Lipid Accumulation Response Research Articles

Regulation of Myocardial Lipid Accumulation in the Development of Obesity

Energy metabolism is important in the development of heart disease, which is the leading cause of death worldwide. The importance of energy metabolism in disease is becoming even more clear with the rise in obesity, metabolic diseases, and the recognition that they all fit together as with the recent offcial report of a new syndrome named Cardiovascular-Kidney-Metabolic (CKM) Syndrome. A complete understanding of myocardial lipid storage and subsequent utilization of lipids is essential to understanding the impact of these metabolic diseases in the heart and on the development of cardiac dysfunction and hypertrophy. In this study we investigate how the heart controls fuel storage by taking advantage of the impact of the transcription factor peroxisome proliferator activated receptor α (PPARα) on 1) fatty acid metabolism and lipid accumulation, 2) its key role in sex differences in cardiac hypertrophy which we previously reported, and 3) the regulation of lipid droplets. It is controversial whether a decline in myocardial PPARα mediates cardiac hypertrophy and whether it increases or reduces lipid accumulation. Using our tamoxifen inducible cardiac-specific PPARα knockout mouse (cPPAR−/−) we see less cardiac lipid accumulation in response to a 5 week high fat diet. Our data indicate that this difference in triglyceride levels is present in both males and females. Importantly, this is not driven by a difference in fatty acid supply to these hearts. Serum fatty acid levels are not lower in cPPAR−/− vs Control mice fed a high fat diet. Further, we have not observed a difference in high fat diet induced body weight gain between cPPAR−/− and Control mice. We have begun to examine how these differences in high fat diet-induced lipid accumulation correlate with cardiac hypertrophy by looking at heart weight normalized to tibia length. While we did not expect 5 weeks of a high fat diet to induce cardiac hypertrophy, it is possible that the difference in cardiac lipid storage in the cPPAR−/− hearts could impact this. This measurement did not indicate a difference in cardiac hypertrophy between high fat diet fed cPPAR−/− and Control mice. We are utilizing this mouse as a model to investigate the regulation of lipid droplets and the lipid droplet proteome. So far, our results are indicating that the regulation of lipid droplet proteins is not affected in a uniform manner. For example, our data is suggesting that the expression of different members of the perilipin family are affected in distinct ways. Overall, these data indicate that a reduction in cardiac PPARα protects against obesity induced cardiac lipid accumulation. Going forward, it will be interesting to see how these changes occur in relation to the lipid droplet proteome and whether any changes that occur differentially in males vs female may be involved in sex differences in cardiac hypertrophy. Better understanding these factors will be essential for the development of safe and effective future therapies for CKM syndrome and other cardiovascular diseases. ND-EPSCoR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Toll-like receptor stimulants in processed meats promote lipid accumulation in macrophages and atherosclerosis in Apoe−/− mice

Dietary intake of processed meat is a risk factor for cardiovascular disease. However, the effects of processed meats on lipid metabolism in macrophages, a key regulator of cardiovascular risk, have remained largely unexplored.Extracts of processed meats, but not their fresh non-processed equivalents, were found to promote a significant increase in macrophage lipid accumulation in vitro. Calibrated receptor-dependent reporter assays revealed that pro-inflammatory stimulants of Toll-like receptor (TLR)-2 and TLR4 were low or undetectable in fresh meats, but rose dramatically following chopping and storage at 4 °C. Lipid accumulation in response to processed meats correlated well with TLR-stimulant content, was significantly reduced in TLR4-deficient macrophages, and was absent in response to meats stored frozen to prevent bacterial growth. TLR-stimulation significantly increased the incorporation of 14C-acetate into cellular lipids, and induced lipid accumulation in the absence of exogenous lipoproteins, suggesting a key role for de novo lipid synthesis in this process. Aortic atherosclerosis was also significantly accelerated in Apoe−/− mice receiving a diet supplemented with TLR-stimulants at concentrations relevant to those measured in processed meats, compared to normal chow.The findings reveal novel mechanisms which may be of relevance to the observed connections between processed meat consumption, inflammatory markers and cardiovascular risk.

Expression Profiling of Adipogenic and Anti-Adipogenic MicroRNA Sequences following Methylmercury Exposure in Caenorhabditis elegans.

MicroRNA (miRNA) are important regulators of gene expression that respond not only to developmental and pathological cues, but also to environmental stimuli. Dyslipidemia is a hallmark of metabolic conditions and has been shown to significantly affect the expression of circulating miRNA sequences. Recently, our lab has shown that the environmental toxicant methylmercury (MeHg) causes dyslipidemia in the Caenorhabditis elegans model organism. While 10 and 20 μM MeHg increases the expression of adipogenic transcription factors and lipid-binding proteins in worms, there is limited information on how the toxicant affects the miRNA regulators of these genes. We hypothesized that MeHg would increase the expression of adipogenic miRNA sequences and/or decrease the expression of anti-adipogenic miRNA sequences. We further hypothesized that the target mRNA sequences for the miRNAs affected by MeHg would be consequently altered. We selected three potentially adipogenic (mir-34, mir-124, and mir-355) and three potentially anti-adipogenic (mir-240, mir-786, and let-7) miRNA sequences homologous to known human miRNA sequences altered in obesity, and quantified their levels 24 h and 48 h post MeHg treatment. At 24 h post exposure, MeHg significantly increased expression of both the adipogenic and anti-adipogenic miRNA sequences 1.5-3x above untreated control. By 48 h post exposure, only the adipogenic miRNA sequences were elevated, while the anti-adipogenic miRNA sequences were decreased by 50% compared to untreated control. These data suggest that there are developmental changes in miRNA expression over time following MeHg exposure. We next selected one target mRNA sequence for each miRNA sequence based on miRNA-mRNA relationships observed in humans. MeHg altered the gene expression of all the target genes assayed. Except for mir-34, all the tested miRNA-mRNA sequences showed a conserved relationship between nematode and humans. To determine whether the selected miRNA sequences were involved in lipid accumulation in response to MeHg, lipid storage was investigated in transgenic worm strains that lacked the specific miRNA strains. Of the six strains investigated, only the mir-124 and let-7 mutant worms had lipid storage levels that were statistically different from wild type, suggesting that these two sequences can be potential mediators of MeHg-induced lipid dysregulation.

A Comparison of Primary Human Hepatocytes and Hepatoma Cell Lines to Model the Effects of Fatty Acids, Fructose and Glucose on Liver Cell Lipid Accumulation.

Non-alcoholic fatty liver disease (NAFLD) begins with lipid accumulation within hepatocytes, but the relative contributions of different macronutrients is still unclear. We investigated the impact of fatty acids, glucose and fructose on lipid accumulation in primary human hepatocytes (PHH) and three different cell lines: HepG2 (human hepatoblastoma-derived cell line), Huh7 (human hepatocellular carcinoma cell line) and McA-RH7777 (McA, rat hepatocellular carcinoma cell line). Cells were treated for 48 h with fatty acids (0 or 200 μM), glucose (5 mM or 11 mM) and fructose (0 mM, 2 mM or 8 mM). Lipid accumulation was measured via Nile Red staining. All cell types accumulated lipid in response to fatty acids (p < 0.001). PHH and McA, but not HepG2 or Huh7 cells, accumulated more lipid with 11 mM glucose plus fatty acids (p = 0.004, fatty acid × glucose interaction, for both), but only PHH increased lipid accumulation in response to fructose (p < 0.001). Considerable variation was observed between PHH cells from different individuals. Lipid accumulation in PHH was increased by insulin (p = 0.003) with inter-individual variability. Similarly, insulin increased lipid accumulation in both HepG2 and McA cells, with a bigger response in McA in the presence of fatty acids (p < 0.001 for fatty acid × insulin). McA were more insulin sensitive than either HepG2 or Huh7 cells in terms of AKT phosphorylation (p < 0.001 insulin × cell type interaction). Hence, glucose and fructose can contribute to the accumulation of lipid in PHH with considerable inter-individual variation, but hepatoma cell lines are not good models of PHH.

Intratracheal Administration of Acyl Coenzyme A Acyltransferase-1 Inhibitor K-604 Reduces Pulmonary Inflammation Following Bleomycin-Induced Lung Injury.

Acute lung injury (ALI) is characterized by epithelial damage, barrier dysfunction, and pulmonary edema. Macrophage activation and failure to resolve play a role in ALI; thus, macrophage phenotype modulation is a rational target for therapeutic intervention. Large, lipid-laden macrophages have been observed in various injury models, including intratracheal bleomycin (ITB), suggesting that lipid storage may play a role in ALI severity. The endoplasmic reticulum-associated enzyme acyl coenzyme A acyltransferase-1 (Acat-1/Soat1) is highly expressed in macrophages, where it catalyzes the esterification of cholesterol, leading to intracellular lipid accumulation. We hypothesize that inhibition of Acat-1 will reduce macrophage activation and improve outcomes of lung injury in ITB. K-604, a selective inhibitor of Acat-1, was used to reduce cholesterol esterification and hence lipid accumulation in response to ITB. Male and female C57BL6/J mice (n = 16-21/group) were administered control, control + K-604, ITB, or ITB + K-604 on d0, control or K-604 on d3, and were sacrificed on day 7. ITB caused significant body weight loss and an increase in cholesterol accumulation in bronchoalveolar lavage cells. These changes were mitigated by Acat-1 inhibition. K-604 also significantly reduced ITB-induced alveolar thickening. Surfactant composition was normalized as indicated by a significant decrease in phospholipid: SP-B ratio in ITB+K-604 compared with ITB. K-604 administration preserved mature alveolar macrophages, decreased activation in response to ITB, and decreased the percentage mature and pro-fibrotic interstitial macrophages. These results show that inhibition of Acat-1 in the lung is associated with reduced inflammatory response to ITB-mediated lung injury.SIGNIFICANCE STATEMENTAcyl coenzyme A acyltransferase-1 (Acat-1) is critical to lipid droplet formation, and thus inhibition of Acat-1 presents as a pharmacological target. Intratracheal administration of K-604, an Acat-1 inhibitor, reduces intracellular cholesterol ester accumulation in lung macrophages, attenuates inflammation and macrophage activation, and normalizes mediators of surface-active function after intratracheal bleomycin administration in a rodent model. The data presented within suggest that inhibition of Acat-1 in the lung improves acute lung injury outcomes.

Long non-coding RNA G23Rik attenuates fasting-induced lipid accumulation in mouse liver

Peroxisome proliferator-activated receptor α (PPARα) is a key mediator of lipid metabolism and metabolic stress in the liver. A recent study revealed that PPARα-dependent long non-coding RNAs (lncRNAs) play an important role in modulating metabolic stress and inflammation in the livers of fasted mice. Here hepatic lncRNA 3930402G23Rik (G23Rik) was found to have active peroxisome proliferator response elements (PPREs) within its promoter and is directly regulated by PPARα. Although G23Rik RNA was expressed to varying degrees in several tissues, the PPARα-dependent regulation of this lncRNA was only observed in the liver. Pharmacological activation of PPARα induced PPARα recruitment at the G23Rik promoter and a pronounced increase in hepatic G23Rik lncRNA expression. A G23Rik-null mouse line was developed to further characterize the function of this lncRNA in the liver. G23Rik-null mice were more susceptible to hepatic lipid accumulation in response to acute fasting. Histological analysis further revealed a pronounced buildup of lipid droplets and a significant increase in neutral triglycerides and lipids as indicated by enhanced oil red O staining of liver sections. Hepatic cholesterol, non-esterified fatty acid, and triglyceride levels were significantly elevated in G23Rik-null mice and associated with induction of the lipid-metabolism related gene Cd36. These findings provide evidence for a lncRNA dependent mechanism by which PPARα attenuates hepatic lipid accumulation in response to metabolic stress through lncRNA G23Rik induction.

The benzoate plant metabolite ethyl gallate prevents cellular- and vascular-lipid accumulation in experimental models of atherosclerosis

Ethyl gallate (EG) is a well-known constituent of medicinal plants, but its effects on atherosclerosis development are not clear. In the present study, the anti-atherosclerosis effects of EG and the underlying mechanisms were explored using macrophage cultures, zebrafish and apolipoprotein (apo) E deficient mice. Treatment of macrophages with EG (20 μM) enhanced cellular cholesterol efflux to HDL, and reduced net lipid accumulation in response to oxidized LDL. Secretion of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from activated macrophages was also blunted by EG. Fluorescence imaging techniques revealed EG feeding of zebrafish reduced vascular lipid accumulation and inflammatory responses in vivo. Similar results were obtained in apoE-/- mice 6.5 months of age, where plaque lesions and monocyte infiltration into the artery wall were reduced by 70% and 42%, respectively, after just 6 weeks of injections with EG (20 mg/kg). HDL-cholesterol increased 2-fold, serum cholesterol efflux capacity increased by ∼30%, and the levels of MCP-1 and IL-6 were reduced with EG treatment of mice. These results suggest EG impedes early atherosclerosis development by reducing the lipid and macrophage-content of plaque. Underlying mechanisms appeared to involve HDL cholesterol efflux mechanisms and suppression of pro-inflammatory cytokine secretion.

Carbon flow conversion induces alkali resistance and lipid accumulation under alkaline conditions based on transcriptome analysis in Chlorella sp. BLD

Alkaline environments are abundant globally and cause damage to most organisms, while some microalgae can grow well and accumulate lipids under those conditions. Here the mechanisms of alkali resistance and lipid accumulation in the alkaliphilic microalgae Chlorella sp. BLD were explored using physiological-biochemical and transcriptome analysis. When cultivated at alkaline pH, Chlorella sp. BLD exhibited good alkali-resistance ability and increased biomass (0.97 g L−1). The biochemical composition of Chlorella sp. BLD changed significantly (lipid content increased 39% and protein content decreased 19.5%) compared with pH 7.5. Through transcriptome analysis, we found that pathways related to carbon metabolism such as photosynthesis, glycolysis, and the TCA cycle were significantly regulated under alkaline conditions. Genes that encoding the key enzyme in carbon-related metabolism such as Rubisco, AMY, PK, ME, CS, ACAT, KAS, and DGAT were identified. Transcriptional regulation of these genes results in carbon flow switching from starch and protein to cell wall metabolism, organic acid synthetic and lipid accumulation in response to alkaline conditions. These results reveal the alkali resistance mechanism of Chlorella sp. BLD and provide a theoretical basis for microalgae oil production under alkaline conditions.

A Low Protein Diet during Gestation and Lactation Increases Hepatic Lipid Accumulation through Autophagy and Histone Deacetylase.

The present study examined the mechanism of a low protein (LP) diet on hepatic lipid metabolism during gestation and lactation. Timed-pregnant Sprague-Dawley rats were fed a control or an LP diet during gestation and lactation. LP dams had increased hepatic triglyceride accumulation and significantly higher aspartate/alanine transaminase ratio, accompanied by a decrease in circulating very low-density/low-density lipoprotein ratio. LC3B (Microtubule Associated Protein 1 Light Chain 3 Beta) expression was stimulated in LP dams along with increased histone acetylation. LP diet-induced co-localization of the LC3 binding motif-interacting proteins APOB or MTTP with LC3B, suggesting autophagic degradation. HDAC3 is found necessary to prevent lipid accumulation in response to amino acid deprivation in HepG2 cells. LC3B-mediated APOB protein degradation is related to increases in lipid accumulation. Conclusion: HDAC3 regulated LC3B-induced lipid accumulation potentially through autophagic degradation of APOB and MTTP in response to amino acid limitation caused by a low protein diet.

A Subfornical Organ→Paraventricular Nucleus Neuronal Network Contributes to Non‐Alcoholic Fatty Liver Disease via Hepatic Sympathetic Outflow

Non‐alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, affects 1 in 3 adults and leads to an increased risk for metabolic and cardiovascular diseases. Our recent work indicates that obesity‐induced NAFLD is mediated by elevations in hepatic sympathetic nerve activity. However, the neural pathways that contribute to hepatic sympathetic overactivity and subsequent NAFLD development remain unknown. The subfornical organ (SFO) ‐ a forebrain circumventricular region that lies outside the blood brain barrier ‐ senses, integrates and responds to circulating stimuli. Anatomical and electrophysiological data support dense, excitatory projections from the SFO to the paraventricular nucleus of the hypothalamus (SFO→PVN), an integrative nucleus with direct hepatic spinal projections. Taken together, we hypothesized that an SFO→PVN excitatory neuronal network causes hepatic steatosis via elevations in liver sympathetic outflow. An intersectional viral strategy was used in which a retrograde transported canine adenovirus was targeted to the PVN to express Cre‐recombinase in SFO→PVN neurons (CAV2‐Cre‐GFP). This was combined with SFO‐targeted delivery of a Cre‐inducible designer receptors engineered against designer drugs (DREADDs) excitatory construct (AAV2‐DIO‐hM3Gq‐mCherry). With this approach, the pharmacological ligand clozapine‐N‐oxide (CNO; 3 mg/kg i.p.) was administered once daily over 6 days to activate SFO→PVN neurons (n=4). Short‐term activation of SFO→PVN neurons resulted in hepatic steatosis (2.6±0.02 vs 2.9±0.02 density*107, saline vs CNO, p<0.05) that was paralleled by an elevation in liver tyrosine hydroxylase protein (1.8±0.4 CNO fold saline, p<0.05), the rate‐limiting enzyme for catecholamine synthesis within postganglionic nerve terminals. Based on this, we combined the above viral strategy with selective hepatic denervation (phenol application) or sham surgery (n=5–6). Oil Red O staining (Figure 1) demonstrated that hepatic denervation prevented liver lipid accumulation in response to 6‐day activation of SFO→PVN neurons (1.9±0.01 vs 1.7±0.01 density*107, CNO sham vs CNO denervation, p<0.05). Importantly, this occurred independent of changes in body weight and food intake. Hepatic denervation was confirmed by a reduction in liver tyrosine hydroxlase protein expression (0.3±0.1 vs 1.8±0.3 vs 0.4±0.1 fold saline sham, CNO sham vs saline denervation vs CNO denervation, all p<0.05). Lastly, male C57Bl/6 mice that were fed a high fat diet (10 wks) underwent intersectional viral targeting for expression of an inhibitory DREADDs construct (AAV2‐DIO‐hM4Gi‐mCherry) in SFO→PVN neurons. Remarkably, acute inhibition of SFO→PVN neurons in obese mice resulted in an approximate 45% reduction in hepatic steatosis (Figure 2, n=4/group). Collectively, these findings indicate that activation of SFO→PVN neurons causes liver triglyceride deposition via elevations in hepatic sympathetic outflow. Furthermore, in the context of obesity, inhibition of this forebrain‐hypothalamic‐autonomic circuit results in a marked reduction in NAFLD.Support or Funding InformationR01DK117007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipid accumulation responses in the liver of Rana nigromaculata induced by perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is a perfluorinated compound that is widely distributed, is persistent in the environment, and has a low-level chronic exposure effect on human health. The aim of this study was to investigate the peroxisome proliferator activated receptors γ (PPARγ) and the sterol regulatory element-binding protein 2 (SREBP2) signaling pathways in regulating the lipid damage response to PFOA in the livers of amphibians. Male and female frogs (Rana nigromaculata) were exposed to 0, 0.01, 0.1, 0.5 and 1 mg/L PFOA. After treatment, we evaluated the pathological changes in the liver by Oil Red O, staining and examined the total cholesterol (T-CHO) and triglyceride (TG) contents. The mRNA expression levels of PPARγ, Fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), Glycerol-3-phosphate acyltransferase (GPAT), SREBP2 and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The administration of PFOA caused marked lipid accumulation damage in the amphibian livers. The T-CHO contents were elevated significantly after PFOA treatment; these results show a dose-dependent manner in both sexes. The TG content showed a significant increase in male livers, while it was elevated significantly in female livers. The RT-PCR results showed that the mRNA expression levels of PPARγ, ACC, FAS, GPAT, SREBP2 and HMG-CoA were significantly dose-dependently increased in the PFOA-treated groups compared with those of the control group. Our results demonstrated that PFOA-induced lipid accumulation also affected the expression levels of genes FAS, ACC, GPAT and HMG-CoA in the PPARγ and SREBP2 signaling pathways in the liver. These finding will provide a scientific theoretical basis for the protection of Rana nigromaculata against PFOA effects.

De novo transcriptome analysis and gene expression profiling of an oleaginous microalga Scenedesmus acutus TISTR8540 during nitrogen deprivation-induced lipid accumulation

Nitrogen deprivation (−N) has been used as a technique to promote lipid accumulation in various microalgae. Scenedesmus acutus is a promising oleaginous green microalga that can be cultivated in organic wastewater for biodiesel production. Nevertheless, the molecular mechanisms controlling S. acutus lipid accumulation in response to −N remain unidentified. Physiological study determined that −N reduced cell growth and photosynthetic pigments. On the other hand, it promoted carbohydrate and neutral lipid accumulation. To find the mechanisms underlying lipid accumulation, we performed de novo transcriptome profiling of the non-model S. acutus in response to −N. The transcriptome analysis revealed that glycolysis and starch degradation were up-regulated; on the contrary, gluconeogenesis, photosynthesis, triacylglycerol (TAG) degradation and starch synthesis were down-regulated by −N. Under −N, the carbon flux was shifted toward fatty acid and TAG synthesis, and the down regulation of TAG lipase genes may contribute to TAG accumulation. A comparative analysis of the −N transcriptomes of oleaginous microalgae identified that the down-regulation of multiple lipase genes was a specific mechanism found only in the −N transcriptome of S. acutus. Our study unraveled the mechanisms controlling −N-induced lipid accumulation in S. acutus, and provided new perspectives for the genetic manipulation of biodiesel-producing microalgae.

Physiological plasticity of a Chilean strain of the diatom Phaeodactylum tricornutum: the effect of culture conditions on the quantity and quality of lipid production

Microalgal biotechnology is always in demand for new fast-growing and oil-rich microalgal strains for different industries. A fusiform diatom strain with robust growth attributes was isolated from Coliumo’s Bay in the Biobio Region, Chile, in 2005. In order to determine the taxonomic identity of the strain, partial sequences of 18S rDNA and ITS region were obtained. Sequence analysis revealed 99% identity with strains of Phaeodactylum tricornutum Bohlin emend. The effect of autotrophic (combination of irradiances, nitrate, and phosphate concentrations) and mixotrophic (addition of sodium acetate under low light) culture conditions was evaluated on growth and lipid accumulation. Hierarchical analysis clustered the autotrophic culture conditions in three main groups of common growth and lipid accumulation responses. Independently on the irradiance, the lowest nitrate and phosphate concentrations induced the highest total lipid accumulation (26–29% lipids per biomass dry weight) and the maximum saturated fatty acid ratio (ca. 42% of total fatty acids), without negative effect on growth; conversely, the highest nitrate and phosphate concentrations stimulated growth and PUFA proportion (20–25% of total fatty acids), but not total lipid accumulation (ca. 15% lipids per biomass dry weight). On the other hand, mixotrophic conditions stimulated growth but not lipid accumulation. Saturated and monounsaturated fatty acid fractions increased as acetate was added while PUFA fraction decreased. Physiological plasticity demonstrated by the Chilean strain of P. tricornutum studied, when faced with different growing conditions, makes it a potential candidate for commercial oil production for different markets, such as biodiesel or feed/food.

Protein Tyrosine Phosphatase 1B (PTP1B) Deficiency in Proopiomelanocortin (POMC) Neurons Attenuates Body Weight, Fat Mass and Liver Lipid Accumulation in Mice Fed a High Fat Diet

Leptin acts on the central nervous system (CNS) to reduce appetite and body weight, improve glucose regulation, and increase energy expenditure and sympathetic nervous system (SNS) activity. Central leptin has also been shown to exert antisteatotic effects on non‐adipose tissues. However, in severe obesity there may be resistance to leptin's anorexic, antidiabetic and antisteatotic effects but sustained effects to increase SNS activity, blood pressure (BP), and heart rate (HR). Our previous studies showed that POMC neurons are important for mediating the BP, HR and glucose effects of leptin but the role of POMC neurons in mediating leptin's effects on liver lipid accumulation remain less well defined. In this study we examined if deleting PTP1B, a negative regulator of leptin signaling, specifically in POMC neurons or in the entire forebrain, would protect mice against the adverse metabolic effects of a high fat diet (HFD). Male and female mice with forebrain (PTP1Bflox/flox/CamK2R5‐Cre) or POMC specific (PTP1Bflox/flox/POMC‐Cre) PTP1B deficiency and littermate controls (PTP1Bfox/flox) were fed a HFD from 6–20 weeks of age. Compared to PTP1Bflox/flox mice, mice with PTP1B deficiency in POMC neurons or in the entire forebrain had reduced body weight gain between 6 and 20 weeks of age when fed a HFD (Δ19.1 ±1.7g vs. Δ13.7±1.1g and Δ15.3±1.4g, respectively), and attenuated increase in fat mass (Δ13.3±1.2g vs. Δ7.3±0.7g and Δ10.2±1.0g, respectively). PTP1B deficiency attenuated food intake in forebrain specific PTP1B deficient mice compared to PTP1Bflox/flox and POMC specific PTP1B deficient mice (2.0±0.1g, 2.7±0.1g and 2.5±0.1 g, respectively). Glucose tolerance, expressed as area under the curve (AUC), was improved by PTP1B deficiency in forebrain and POMC specific neurons compared to PTP1Bflox/flox mice fed a HFD (AUC 19016±1172, 23207±1534 and 27561±3088, respectively). Liver fat mass, assessed by EchoMRI at 29±0.5 weeks of age, was reduced and liver lean mass was increased in mice with PTP1B deficiency in POMC neurons or in the entire forebrain (Fat: 8.8±1.8 and 6.0±3.3 mg/g, Lean: 93.6±2.8 and 97.2±5.6 mg/g, respectively) compared to PTP1Bflox/flox mice (Fat: 14.2±2., Lean: 79.4±10.3 mg/g), indicating a healthier liver profile. These findings indicate that PTP1B signaling in POMC neurons plays an important role in regulating body weight, fat mass, glucose tolerance and liver lipid accumulation in response to a chronic HFD but effects of PTP1B on food intake are mediated mainly in forebrain neurons other than POMC.Support or Funding Informationsupported by NHLBI‐PO1HL51971 and NIGMS P20GM104357.

BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana

Brominated flame retardant, 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47), has received grave concerns as a persistent organic pollutant, which is toxic to marine organisms, and a suspected link to endocrine abnormalities. Despite the wide distribution in the marine ecosystem, very little is known about the toxic impairments on marine organisms, particularly on invertebrates. Thus, we examined the adverse effects of BDE-47 on life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in response to BDE-47-induced stress in the marine copepod Paracyclopina nana. Also, activation level of mitogen-activated protein kinase (MAPK) signaling pathways along with the gene expression profile of de novo lipogenesis (DNL) pathways were addressed. As a result, BDE-47 induced oxidative stress (e.g. reactive oxygen species, ROS) mediated activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induced signal molecules that bind to the transcription factors (TFs) responsible for lipogenesis to EcR, SREBP, ChREBP promoters. Also, the stress stimulated the conversion of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs), a preparedness of the organism to adapt the observed stress, which could be correlated with the elongase and desaturase gene (e.g. ELO3, Δ5-DES, Δ9-DES) expressions, and then extended to the delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana. This study will provide a better understanding of how BDE-47 effects on marine invertebrates particularly on the copepods, an important link in the marine food chain.

Improving productivity and quality of biodiesel from Chlorella vulgaris SDEC-3M through customized process designs

In this study, temporal variations of microalgae growth and lipid accumulation in response to different nitrogen concentrations in media were investigated and discussed in detail, using a mutant Chlorella vulgaris SDEC-3M as a model. The results show that a short-term of dramatic lipid accumulation and nonsignificant descent of biomass productivities can be concomitant in SDEC-3M during early N-deficient stage (buffer period). It was found that the maximum lipid productivities of 63.52mgL−1d−1 occurred after 24hN-shift. It means that, obtained lipid productivity under two-stage strategy with 24hN-deficient stage was approximately three times as high as the maximum one obtained under N-rich condition and the final one obtained during longer N-deficient culture. Batch culture mode, higher light intensity and light/dark cycle in cooperation with N-rich/N-deficient cycle were also suggested as feasible scheme to improve lipid productivity in large scale culture. Additionally, the fatty acid profiles analysis indirectly showed that the properties of biodiesel from SDEC-3M majorly satisfied biodiesel standards. The fuel performances varied with N-deficient culture time, meaning that better combustion performance, oxidation stability, and exhaust emissions level related to longer N-deficient stage, while the biodiesel produced form N-deficient would perform well under low temperature. The results mean that SDEC-3M-derived biodiesels with customized process designs have commercial potential in the aspects of productivity and quality.

Characterization and nitrogen deficiency response of ATP-citrate lyase from unicellular alga Dunaliella tertiolecta

ATP-citrate lyase (ACL) catalyzes the formation of cytosolic acetyl-CoA, which is responsible for the biosynthesis of fatty acids, lipids, and flavonoid. Here, cDNAs of the small and large subunits of ACL (DtACLA and DtACLB) and their genomic sequences were isolated from Dunaliella tertiolecta using RT-PCR, RACEs and genomic walking techniques. It was found that there were 12 exons and 11 introns in DtACLA, and 20 exons and 19 introns in DtACLB. PlantPAN and PlantCARE revealed a number of putative transcription factor binding sites in the 5′-flanking regions of DtACLA and DtACLB. Conserved domain analysis and phylogenetic analysis showed that ACLA and SCSβ may share the common evolutionary origin, and ACLB may have a common origin from the fusion and divergence of SCSα and CS. The recombinant DtACLA and DtACLB in E. coli BL21 (DE3) seemed to be expressed mainly in the form of insoluble fraction. The transcription levels of DtACLA and DtACLB were fairly consistent with lipid accumulation in response to nitrogen deficiency, suggesting that both subunits of ACL from D. tertiolecta may be coordinate to function in the catalysis, and their activity was related to the lipid accumulation in D. tertiolecta.

Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica.

BackgroundYarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. While the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to better understand how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield.ResultsWe found that ribosome structural genes are down-regulated under nitrogen limitation, during which nitrogen containing compounds (alanine, putrescine, spermidine and urea) are depleted and sugar alcohols and TCA cycle intermediates accumulate (citrate, fumarate and malate). We identified 1219 novel phosphorylation sites in Y. lipolytica, 133 of which change in their abundance during nitrogen limitation. Regulatory proteins, including kinases and DNA binding proteins, are particularly enriched for phosphorylation. Within lipid synthesis pathways, we found that ATP-citrate lyase, acetyl-CoA carboxylase and lecithin cholesterol acyl transferase are phosphorylated during nitrogen limitation while many of the proteins involved in β-oxidation are down-regulated, suggesting that storage lipid accumulation may be regulated by phosphorylation of key enzymes. Further, we identified short DNA elements that associate specific transcription factor families with up- and down-regulated genes.ConclusionsIntegration of metabolome, proteome and phosphoproteome data identifies lipid accumulation in response to nitrogen limitation as a two-fold result of increased production of acetyl-CoA from excess citrate and decreased capacity for β-oxidation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2471-2) contains supplementary material, which is available to authorized users.

FABP4 Regulates Fatty Acid Transfer from Bone Marrow Adipocytes to Acute Myeloid Leukemia Blasts

IntroductionThe incidence of acute myeloid leukemia (AML) is strongly related to age with poor survival rates in the elderly population. AML blasts isolated from the bone marrow undergo spontaneous apoptosis in culture suggesting its microenvironment in the bone marrow (BM) contains cells and factors favorable to the progression of the disease. The BM microenvironment consists of many cell types not directly of the hematopoietic lineage, including bone marrow adipose tissue (MAT), which accounts for circa 50% of the BM volume in the axial skeleton of adults. We hypothesize that MAT may contribute to the cancerous hallmarks exhibited by the AML blasts in the bone marrow. Here we examine the relationship between cancer associated MAT and AML blast survival and proliferation. We describe how this association favors the survival of AML blasts by the transfer of free fatty acids (FFA) released from MAT to AML blasts via the chaperone protein fatty acid binding protein 4 (FABP4). We also show FABP4 to be a critical player in fatty acid transport and therefore the survival of AML blasts in the bone marrow.MethodsTo investigate the role of MAT in regulating AML survival we used primary AML blasts and marrow derived adipose tissue obtained from patient's bone marrow following an informed consent. MAT was co-cultured with primary AML blasts in vitro and the proliferation and survival of the blasts was determined by BrdU incorporation and Annexin V/PI staining respectively. Immunocytochemistry using lipid specific dye, CD34 antibody and nuclear staining was performed to determine lipid storage in primary AML blasts. Western blot analysis was used to determine the level of phosphorylated hormone sensitive lipase (HSL) and fatty acid binding protein 4 (FABP4) in adipocytes. ß oxidation in AML blasts was determined by transcript levels of CPT-1a and ACOX-1. FABP4 concentration was also assayed in MAT and MAT/AML co-cultures by ELISA. Adipocyte FABP4 was knocked down by shRNA and a small molecule inhibitor (BMS309403) was used for pharmacological inhibition.ResultsResults show that in vitro primary AML blasts demonstrate increased survival and proliferation when co-cultured on MAT. Immunocytochemistry using lipid specific dye on CD34+ primary AML blasts revealed presence of neutral lipids within the blasts, which following a 48 hour incubation in monoculture was significantly depleted. Primary AML blasts/MAT co-cultivation caused lipid accumulation in AML blasts and lipolysis of MAT indicated by increased levels of glycerol and FFA in the co-culture media compared to control. Immunoblotting of co-cultured MAT showed increased levels of lipolysis associated factors, phosphorylated HSL and perilipin with a decreased expression of FABP4 compared to MAT monocultures. An apparent increase in ß oxidation was also revealed in AML blasts indicated by the transcriptional activation of ß oxidation related genes. FABP4 MAT transcript levels were shown to increase significantly suggesting an increased production and subsequent trafficking of FABP4 from MAT. Pharmacological inhibition and shRNA mediated knock-down of FABP4 showed a significant decrease in survival and proliferation of AML blasts cultured with adipocytes. Finally investigations also revealed that AML blasts cultured with adipocytes had depleted lipid accumulation in response to FABP4 inhibition.ConclusionHere we report that lipid trafficking between MAT and AML supports survival and proliferation of the leukemic blasts in-vitro. We show that FABP4 is transcriptionally up-regulated in both AML and MAT which mediates the transport of FFA from adipocytes to the leukemic blast. Furthermore, the inhibition of FABP4 significantly reduces AML survival. Together, our data shows evidence for the relationship between MAT and AML blasts by fatty acid transfer identifying FA metabolism as a potential therapeutic target for this aggressive disease. DisclosuresNo relevant conflicts of interest to declare.

Lipid accumulation in response to nitrogen limitation and variation of temperature in Nannochloropsis salina.

BackgroundThis batch study deals with the relation between lipid as well as triglyceride contents in Nannochloropsis salina and variation in culture conditions such as nitrogen concentration and temperature.ResultsThe tested parameters caused reduction in growth expressed as cell count, optical density and dry weight, as well strongly involved in lipids and triglycerides accumulation and significantly affected the lipid productivity. At the beginning of the work, the concentration of nitrogen in the medium was reduced to three quarter, half and quarter of the original f2 medium while the temperature kept constant. After that, the optimal nitrogen concentration (quarter of the original media) giving high lipid yield was tested with different temperature degrees from 15 to 35°C with five degree intervals. Although the growth was insignificantly influenced, a considerable increase in lipid and triglyceride (56.1 and 15.1% of dry weight respectively) was observed when the concentration of nitrogen in the medium was reduced to the quarter. Moreover, 59.3% lipid and 17.1% triglyceride on the basis of dry weight were obtained by the combination of 25% nitrogen concentration and 30°C. Simple regressions recommended that the interaction effect of nitrogen limitation and temperature on lipid and triglyceride accumulation was not as fundamental as for nitrogen limitation stress.ConclusionThe degree of nitrogen availability in the combination of temperature effect has been identified as the critical determinant for the maximal production of lipid in N. salina. Nevertheless, major advances in this field can be considered by studying more stresses techniques and genetic strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-015-0085-7) contains supplementary material, which is available to authorized users.