Mechanism Of Lipid Accumulation Research Articles

Prostaglandin D2 enhances lipid accumulation through suppression of lipolysis via DP2 (CRTH2) receptors in adipocytes

Prostaglandin (PG) D2 enhanced lipid accumulation in adipocytes. However, its molecular mechanism remains unclear. In this study, we investigated the regulatory mechanisms of PGD2-elevated lipid accumulation in mouse adipocytic 3T3-L1 cells. The Gi-coupled DP2 (CRTH2) receptors (DP2R), one of the two-types of PGD2 receptors were dominantly expressed in adipocytes. A DP2R antagonist, CAY10595, but not DP1 receptor antagonist, BWA868C cleared the PGD2-elevated intracellular triglyceride level. While, a DP2R agonist, 15R-15-methyl PGD2 (15R) increased the mRNA levels of the adipogenic and lipogenic genes, and decreased the glycerol release level. In addition, the forskolin-mediated increase of cAMP-dependent protein kinase A (PKA) activity and phosphorylation of hormone-sensitive lipase (HSL) was repressed by the co-treatment with 15R. Moreover, the lipolysis was enhanced in the adipocyte-differentiated DP2R gene-knockout mouse embryonic fibroblasts. These results indicate that PGD2 suppressed the lipolysis by repression of the cAMP-PKA-HSL axis through DP2R in adipocytes.

Evolutionary engineering of salt-resistant Chlamydomonas sp. strains reveals salinity stress-activated starch-to-lipid biosynthesis switching

The aim of this study was to improve biomass production of the green microalga Chlamydomonas sp. JSC4 under high salinity conditions. For this purpose, heavy ion beam-coupled mutagenesis and evolutionary engineering were performed using JSC4 as the parent strain. After long-term and continuous cultivation with high salinity, salt-resistant strains that grow well even in the presence of 7% sea salt were successfully obtained. Transcriptional analysis revealed inactivation of starch-to-lipid biosynthesis switching, which resulted in delayed starch degradation and decreased lipid content in the salt-resistant strains. Cellular aggregation and hypertrophy during high salinity were relieved in these strains, indicating strong resistance to salt stress. These results suggest that high salinity stress, not the salinity condition itself, is important for activating lipid accumulation mechanisms in microalgae.

RNA-Seq and metabolic flux analysis of Tetraselmis sp. M8 during nitrogen starvation reveals a two-stage lipid accumulation mechanism

To map out key lipid-related pathways that lead to rapid triacylglyceride accumulation in oleaginous microalgae, RNA-Seq was performed with Tetraselmis sp. M8 at 24h after exhaustion of exogenous nitrogen to reveal molecular changes during early stationary phase. Further gene expression profiling by quantitative real-time PCR at 16–72h revealed a distinct shift in expression of the fatty acid/triacylglyceride biosynthesis and β-oxidation pathways, when cells transitioned from log-phase into early-stationary and stationary phase. Metabolic reconstruction modeling combined with real-time PCR and RNA-Seq gene expression data indicates that the increased lipid accumulation is a result of a decrease in lipid catabolism during the early-stationary phase combined with increased metabolic fluxes in lipid biosynthesis during the stationary phase. During these two stages, Tetraselmis shifts from reduced lipid consumption to active lipid production. This process appears to be independent from DGAT expression, a key gene for lipid accumulation in microalgae.

Fatty Acid Binding Protein 3 And Transzonal Projections Are Involved In Lipid Accumulation During In Vitro Maturation Of Bovine Oocytes

Oocytes that undergo in vitro maturation (IVM) are metabolically abnormal and accumulate excess lipid content. However, the mechanism of lipid accumulation and the role of cumulus cells in this process are unclear. Recently, it was shown that fatty acid binding proteins (FABPs) performed intra- and extracellular fatty acid transport. We postulated that FABP3 might be responsible for fatty acid transport from cumulus cells to the oocytes via transzonal projections (TZPs) during IVM. Transcript and protein levels of FABP3 were analyzed in both in vivo- and in vitro-matured cumulus-oocyte-complexes and were increased in IVM samples. Further analysis showed increased lipid content in oocytes and cumulus cells in IVM samples compared to in vivo-derived. We therefore speculated that altered traffic of fatty acids via FABP3 during IVM was the mechanism leading to the excess of lipids accumulated within IVM oocytes. Furthermore, we demonstrated an increase in FABP3 levels and lipid content during the first 9 h of IVM, further strengthening the possibility of fatty acid transport via FABP3 and TZPs. Additionally, disruptions of TZPs during IVM decreased lipid accumulation in oocytes. Our results shed light on a possible mechanism involving FABP3 and TZPs that causes excess lipid accumulation in oocytes during IVM.

Characterization of lipid accumulation and lipidome analysis in the oleaginous yeasts Rhodosporidium azoricum and Trichosporon oleaginosus

The influence of cultural conditions on lipid production was investigated in two species, Trichosporon oleaginosus and Rhodosporidium azoricum. We showed that nitrogen limitation is not the main factor triggering the mechanism of lipid accumulation in T. oleaginosus. Moreover, a scarce availability of oxygen negatively affected lipid synthesis to a lesser extent in T. oleaginosus than in R. azoricum. This highlights how the importance of controlling fermentation parameters is strictly linked to the yeast species employed. We showed that these parameters affect the activity of important enzymes, influencing the metabolic fluxes into different pathways, in particular pentose phosphate pathway and cytoplasmic pyruvate bypass. Furthermore, T. oleaginosus exhibited wider substrate flexibility, faster growth and higher lipid accumulation in fed-batch cultivation. Microbial oils obtained from both yeasts proved a valuable feedstock, alternative to vegetable oils, for advanced diesel biofuel production.

In Vivo Inhibition of Lipid Accumulation in Caenorhabditis elegans

This is a preliminary research report on the use of Caenorhabditis elegans as a model to establish anti-obesity screening assay of the natural plant resources. Nematode C. elegans has been used as experimental animal model for understanding lipid accumulation. The objective of this research was to investigate the effect of selected plant extracts on lipid accumulation in C. elegans. Currently no report could be found regarding lipid accumulation in C.elegans treated with ethanolic leaf extracts of jabon merah (Anthocephalus macrophyllus), jati belanda (Guazuma ulmifolia), and Mindi (Melia Azedarach) plants. Lipid accumulation was determined qualitatively using lipid staining method and quantitatively by colorimetry using sulpho-phospho-vanillin reagent. Data showed that lipid accumulation was inhibited up to 72% by extract of M. azedarach, about 35% by both of A. macrophyllus and G. ulmifolia extracts, and up to 25% by orlistat (a synthetic slimming drug). Ethanolic extract of A. macrophyllus, G. ulmifolia, and M. azedarach leaves were shown to inhibit lipid accumulation in C. elegans and M. azedarach leaves extracts was the most effective inhibitor. C.elegans were shown to be an effective model for in vivo lipid accumulation mechanism and potential to be used as a rapid screening assay for bioactive compounds with lipid accumulation inhibitory activity.

The boosted lipid accumulation in microalga Chlorella vulgaris by a heterotrophy and nutrition-limitation transition cultivation regime.

A model of heterotrophy and nutrition-limitation transition cultivation for efficient algal biomass and lipid production was proposed in this study, wherein sufficient robust heterotrophic-seed cells submitted into nitrogen-starvation induction for boosted lipid accumulation. The results demonstrated that heterotrophic-seed (HS) achieved specific growth rate of 1.35day-1 and biomass productivity of 1.93mg/L/d, representing 6.42- and 32.16-fold, 2.01- and 2.75-fold more than that of photoautotrophic-seed (PS) and mixtrophic-seed (MS). Even though subsequent nutrition-limitation cultivation repressed the growth of HS, the overall lipid productivity caused by nitrogen-starvation was not offset by biomass loss. The most favorable lipid productivity (465.61mg/L/d) of HS was 3.25 and 52.31 times higher than that of MS and PS. The high content of monounsaturated fatty acids (50.13%) over saturated and polyunsaturated fatty acids (totally 47.39%) in HS cells could provide superior oxidation stability and lower viscosity for biofuels generated from algal biomass feedstock. These findings suggested the feasibility of using heterotrophy and nutrition-limitation transition cultivation for enhancing the overall lipid productivity. Further, several critical enzymes (i.e. G3PDH, ME, and ACAD) were highly related to lipid accumulation and showed especially pronounced up-regulation or down-regulation expression in HS, which provide indications for shedding light on the molecular mechanisms of lipid accumulation and a prospective metabolic engineering for lipid production.

Maternal investment in fish oocytes and eggs: The molecular cargo and its contributions to fertility and early development

The production of fertile eggs with the capacity to develop into larvae and subsequently into marketable fish is centrally important to the aquaculture industry. This entails not only the programmed production of large numbers of eggs, but also high quality eggs with the potential to support normal development and high survival of offspring to juvenile and later stages of development and growth. Numerous studies highlight the maternal contributions to the development of embryos, including transcripts that regulate cell division and determine oocyte polarity, pattern development during early and late embryonic stages and the transition from maternal to zygotic gene expression and translation. Since most fish embryos develop independently within an enclosed egg envelope, they rely on compounds deposited within the oocytes during their various stages of development. In addition to regulatory nucleic acids (maternal DNA and RNA), these include proteins and other compounds that contribute to the structure and function of the egg envelope and the bulk molecular cargo that will be used as a source of cellular energy and structural components for formation of embryos and larvae. These latter components notably include yolk lipids and proteins deposited during oocyte growth and water acquired at the same time and during cytoplasmic maturation. In this review we highlight recent advances made in revealing the transcripts deposited within the oocyte that contribute to the structural and morphological development of the embryo, and to the regulation of gene expression and translation during oocyte development. Significant advances have been made in revealing the molecular mechanisms of lipid accumulation and metabolism within the oocyte, the intricacies of yolk protein formation via endocytosis of multiple yolk precursor proteins by multiple oocyte receptors, and the complex machinery supporting massive accumulation of water by maturing oocytes of many species. Additionally, many advances have been made in our understanding of the endocrine regulation of all of these processes during oogenesis. We provide here an overview of recent advances in our knowledge on these various aspects of oogenesis and identify several gaps in our knowledge for future studies.

Experimental Models of Foamy Macrophages and Approaches for Dissecting the Mechanisms of Lipid Accumulation and Consumption during Dormancy and Reactivation of Tuberculosis.

Despite a slight decline since 2014, tuberculosis (TB) remains the major deadly infectious disease worldwide with about 1.5 million deaths each year and with about one-third of the population being latently infected with Mycobacterium tuberculosis, the etiologic agent of TB. During primo-infection, the recruitment of immune cells leads to the formation of highly organized granulomas. Among the different cells, one outstanding subpopulation is the foamy macrophage (FM), characterized by the abundance of triacylglycerol-rich lipid bodies (LB). M. tuberculosis can reside in FM, where it acquires, from host LB, the neutral lipids which are subsequently processed and stored by the bacilli in the form of intracytosolic lipid inclusions (ILI). Although host LB can be viewed as a reservoir of nutrients for the pathogen during latency, the molecular mechanisms whereby intraphagosomal mycobacteria interact with LB and assimilate the LB-derived lipids are only beginning to be understood. Past studies have emphasized that these physiological processes are critical to the M. tuberculosis infectious-life cycle, for propagation of the infection, establishment of the dormancy state and reactivation of the disease. In recent years, several animal and cellular models have been developed with the aim of dissecting these complex processes and of determining the nature and contribution of their key players. Herein, we review some of the in vitro and in vivo models which allowed to gain significant insight into lipid accumulation and consumption in M. tuberculosis, two important events that are directly linked to pathogenicity, granuloma formation/maintenance and survival of the tubercle bacillus under non-replicative conditions. We also discuss the advantages and limitations of each model, hoping that this will serve as a guide for future investigations dedicated to persistence and innovative therapeutic approaches against TB.

Mechanisms of Lipid Accumulation in the Bone Morphogenetic Protein Receptor Type 2 Mutant Right Ventricle.

In heritable pulmonary arterial hypertension with germline mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene, right ventricle (RV) dysfunction is associated with RV lipotoxicity; however, the underlying mechanism for lipid accumulation is not known. We hypothesized that lipid accumulation in cardiomyocytes with BMPR2 mutation occurs owing to alterations in lipid transport and impaired fatty acid oxidation (FAO), which is exacerbated by a high-lipid (Western) diet (WD). We used a transgenic mouse model of pulmonary arterial hypertension with mutant BMPR2 and generated a cardiomyocyte cell line with BMPR2 mutation. Electron microscopy and metabolomic analysis were performed on mouse RVs. By metabolomics analysis, we found an increase in long-chain fatty acids in BMPR2 mutant mouse RVs compared with controls, which correlated with cardiac index. BMPR2-mutant cardiomyocytes had increased lipid compared with controls. Direct measurement of FAO in the WD-fed BMPR2-mutant RV showed impaired palmitate-linked oxygen consumption, and metabolomics analysis showed reduced indices of FAO. Using both mutant BMPR2 mouse RVs and cardiomyocytes, we found an increase in the uptake of (14)C-palmitate and fatty acid transporter CD36 that was further exacerbated by WD. Taken together, our data suggest that impaired FAO and increased expression of the lipid transporter CD36 are key mechanisms underlying lipid deposition in the BMPR2-mutant RV, which are exacerbated in the presence of dietary lipids. These findings suggest important features leading to RV lipotoxicity in pulmonary arterial hypertension and may point to novel areas of therapeutic intervention.

Different mechanism of lipid accumulation in embryoblast and trophoblast cells trigger by lipid excess

Different mechanism of lipid accumulation in embryoblast and trophoblast cells trigger by lipid excess

Effect of diurnal temperature difference on lipid accumulation and development in Calanus sinicus (Copepoda: Calanoida)

Calanus sinicus, the dominant copepod in the Yellow Sea, develops a large oil sac in late spring to prepare for over-summering in the Yellow Sea Cold Water Mass (YSCWM). The lipid accumulation mechanism for the initiation of over-summering is unknown. Here, we cultured C3 copepodites at four constant temperatures (10, 13, 16, and 19°C) and at three temperature regimes that mimicked the temperature variations experienced during diurnal vertical migration (10–13°C, 10–16°C, and 10–19°C) for 18 days to explore the effects of temperature differences on copepod development and lipid accumulation. C. sinicus stored more lipid at low than at high temperatures. A diurnal temperature difference (10–16°C and 10–19°C) promoted greater lipid accumulation (1.9–2.1 times) than a constant temperature of either 16°C or 19°C, by reducing the energy cost at colder temperatures and lengthening copepodite development. Thereafter, the lipid reserve supported gonad development after final molting. Only one male developed in these experiments. This highly female-skewed sex ratio may have been the result of the monotonous microalgae diet fed to the copepodites. This study provides the first evidence that diurnal temperature differences may promote lipid accumulation in C. sinicus, and provides a foundation for future investigations into the mechanisms involved in over-summering in the YSCWM.

SF-1 deficiency causes lipid accumulation in Leydig cells via suppression of STAR and CYP11A1.

Genetic mutations of steroidogenic factor 1 (also known as Ad4BP or Nr5a1) have increasingly been reported in patients with 46,XY disorders of sex development (46,XY disorders of sex development). However, because the phenotype of 46,XY disorders of sex development with a steroidogenic factor 1 mutation is wide-ranging, its precise diagnosis remains a clinical problem. We previously reported the frequent occurrence of lipid accumulation in Leydig cells among patients with 46,XY disorders of sex development with a steroidogenic factor 1 mutation, an observation also reported by other authors. To address the mechanism of lipid accumulation in this disease, we examined the effects of steroidogenic factor 1 deficiency on downstream targets of steroidogenic factor 1 in in vitro and in vivo. We found that lipid accumulation in Leydig cells was enhanced after puberty in heterozygous steroidogenic factor 1 knockout mice compared with wild-type mice, and was accompanied by a significant decrease in steroidogenic acute regulatory protein and CYP11A1 expression. In mouse Leydig cell lines, steroidogenic factor 1 knockdown induced a remarkable accumulation of neutral lipids and cholesterol with reduced androgen levels. Steroidogenic factor 1 knockdown reduced the expression of steroidogenic acute regulatory protein and CYP11A1, both of which are transcriptional targets of steroidogenic factor 1 and key molecules for steroidogenesis from cholesterol in the mitochondria. Knockdown of either steroidogenic acute regulatory protein or CYP11A1 also induced lipid accumulation, and knockdown of both had an additive effect. Our data suggested that lipid accumulation in the Leydig cells of the 46,XY disorders of sex development phenotype with a steroidogenic factor 1 mutation is due, at least in part, to the suppression of steroidogenic acute regulatory protein and CYP11A1, and a resulting increase in unmetabolized cholesterol.

Modulation of gluconeogenesis and lipid production in an engineered oleaginous Saccharomyces cerevisiae transformant.

We previously created an oleaginous Saccharomyces cerevisiae transformant as a dga1 mutant overexpressing Dga1p lacking 29 amino acids at the N-terminal (Dga1∆Np). Because we have already shown that dga1 disruption decreases the expression of ESA1, which encodes histone acetyltransferase, the present study was aimed at exploring how Esa1p was involved in lipid accumulation. We based our work on the previous observation that Esa1p acetylates and activates phosphoenolpyruvate carboxykinase (PEPCK) encoded by PCK1, a rate-limiting enzyme in gluconeogenesis, and subsequently evaluated the activation of Pck1p by yeast growth with non-fermentable carbon sources, thus dependent on gluconeogenesis. This assay revealed that the ∆dga1 mutant overexpressing Dga1∆Np had much lower growth in a glycerol-lactate (GL) medium than the wild-type strain overexpressing Dga1∆Np. Moreover, overexpression of Esa1p or Pck1p in mutants improved the growth, indicating that the ∆dga1 mutant overexpressing Dga1∆Np had lower activities of Pck1p and gluconeogenesis due to lower expression of ESA1. In vitro PEPCK assay showed the same trend in the culture of the ∆dga1 mutant overexpressing Dga1∆Np with 10% glucose medium, indicating that Pck1p-mediated gluconeogenesis decreased in this oleaginous transformant under the lipid-accumulating conditions introduced by the glucose medium. The growth of the ∆dga1 mutant overexpressing Dga1∆Np in the GL medium was also improved by overexpression of acetyl-CoA synthetase, Acs1p or Acs2p, indicating that supply of acetyl-CoA was crucial for Pck1p acetylation by Esa1p. In addition, the ∆dga1 mutant without Dga1∆Np also showed better growth in the GL medium, indicating that decreased lipid accumulation was enhancing Pck1p-mediated gluconeogenesis. Finally, we found that overexpression of Ole1p, a fatty acid ∆9-desaturase, in the ∆dga1 mutant overexpressing Dga1∆Np improved its growth in the GL medium. Although the exact mechanisms leading to the effects of Ole1p were not clearly defined, changes of palmitoleic and oleic acid contents appeared to be critical. This observation was supported by experiments using exogenous palmitoleic and oleic acids or overexpression of elongases. Our findings provide new insights on lipid accumulation mechanisms and metabolic engineering approaches for lipid production.

Life Long High Fat Diet Causes Hepatic Lipid Accumulation by Programming Lipid Synthesis Genes in Liver of Male Rat

Life long high fat (HF) diet has been shown to cause more hepatic lipid accumulation in offspring as compared to a post‐weaning HF diet. However, it is still unclear what the epigenetic mechanisms are. We hypothesize that a life long HF diet will cause greater hepatic lipid accumulation as compared to a post‐weaning HF diet. The objective of this study is to understand the epigenetic mechanisms of hepatic lipid accumulation as an outcome of life long HF diet. Timed‐pregnant Sprague‐Dawley rats were given either a control (C, 16% fat) or HF (HF, 45% fat) diet during gestation and lactation. Pups from both C and HF mothers were weaned onto the same HF diet, creating two offspring groups: C/HF and HF/HF. Rats were sacrificed at 12 weeks of age and the left lobe of the liver was used for further analysis. Liver histology showed greater hepatic fat accumulation in the HF/HF group. Gene expression data was measured for multiple genes in the following pathways: glycolysis, gluconeogenesis, fatty acid synthesis, and triacylglycerol synthesis. Only glycerol‐3‐phosphate acyltransferase (GPAM) and fatty acid synthase (FASN) showed higher levels of mRNA. DNA methylation of specific genes was analyzed based on data obtained from methyl‐DNA immunoprecipitation with high‐throughput sequencing. Differential methylation was observed for both GPAM and FASN. Our data suggests that life long HF diet increases lipid storage by increased hepatic GPAM and FASN gene expression in male rats, potentially through epigenetic regulation.

A rapid and accurate quantification method for real-time dynamic analysis of cellular lipids during microalgal fermentation processes in Chlorella protothecoides with low field nuclear magnetic resonance

The rapid and real-time lipid determination can provide valuable information on process regulation and optimization in the algal lipid mass production. In this study, a rapid, accurate and precise quantification method of in vivo cellular lipids of Chlorella protothecoides using low field nuclear magnetic resonance (LF-NMR) was newly developed. LF-NMR was extremely sensitive to the algal lipids with the limits of the detection (LOD) of 0.0026g and 0.32g/L in dry lipid samples and algal broth, respectively, as well as limits of quantification (LOQ) of 0.0093g and 1.18g/L. Moreover, the LF-NMR signal was specifically proportional to the cellular lipids of C. protothecoides, thus the superior regression curves existing in a wide detection range from 0.02 to 0.42g for dry lipids and from 1.12 to 8.97gL−1 of lipid concentration for in vivo lipid quantification were obtained with all R2 higher than 0.99, irrespective of the lipid content and fatty acids profile variations. The accuracy of this novel method was further verified to be reliable by comparing lipid quantification results to those obtained by GC–MS. And the relative standard deviation (RSD) of LF-NMR results were smaller than 2%, suggesting the precision of this method. Finally, this method was successfully used in the on-line lipid monitoring during the algal lipid fermentation processes, making it possible for better understanding of the lipid accumulation mechanism and dynamic bioprocess control.

Proteomics analysis of high lipid-producing strain Mucor circinelloides WJ11: an explanation for the mechanism of lipid accumulation at the proteomic level.

BackgroundThe oleaginous fungus, Mucor circinelloides, is attracting considerable interest as it produces oil rich in γ-linolenic acid. Nitrogen (N) deficiency is a common strategy to trigger the lipid accumulation in oleaginous microorganisms. Although a simple pathway from N depletion in the medium to lipid accumulation has been elucidated at the enzymatic level, global changes at protein levels upon N depletion have not been investigated. In this study, we have systematically analyzed the changes at the levels of protein expression in M. circinelloides WJ11, a high lipid-producing strain (36 %, lipid/cell dry weight), during lipid accumulation.ResultsProteomic analysis demonstrated that N depletion increased the expression of glutamine synthetase, involved in ammonia assimilation, for the supply of cellular nitrogen but decreased the metabolism of amino acids. Upon N deficiency, many proteins (e.g., fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase) involved in glycolytic pathway were up-regulated while proteins involved in the tricarboxylic acid cycle (e.g., isocitrate dehydrogenase, succinyl-CoA ligase, succinate dehydrogenase, fumarate hydratase) were down-regulated, indicating this activity was retarded thereby leading to a greater flux of carbon into fatty acid biosynthesis. Moreover, glucose-6-phosphate dehydrogenase, transaldolase and transketolase, which participate in the pentose phosphate pathway, were up-regulated, leading to the increased production of NADPH, the reducing power for fatty acid biosynthesis. Furthermore, protein and nucleic acid metabolism were down-regulated and some proteins involved in energy metabolism, signal transduction, molecular chaperone and redox homeostasis were up-regulated upon N depletion, which may be the cellular response to the stress produced by the onset of N deficiency.ConclusionN limitation increased those expressions of the proteins involved in ammonia assimilation but decreased that involved in the biosynthesis of amino acids. Upon N deprivation, the glycolytic pathway was up-regulated, while the activity of the tricarboxylic acid cycle was retarded, thus, leading more carbon flux to fatty acid biosynthesis. Moreover, the pentose phosphate pathway was up-regulated, then this would increase the production of NADPH. Together, coordinated regulation of central carbon metabolism upon N limitation, provides more carbon flux to acetyl-CoA and NADPH for fatty acid biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0428-4) contains supplementary material, which is available to authorized users.

Palmitate-induced Regulation of PPARγ via PGC1α: a Mechanism for Lipid Accumulation in the Liver in Nonalcoholic Fatty Liver Disease.

The aim was to examine the effect of free fatty acids on the regulation of PPARγ-PGC1α pathway, and the effect of PPARγ/PGC1α in NAFLD. The mRNA and protein expression of PGC1α and phospho/total PPARγ were examined in Huh7 cells after the palmitate/oleate treatment with/without the transfection with siRNA against PGC1a. The palmitate content, mRNA and protein expression of PGC1α and PPARγ in the liver were examined in the control and NAFLD mice. Palmitate (500 μM), but not oleate, increased protein expression of PGC1α and phospho PPARγ (PGC1α, 1.42-fold, P=0.038; phospho PPARγ, 1.56-fold, P=0.022). The palmitate-induced PPARγ mRNA expression was reduced after the transfection (0.46‑fold), and the protein expressions of PGC1α (0.52-fold, P=0.019) and phospho PPARγ (0.43-fold, P=0.011) were suppressed in siRNA-transfected cells. The palmitate (12325.8 ± 1758.9 μg/g vs. 6245.6 ± 1182.7 μg/g, p=0.002), and mRNA expression of PGC1α (11.0 vs. 5.5, p=0.03) and PPARγ (4.3 vs. 2.2, p=0.0001) in the liver were higher in high-triglyceride liver mice (>15.2 mg/g) than in low-triglyceride liver mice (<15.2 mg/g). The protein expressions of both PGC1α and PPARγ were higher in the NAFLD group than in the controls (PGC1α, 1.41-fold, P=0.035; PPARγ, 1.39-fold, P=0.042), and were higher in the high-triglyceride liver group (PGC1α, 1.52-fold, p=0.03; PPARγ, 1.22-fold, p=0.05) than in the low-triglyceride liver group. In conclusion, palmitate appear to up-regulate PPARγ via PGC1α in Huh7 cells, and both PGC1α and PPARγ are up-regulated in the NAFLD mice liver, suggesting an effect on lipid metabolism leading to intrahepatic triglyceride accumulation.

Bioinformatical analysis and preliminary study of the role of lipase in lipid metabolism in Mucor circinelloides

The filamentous fungusMucor circinelloideshas been widely used as a model organism to investigate the mechanisms of lipid accumulation.

LipiD-QuanT: a novel method to quantify lipid accumulation in live cells

Lipid droplets (LDs) are the main storage organelles for triglycerides. Elucidation of lipid accumulation mechanisms and metabolism are essential to understand obesity and associated diseases. Adipogenesis has been well studied in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell lines. However, most techniques for measuring LD accumulation are either not quantitative or can be destructive to samples. Here, we describe a novel, label-free LD quantification technique (LipiD-QuanT) to monitor lipid dynamics based on automated image analysis of phase contrast microscopy images acquired during in vitro human adipogenesis. We have applied LipiD-QuanT to measure LD accumulation during differentiation of SGBS cells. We demonstrate that LipiD-QuanT is a robust, nondestructive, time- and cost-effective method compared with other triglyceride accumulation assays based on enzymatic digest or lipophilic staining. Further, we applied LipiD-QuanT to measure the effect of four potential pro- or antiobesogenic substances: DHA, rosiglitazone, elevated levels of D-glucose, and zinc oxide nanoparticles. Our results revealed that 2 µmol/l rosiglitazone treatment during adipogenesis reduced lipid production and caused a negative shift in LD diameter size distribution, but the other treatments showed no effect under the conditions used here.