Redundant roles of the phosphatidate phosphatase family in triacylglycerol synthesis in human adipocytes.

AIMS/HYPOTHESIS: In mammals, the evolutionary conserved family of Mg(2+)-dependent phosphatidate phosphatases (PAP1), involved in phospholipid and triacylglycerol synthesis, consists of lipin-1, lipin-2 and lipin-3. While mutations in the murine Lpin1 gene cause lipodystrophy and its knockdown in mouse 3T3-L1 cells impairs adipogenesis, deleterious mutations of human LPIN1 do not affect adipose tissue distribution. However, reduced LPIN1 and PAP1 activity has been described in participants with type 2 diabetes. We aimed to characterise the roles of all lipin family members in human adipose tissue and adipogenesis. METHODS: The expression of the lipin family was analysed in adipose tissue in a cross-sectional study. Moreover, the effects of lipin small interfering RNA (siRNA)-mediated depletion on in vitro human adipogenesis were assessed. RESULTS: Adipose tissue gene expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell line, alters expression levels of adipogenic transcription factors and lipid biosynthesis genes in early stages of differentiation. Lipin-1 knockdown alone causes a 95% depletion of PAP1 activity. Despite the reduced PAP1 activity and alterations in early adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids. Even combinatorial knockdown of lipins shows mild effects on triacylglycerol accumulation in mature adipocytes. CONCLUSIONS/INTERPRETATION: Overall, our data support the hypothesis of alternative pathways for triacylglycerol synthesis in human adipocytes under conditions of repressed lipin expression. We propose that induction of alternative lipid phosphate phosphatases, along with the inhibition of lipid hydrolysis, contributes to the maintenance of triacylglycerol content to near normal levels.

Distinct roles of the phosphatidate phosphatases lipin 1 and 2 during adipogenesis and lipid droplet biogenesis in 3T3-L1 cells.

Lipins are evolutionarily conserved Mg(2+)-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

Hypoxia causes triglyceride accumulation by HIF-1-mediated stimulation of lipin 1 expression.

Adaptation to hypoxia involves hypoxia-inducible transcription factors (HIFs) and requires reprogramming of cellular metabolism that is essential during both physiological and pathological processes. In contrast to the established role of HIF-1 in glucose metabolism, the involvement of HIFs and the molecular mechanisms concerning the effects of hypoxia on lipid metabolism are poorly characterized. Here, we report that exposure of human cells to hypoxia causes accumulation of triglycerides and lipid droplets. This is accompanied by induction of lipin 1, a phosphatidate phosphatase isoform that catalyzes the penultimate step in triglyceride biosynthesis, whereas lipin 2 remains unaffected. Hypoxic upregulation of lipin 1 expression involves predominantly HIF-1, which binds to a single distal hypoxia-responsive element in the lipin 1 gene promoter and causes its activation under low oxygen conditions. Accumulation of hypoxic triglycerides or lipid droplets can be blocked by siRNA-mediated silencing of lipin 1 expression or kaempferol-mediated inhibition of HIF-1. We conclude that direct control of lipin 1 transcription by HIF-1 is an important regulatory feature of lipid metabolism and its adaptation to hypoxia.

The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization.

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

The conserved translocase Tim17 prevents mitochondrial DNA loss.

Maintenance of an intact mitochondrial genome is essential for oxidative phosphorylation in all eukaryotes. Depletion of mitochondrial genome copy number can have severe pathological consequences due to loss of respiratory capacity. In Saccharomyces cerevisiae, several bifunctional metabolic enzymes have been shown to be required for mitochondrial DNA (mtDNA) maintenance. For example, Ilv5 is required for branched chain amino acid biosynthesis and mtDNA stability. We have identified OXA1 and TIM17 as novel multicopy suppressors of mtDNA instability in ilv5 cells. In addition, overexpression of TIM17, but not OXA1, prevents the complete loss of mtDNA in cells lacking the TFAM homologue Abf2. Introduction of the disease-associated A3243G mutant mtDNA into human NT2 teratocarcinoma cells frequently causes mtDNA loss. Yet when human TIM17A is overexpressed in NT2 cybrids carrying A3243G mtDNA, the proportion of cybrid clones maintaining mtDNA increases significantly. TIM17A overexpression results in long-term mtDNA stabilization, since NT2 cybrids overexpressing TIM17A maintain mtDNA at levels similar to controls for several months. Tim17 is a conserved suppressor of mtDNA instability and is the first factor to be identified that can prevent mtDNA loss in a human cellular model of mitochondrial disease.

Mammalian mitochondrial nucleoids: organizing an independently minded genome.

Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids. Nucleoid proteins include not only factors involved in replication and transcription but also structural proteins required for mitochondrial DNA maintenance. Although several nucleoid proteins have been identified and characterized in yeast over the course of the past decade, little was known of mammalian mitochondrial nucleoids until recently. Two publications in the past year have expanded considerably the pool of putative mammalian mitochondrial nucleoid proteins; and analysis of one of the candidates, ATAD3p, suggests that mitochondrial nucleoid formation and division are orchestrated, not random, events.

Performance of the SAMBA I and II HIV-1 Semi-Q Tests for viral load monitoring at the point-of-care.

Although access to antiretroviral therapy for HIV infection is increasing in resource-poor countries, viral load testing for monitoring of treatment efficacy remains limited, expensive, and confined to centralized laboratories. The SAMBA HIV-1 Semi-Q Test is a nucleic acid-based amplification assay developed for viral load monitoring performed on either the semi-automated SAMBA I system for laboratory use or the fully automated SAMBA II system for point-of care use. We have assessed the performance characteristics of the SAMBA HIV-1 Semi-Q Test on SAMBA I and SAMBA II systems according to the Common Technical Specifications of the European Community's 98/79 In Vitro Diagnostic Medical Devices Directive. The sensitivity, specificity, reproducibility, and viral subtype coverage of the test were similar on the SAMBA I and SAMBA II platforms. The clinical performance on the SAMBA I system was compared with the Roche CAP/CTM assay and evaluated in-house with 130 patient specimens from London as well as in the field with 390 specimens in Kenya and Zimbabwe. The overall concordance between the SAMBA and CAP/CTM assays was 98.1%. The clinical performance of the test on the SAMBA II platform in comparison with the Abbott HIV-1 RealTime Assay was evaluated in-house with 150 specimens from Ukraine, yielding a concordance of 98.0%. The results thus show that the SAMBA HIV-1 Semi-Q Test performs equivalently on SAMBA I and SAMBA II, and they suggest that the test is suitable for implementation at the point-of-care in resource-poor regions where viral load testing is desperately needed but often unavailable.

Performance evaluation of the point-of-care SAMBA I and II HIV-1 Qual whole blood tests.

The SAMBA HIV-1 Qual Whole Blood Test is a nucleic acid-based amplification assay for the qualitative detection of HIV-1 in whole blood of adults or infants. The test can be run on either the semi-automated SAMBA I system for clinical use or the fully automated, including readout, SAMBA II system for point-of-care use in resource-limited settings. We have assessed the performance characteristics of the SAMBA HIV-1 Qual Whole Blood Test on SAMBA I and SAMBA II. The limit of detection obtained for the two tests were 518IU/ml and 399copies/ml on SAMBA I and 457IU/ml and 433copies/ml on SAMBA II. Test specificity on both systems was 100% with a panel of 503 HIV-1 negative samples. Evaluation of test reproducibility showed 100% concordance with expected gold standard results as well as 100% agreement between operators, days, and runs as well as within runs on both SAMBA I and SAMBA II. Our results thus show that the SAMBA HIV-1 Qual Whole Blood Test performs equivalently on SAMBA I and SAMBA II, and also suggest that the test is suitable for implementation in medium-throughput clinical facilities (SAMBA I) or low-throughput point-of-care (POC) settings (SAMBA II) in resource-poor regions.

Lipid partitioning at the nuclear envelope controls membrane biogenesis.

Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage.

Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p.

Lipins are evolutionarily conserved phosphatidate phosphatases that perform key functions in phospholipid, triglyceride, and membrane biogenesis. Translocation of lipins on membranes requires their dephosphorylation by the Nem1p-Spo7p transmembrane phosphatase complex through a poorly understood mechanism. Here we identify the carboxy-terminal acidic tail of the yeast lipin Pah1p as an important regulator of this step. Deletion or mutations of the tail disrupt binding of Pah1p to the Nem1p-Spo7p complex and Pah1p membrane translocation. Overexpression of Nem1p-Spo7p drives the recruitment of Pah1p in the vicinity of lipid droplets in an acidic tail-dependent manner and induces lipid droplet biogenesis. Genetic analysis shows that the acidic tail is essential for the Nem1p-Spo7p-dependent activation of Pah1p but not for the function of Pah1p itself once it is dephosphorylated. Loss of the tail disrupts nuclear structure, INO1 gene expression, and triglyceride synthesis. Similar acidic sequences are present in the carboxy-terminal ends of all yeast lipin orthologues. We propose that acidic tail-dependent binding and dephosphorylation of Pah1p by the Nem1p-Spo7p complex is an important determinant of its function in lipid and membrane biogenesis.

The human lipodystrophy protein seipin is an ER membrane adaptor for the adipogenic PA phosphatase lipin 1.

Disruption of the gene BSCL2 causes a severe, generalised lipodystrophy, demonstrating the critical role of its protein product, seipin, in human adipose tissue development. Seipin is essential for adipocyte differentiation, whilst the study of seipin in non-adipose cells has suggested a role in lipid droplet formation. However, its precise molecular function remains poorly understood. Here we demonstrate that seipin can inducibly bind lipin 1, a phosphatidic acid (PA) phosphatase important for lipid synthesis and adipogenesis. Knockdown of seipin during early adipogenesis decreases the association of lipin 1 with membranes and increases the accumulation of its substrate PA. Conversely, PA levels are reduced in differentiating cells by overexpression of wild-type seipin but not by expression of a mutated seipin that is unable to bind lipin 1. Together our data identify lipin as the first example of a seipin-interacting protein and reveals a novel molecular function for seipin in developing adipocytes.

The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA.

Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.

Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number.

Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.