulates sphingomyelin and cholesterol metabolism in growing 293 cells.

Biosynthesis of lipids was investigated in growing 293 cells stably expressing fatty acid (FA) transport protein 1 (FATP1), a bifunctional polypeptide with FA transport as well as fatty acyl-CoA synthetase activity. In short-term (30 s) incubations, FA uptake was increased in FATP1 expressing cells (C8 cells) compared with the vector (as determined by BODIPY 3823 staining and radioactive FA uptake). In long-term (4 h) incubations, incorporation of [(14)C]acetate, [3H]oleic acid, or [(14)C]lignoceric acid into 1,2,3-triacyl-sn-glycerol (TG) was elevated in C8 cells compared with vector, whereas incorporation of radiolabel into glycerophospholipids was unaltered. The increase in TG biosynthesis correlated with an increase in 1,2-diacyl-sn-glycerol acyltransferase activity in C8 cells compared with vector. In contrast, incorporation of [(14)C]acetate into sphingomyelin (SM) and cholesterol, and [3H]oleic acid or [(14)C]lignoceric acid into SM was reduced due to a reduction in de novo biosynthesis of these lipids in C8 cells compared with vector. The results indicate that exogenously supplied FAs, and their subsequently produced acyl-CoAs, are preferentially channeled by an FATP1 linked mechanism into the TG biosynthetic pathway and that such internalized lipids down-regulate de novo SM and cholesterol metabolism in actively growing 293 cells.

Expression of monolysocardiolipin acyltransferase activity is regulated in concert with the level of cardiolipin and cardiolipin biosynthesis in the mammalian heart.

BACKGROUND: Monolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation. RESULTS: In rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil in the drinking water for 34 days, cardiac cardiolipin content was decreased 29% (p < 0.025) and this was associated with a 32% decrease (p < 0.025) in cardiolipin synthase and a 35% reduction (p < 0.025) in MLCL AT activities. Streptozotocin-induced diabetes or hyperinsulinemia in rats did not affect cardiac cardiolipin content nor MLCL AT and cardiolipin synthase activities. Finally, cardiolipin content, MLCL AT and cardiolipin synthase activities were unaltered during murine P19 teratocarcinoma cell differentiation into cardiac myocytes. In all models, phospholipase A2 activities were unaltered compared with controls. CONCLUSION: We propose a general model in which the expression of MLCL AT activity is regulated in concert with the biosynthesis and level of cardiolipin in the heart.