Hongmei Ren

piRNA-Associated Germline Nuage Formation and Spermatogenesis Require MitoPLD Profusogenic Mitochondrial-Surface Lipid Signaling

The mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmic nuclease, is required for piRNA generation, a critical event in germline development. We show that Zuc localizes to mitochondria and has MitoPLD-like activity. Conversely, MitoPLD(-/-) mice exhibit the meiotic arrest, DNA damage, and male sterility characteristic of mice lacking piRNAs. The primary function of MitoPLD seems to be the generation of mitochondrial-surface PA. This PA in turn recruits the phosphatase Lipin 1, which converts PA to diacylglycerol and promotes mitochondrial fission, suggesting a mechanism for mitochondrial morphology homeostasis. MitoPLD and Lipin 1 have opposing effects on mitochondria length and on intermitochondrial cement (nuage), a structure found between aggregated mitochondria that is implicated in piRNA generation. We propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits or activates nuage components critical for piRNA production.

5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a Phospholipase D Pharmacological Inhibitor That Alters Cell Spreading and Inhibits Chemotaxis

The signaling enzyme phospholipase D (PLD) and the lipid second messenger it generates, phosphatidic acid (PA), are implicated in many cell biological processes, including Ras activation, cell spreading, stress fiber formation, chemotaxis, and membrane vesicle trafficking. PLD production of PA is inhibited by the primary alcohol 1-butanol, which has thus been widely employed to identify PLD/PA-driven processes. However, 1-butanol does not always effectively reduce PA accumulation, and its use may result in PLD-independent deleterious effects. Consequently, identification of potent specific small-molecule PLD inhibitors would be an important advance for the field. We examine one such here, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), which was identified recently in an in vitro chemical screen for PLD2 inhibitors, and show that it rapidly blocks in vivo PA production with subnanomolar potency. We were surprised to find that several biological processes blocked by 1-butanol are not affected by FIPI, suggesting the need for re-evaluation of proposed roles for PLD. However, FIPI does inhibit PLD regulation of F-actin cytoskeleton reorganization, cell spreading, and chemotaxis, indicating potential utility for it as a therapeutic for autoimmunity and cancer metastasis.

Expression of the Splicing Factor Gene SFRS10 is Reduced in Human Obesity and Contributes to Enhanced Lipogenesis

Alternative mRNA splicing provides transcript diversity and may contribute to human disease. We demonstrate that expression of several genes regulating RNA processing is decreased in both liver and skeletal muscle of obese humans. We evaluated a representative splicing factor, SFRS10, downregulated in both obese human liver and muscle and in high-fat-fed mice, and determined metabolic impact of reduced expression. SFRS10-specific siRNA induces lipogenesis and lipid accumulation in hepatocytes. Moreover, Sfrs10 heterozygous mice have increased hepatic lipogenic gene expression, VLDL secretion, and plasma triglycerides. We demonstrate that LPIN1, a key regulator of lipid metabolism, is a splicing target of SFRS10; reduced SFRS10 favors the lipogenic β isoform of LPIN1. Importantly, LPIN1β-specific siRNA abolished lipogenic effects of decreased SFRS10 expression. Together, our results indicate that reduced expression of SFRS10, as observed in tissues from obese humans, alters LPIN1 splicing, induces lipogenesis, and therefore contributes to metabolic phenotypes associated with obesity.

Autotaxin and Its Product Lysophosphatidic Acid Suppress Brown Adipose Differentiation and Promote Diet-Induced Obesity in Mice

Brown adipose tissue is a thermogenic organ that dissipates stored energy as heat to maintain body temperature. This process may also provide protection from development of diet-induced obesity. We report that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, whereas potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibit reduced expression of brown adipose tissue-related genes in peripheral white adipose tissue and accumulate significantly more fat than wild-type controls when fed a high-fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and are consistent with a model in which a decrease in mature peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice.

A Phosphatidic Acid Binding/Nuclear Localization Motif Determines Lipin1 Function in Lipid Metabolism and Adipogenesis

A polybasic motif in the metabolic regulator lipin1 is both a membrane anchor and a nuclear localization sequence required for lipin1 function in phospholipid metabolism and adipogenesis.

Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation

Lipin 1 is a coregulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that catalyzes a critical step in the synthesis of glycerophospholipids. Lipin 1 is highly expressed in adipocytes, and constitutive loss of lipin 1 blocks adipocyte differentiation; however, the effects of Lpin1 deficiency in differentiated adipocytes are unknown. Here we report that adipocyte-specific Lpin1 gene recombination unexpectedly resulted in expression of a truncated lipin 1 protein lacking PAP activity but retaining transcriptional regulatory function. Loss of lipin 1-mediated PAP activity in adipocytes led to reduced glyceride synthesis and increased PA content. Characterization of the deficient mice also revealed that lipin 1 normally modulates cAMP-dependent signaling through protein kinase A to control lipolysis by metabolizing PA, which is an allosteric activator of phosphodiesterase 4 and the molecular target of rapamycin. Consistent with these findings, lipin 1 expression was significantly related to adipose tissue lipolytic rates and protein kinase A signaling in adipose tissue of obese human subjects. Taken together, our findings identify lipin 1 as a reciprocal regulator of triglyceride synthesis and hydrolysis in adipocytes, and suggest that regulation of lipolysis by lipin 1 is mediated by PA-dependent modulation of phosphodiesterase 4.

Lipid phosphate phosphatase (LPP3) and vascular development

Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Blood mononuclear cells and platelets have abnormal fatty acid composition in homozygous sickle cell disease

Leukocyte adhesion to vascular endothelium contributes to vaso-occlusion and widespread organ damage in sickle cell disease (SCD). Previously, we found high expression of the adhesion molecules αMβ2 integrin and L-selectin in HbSS individuals with severe disease. Since membrane n-6 and n-3 polyunsaturated fatty acids modulate cell adhesion, inflammation, aggregation and vascular tone, we investigated the fatty acid composition of mononuclear cells (MNC) and platelets of HbSS patients in steady state (n=28) and racially matched, healthy HbAA controls with similar age and sex distribution living in the same environment (n=13). MNC phospholipids of the patients had lower levels of docosahexaenoic acid (DHA, p<0.01) and increased arachidonic acid (AA, p<0.005) relative to HbAA controls. Similarly, platelets from HbSS patients had less eicosapentaenoic acid (EPA, p<0.05) and more AA (p<0.05) in choline phosphoglycerides (CPG), with reduced DHA (p<0.05) in ethanolamine phosphoglycerides. Platelet CPG had lower DHA levels in SCD patients with complications compared to those without (p<0.05). Reduced cell content of EPA and DHA relative to AA favours the production of aggregatory and proinflammatory eicosanoids that activate leukocytes and platelets. This facilitates inflammation, leukocyte adhesion, platelet aggregation and vaso-occlusion in SCD.

Patients with Sickle Cell Disease have Reduced Blood Antioxidant Protection

In previous studies, we found that homozygous sickle cell (HbSS) patients, compared with their healthy (HbAA) counterparts, had reduced levels of the omega-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, in red cells, platelets, and mononuclear cells. These differences were not due to lower intake of the two fatty acids. We have investigated whether reduced antioxidant status in the patients could help explain the observed phenomenon. Blood specimens previously obtained for fatty acid study from Nigerian (26 HbSS and 30 HbAA) and British (30 HbSS, 9 sickle cell-hemoglobin C/HbSC, and 15 HbAA) subjects were analyzed for antioxidant status. The Nigerian HbSS patients compared with the controls had lower plasma retinol, alpha-tocopherol, and beta-carotene concentrations (p < 0.005) and reduced activity of red cell Cu/Zn-superoxide dismutase (Cu/Zn-SOD) (p < 0.05). Similarly, the British HbSS group had reduced concentrations of plasma alpha-tocopherol (p < 0.005), and activities of red cell Cu/Zn-superoxide dismutase (p < 0.05) and Se-glutathione peroxidase (Se-GPx) (p < 0.005) than the controls. In addition, the British patients in comparison with those who had HbSC, a mild form of the disease, had lower alpha-tocopherol than that of the HbAA controls (p < 0.005). In the British sickle cell patients, there was a positive correlation between red cell ethanolamine phosphoglyceride (EPG) DHA and Cu/Zn-SOD activity (r = 0.700, p < 0.05), choline phosphoglyceride (CPG) DHA and Se-GPx activity (r = 0.605, p < 0.05), and CPG EPA and Se-GPx activity (r = 0.558, p > 0.05). Similarly, the percent DHA in red cell EPG was positively related with the activity of Se-GPx in the patients with HbSC (r = 0.674, p < 0.05). These findings suggest that the lower levels of membrane EPA and DHA in blood cells of the HbSS patients could be due to peroxidation resulting from a compromised antioxidant competence.

Lipin proteins form homo- and hetero-oligomers.

Lipin family members (lipin 1, 2 and 3) are bi-functional proteins that dephosphorylate PA (phosphatidic acid) to produce DAG (diacylglycerol) and act in the nucleus to regulate gene expression. Although other components of the triacylglycerol synthesis pathway can form oligomeric complexes, it is unknown whether lipin proteins also exist as oligomers. In the present study, using various approaches, we revealed that lipin 1 formed stable homo-oligomers with itself and hetero-oligomers with lipin 2/3. Both the N- and C-terminal regions of lipin 1 mediate its oligomerization in a head-to-head/tail-to-tail manner. We also show that lipin 1 subcellular localization can be influenced through oligomerization, and the individual lipin 1 monomers in the oligomer function independently in catalysing dephosphorylation of PA. The present study provides evidence that lipin proteins function as oligomeric complexes and that the three mammalian lipin isoforms can form combinatorial units.