Lipid Export Research Articles

Postural characterization in subjects with Parkinson's disease using a synthetic index based on the Gait Profile Score

How hepatitis C virus (HCV)–associated lipid metabolic alterations recover after sustained virological response (SVR) remains elusive.The aforementioned recovery pattern was investigated.In a prospective cohort study of 438 chronic hepatitis C (CHC) patients with SVR after anti-HCV therapy, 164 sex- and age-matched genotype I (G1) and G2 patients underwent paired-serum liquid chromatography-tandem mass spectrometry analyses before and 24 weeks after therapy. Subjects without CHC served as controls (n = 100).CHC patients had lower baseline lipid levels than controls. Among CHC patients, pre-therapy total cholesterol levels were positively associated with HCV RNA levels; G1 patients had higher pre-therapy HCV RNA levels than G2 patients. Repeated measures analysis of variance of CHC patients showed that lathosterol, lanosterol, total hydroxysphingomyelin, and total phosphatidylcholines levels, and total dicarboxyacylcarnitine/total acylcarnitine (indicators of ω-oxidation) and pre-β-lipoprotein ratios elevated 24 weeks after therapy compared with the levels before therapy. Levels of total lysophosphatidylcholines and α- and β-lipoprotein ratios decreased. Subgroup analyses showed elevated 7-dehydrocholesterol and lanosterol levels, particularly in G2 and male patients, who had broader spectra of altered phosphatidylcholines and acylcarnitines than G1 and female patients, respectively. Compared with controls, CHC patients had higher post-therapy levels of total lysophosphatidylcholines and hydroxysphingomyelins and ratios of total dicarboxyacylcarnitines/total acylcarnitines but lower cholesterol levels.At 24 weeks after therapy, accelerated cholesterol biosynthesis, hepatic lipid export, ω-oxidation, and decreased systemic inflammation were noted in CHC patients with SVR, with greater efficiency in G2 and male patients. Regardless, HCV-associated lipid metabolic alterations required >24 weeks for restoration or were incompletely reversible after SVR.

Structure of the Human Lipid Exporter ABCA1

ABCA1, an ATP-binding cassette (ABC) subfamily A exporter, mediates the cellular efflux of phospholipids and cholesterol to the extracellular acceptor apolipoprotein A-I (apoA-I) for generation of nascent high-density lipoprotein (HDL). Mutations of human ABCA1 are associated with Tangier disease and familial HDL deficiency. Here, we report the cryo-EM structure of human ABCA1 with nominal resolutions of 4.1Å for the overall structure and 3.9Å for the massive extracellular domain. The nucleotide-binding domains (NBDs) display a nucleotide-free state, while the two transmembrane domains (TMDs) contact each other through a narrow interface in the intracellular leaflet of the membrane. In addition to TMDs and NBDs, two extracellular domains of ABCA1 enclose an elongated hydrophobic tunnel. Structural mapping of dozens of disease-related mutations allows potential interpretation of their diverse pathogenic mechanisms. Structural-based analysis suggests a plausible "lateral access" mechanism for ABCA1-mediated lipid export that may be distinct from the conventional alternating-access paradigm.

Hepatic lipid accumulation: cause and consequence of dysregulated glucoregulatory hormones

Fatty liver can be diet, endocrine, drug, virus or genetically induced. Independent of cause, hepatic lipid accumulation promotes systemic metabolic dysfunction. By acting as peroxisome proliferator-activated receptor (PPAR) ligands, hepatic non-esterified fatty acids upregulate expression of gluconeogenic, beta-oxidative, lipogenic and ketogenic genes, promoting hyperglycemia, hyperlipidemia and ketosis. The typical hormonal environment in fatty liver disease consists of hyperinsulinemia, hyperglucagonemia, hypercortisolemia, growth hormone deficiency and elevated sympathetic tone. These endocrine and metabolic changes further encourage hepatic steatosis by regulating adipose tissue lipolysis, liver lipid uptake, de novo lipogenesis (DNL), beta-oxidation, ketogenesis and lipid export. Hepatic lipid accumulation may be induced by 4 separate mechanisms: (1) increased hepatic uptake of circulating fatty acids, (2) increased hepatic de novo fatty acid synthesis, (3) decreased hepatic beta-oxidation and (4) decreased hepatic lipid export. This review will discuss the hormonal regulation of each mechanism comparing multiple physiological models of hepatic lipid accumulation. Nonalcoholic fatty liver disease (NAFLD) is typified by increased hepatic lipid uptake, synthesis, oxidation and export. Chronic hepatic lipid signaling through PPARgamma results in gene expression changes that allow concurrent activity of DNL and beta-oxidation. The importance of hepatic steatosis in driving systemic metabolic dysfunction is highlighted by the common endocrine and metabolic disturbances across many conditions that result in fatty liver. Understanding the mechanisms underlying the metabolic dysfunction that develops as a consequence of hepatic lipid accumulation is critical to identifying points of intervention in this increasingly prevalent disease state.

Abstract P6-02-04: Atorvastatin insensitivity is associated with increased lipid droplets accumulation and fatty acid metabolism in breast cancer cells

Abstract Lipophilic statins, including atorvastatin, may exert significant antiproliferative and proapoptotic effects in breast cancer as demonstrated in both clinical trials and cell models. However, heterogeneity in treatment response still remains a noteworthy challenge to be addressed. In this regard, we previously proposed a multigene signature including genes involved in cholesterol biosynthesis, which were dysregulated upon atorvastatin treatment and was shown to predict statin responsiveness in breast cancer (BC) cell lines and primary tumors. To further delineate the molecular mechanisms underlying this variability, we sought to characterize the differential statin-induced effects on intracellular lipid regulation observed in BC cell lines based on their sensitivity to atorvastatin treatment. BC cells were classified as insensitive (MCF-7 and T47D) or sensitive (MDA-MB-231) to atorvastatin by virtue of growth inhibition rate consequent to treatment with doses ranging up to 10 μM. Under complete culture conditions, atorvastatin-induced decrease in cell proliferation was inversely correlated to a progressive accumulation of neutral lipids in lipid droplets (LDs) in the insensitive cells following 72 hours treatment. Interestingly, in the sensitive MDA-MB-231 cells no significant change in LDs formation was observed despite the very potent antiproliferative effects (60% impairment of growth). However, in correspondence of severe inhibition of growth rate of at least 80% by atorvastatin treatment, MDA-MB-231 cells displayed a consistent reduction in LDs accumulation, suggesting beneficial effects of LDs biosynthesis on proliferation of BC cells. Transcriptional profiling of genes involved in lipid metabolism using microarrays and validated by qRT-PCR ruled out the likelihood that atorvastatin treatment altered lipid uptake and export mechanisms in sensitive cells in the presence of exogenous lipid supply. Nevertheless, gene ontology analysis indicated that an induction of cell stress responses was likely associated to atorvastatin sensitivity. Significant deregulation of genes involved in the fatty acid metabolic process, including biosynthesis of monounsaturated fatty acids (MUFAs), and cholesterol biosynthesis were instead linked to atorvastatin insensitivity. Accordingly, we found that the magnitude of the induction of the mRNAs of stearoyl-CoA desaturase (SCD), the key effector in MUFAs metabolism, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the established target of statins, were consistently lower in sensitive cells compared to the insensitive counterpart in response to atorvastatin treatment. Therefore, we suggest that the ability to significantly increase the number of stored neutral lipids in response to statin treatment may likely confer a proliferative advantage to BC cells. Our results also identified MUFAs metabolism as an attractive pathway to be further investigated in view of finding promising biomarkers for unraveling the molecular basis of statin sensitivity in breast cancer. Citation Format: Lettiero B, Kimbung S, Borgquist S. Atorvastatin insensitivity is associated with increased lipid droplets accumulation and fatty acid metabolism in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-04.

The chloroform extract of Cyclocarya paliurus attenuates high-fat diet induced non-alcoholic hepatic steatosis in Sprague Dawley rats

BackgroundHepatic steatosis (HS) is the early stage of nonalcoholic fatty liver disease which is caused by impaired hepatic lipid homeostasis. Cyclocarya paliurus, an herbal tea consumed in China, has been demonstrated to ameliorate abnormal lipid metabolism for the treatment of metabolic diseases. PurposeWe aimed to investigate the regulative effect of chloroform extract from Cyclocarya paliurus (ChE) on treatment of HS, as well as key factors involved in hepatic lipid metabolism. Study designSprague Dawley rats were fed with high-fat diet (HFD) for 6 weeks to induce HS and treated with or without ChE by gavage for 4 weeks. MethodsThe body weight, relative liver weight and liver fat content were measured. Serum and liver total cholesterol, triglyceride and non-esterified fatty acids, as well as hepatic malonaldehyde levels were accessed by biochemical methods. Serum and liver TNF-α levels were quantified by ELISA kit. Histologic analysis and 1H-MRS study were performed to evaluate HS level. RT-PCR and Western blot were also applied to observe the expression changes of key factors involved in hepatic lipid intake, synthesis, utilization and export. ResultsChE significantly decreased the rats’ body weight, serum lipid and TNF-α level. ChE also reduced their relative liver weight, liver fat content, hepatic oxidative products and TNF-α level. Hepatic steatosis in HFD-fed rats was effectively regressed after 2-weeks administration of ChE. Moreover, ChE treatment remarkably reduced HFD-induced high expression level of fatty acid synthesis genes (including sterol-regulatory element-binding protein 1, acetyl-CoA carboxylase 1 and fatty acid synthase). However, it had no effect on mRNA expression of some genes involved in lipid uptake, β-oxidation and lipid outflow. ConclusionChE exerted a promising regression effect on HS due to a reduced level of serum non-esterified fatty acids which might lead to a decrease in the amount of lipid taken in by the liver, as well as owing to the inhibition of hepatic lipid de novo synthesis to reduce liver lipid production.

Increased susceptibility of prenatal food restricted offspring to high-fat diet-induced nonalcoholic fatty liver disease is intrauterine programmed

The present study aims to explore the mechanisms of fetal origin of high susceptibility to adult high-fat diet induced-nonalcoholic fatty liver disease in rat offspring undergoing intrauterine growth retardation (IUGR) induced by prenatal food restriction (FR) from gestational day 11 until full-term delivery. We observed that adult IUGR offspring rats exhibited gender-dependent catch-up growth with lower serum corticosterone (CORT) but up-regulation of the insulin-like growth factor 1 (IGF1) pathway, higher hepatic Kleiner scores and lower lipid export and oxidation. Furthermore, fetal IUGR offspring rats showed lower body weights with higher serum CORT but down-regulated IGF1 pathway, which was accompanied by enhanced lipid de novo synthetic gene expression, lower lipid output and oxidation gene expression. It is suggested that a “two-programming” mechanism, which refers to the adverse intrauterine programming of hepatic lipid de novo synthesis and glucocorticoid-IGF1 axis programming associated with postnatal catch-up growth, could explain the increased susceptibility.

The LysE Superfamily of Transport Proteins Involved in Cell Physiology and Pathogenesis.

The LysE superfamily consists of transmembrane transport proteins that catalyze export of amino acids, lipids and heavy metal ions. Statistical means were used to show that it includes newly identified families including transporters specific for (1) tellurium, (2) iron/lead, (3) manganese, (4) calcium, (5) nickel/cobalt, (6) amino acids, and (7) peptidoglycolipids as well as (8) one family of transmembrane electron carriers. Internal repeats and conserved motifs were identified, and multiple alignments, phylogenetic trees and average hydropathy, amphipathicity and similarity plots provided evidence that all members of the superfamily derived from a single common 3-TMS precursor peptide via intragenic duplication. Their common origin implies that they share common structural, mechanistic and functional attributes. The transporters of this superfamily play important roles in ionic homeostasis, cell envelope assembly, and protection from excessive cytoplasmic heavy metal/metabolite concentrations. They thus influence the physiology and pathogenesis of numerous microbes, being potential targets of drug action.

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.

Beer Is Less Harmful for the Liver than Plain Ethanol: Studies in Male Mice Using a Binge-Drinking Model.

Mechanisms involved in the less damaging effects of beer in comparison to hard spirits have not yet been fully understood. The aim of the study was to determine if the effect of beer intake on the liver differs from that of plain ethanol and if so to determine mechanisms involved. Male C57BL/6J mice received either ethanol, beer (ethanol content: 6 g/kg body weight) or iso-caloric maltodextrin solution. Markers of steatosis, lipogenesis, activation of the toll-like receptor-4 signaling cascade and lipid export in liver and tight junction proteins in duodenum were measured 6 and 12 h after acute ethanol or beer intake. Alcohol ingestion resulted in a significant increase of hepatic triglyceride accumulation 6 and 12 h after ingestion, respectively, being markedly lower in mice fed beer. Expression of sterol regulatory element-binding protein-1c mRNA was significantly lower 12 h after alcohol or beer exposure, while fatty acid synthase mRNA expression was induced in livers of ethanol-fed mice and to a lesser extent in mice fed beer 6 h after acute alcohol ingestion. Protein levels of tight junction proteins in the small intestine were similar between groups while expression of myeloid differentiation primary response gene 88 in livers was significantly induced in ethanol- but not in beer-fed mice. Concentrations of 4-hydroxynonenal protein adducts and inducible nitric oxide synthase protein were also only induced in livers of mice fed ethanol. Protein levels of apolipoprotein B were induced in livers of beer-fed mice only. Our data suggest that beer is less harmful on the development of acute alcohol-induced liver damage than plain ethanol in male mice.

Dihydroaustrasulfone Alcohol (WA-25) Impedes Macrophage Foam Cell Formation by Regulating the Transforming Growth Factor-β1 Pathway.

Atherosclerosis is considered an inflammatory disease. However, clinically used anti-atherosclerotic drugs, such as simvastatin, have many side effects. Recently, several unique marine compounds have been isolated that possess a variety of bioactivities. In a previous study, we found a synthetic precursor of the marine compound (austrasulfone), which is dihydroaustrasulfone alcohol (WA-25), has anti-atherosclerotic effects in vivo. However, the detailed mechanisms remain unclear. Therefore, to clarify the mechanisms through which WA-25 exerts anti-atherosclerotic activity, we used RAW 264.7 macrophages as an in vitro model to evaluate the effects of WA-25. In lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, WA-25 significantly inhibited expression of the pro-inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In contrast, simvastatin increased the COX-2 expression compared to WA-25. In addition, WA-25 impedes foam cell formation and up-regulated the lysosomal and cyclic adenosine monophosphate (cAMP) signaling pathway. We also observed that transforming growth factor β1 (TGF-β1) was up-regulated by WA-25 and simvastatin in LPS-induced RAW 264.7 cells, and the promising anti-atherosclerosis effects of WA-25 were disrupted by blockade of TGF-β1 signaling. Besides, WA-25 might act through increasing lipolysis than through alteration of lipid export. Taken together, these data demonstrate that WA-25 may have potential as an anti-atherosclerotic drug with anti-inflammatory effects.

Betaine alleviates hepatic lipid accumulation via enhancing hepatic lipid export and fatty acid oxidation in rats fed with a high-fat diet.

To assess the effects of betaine on hepatic lipid accumulation and investigate the underlying mechanism, thirty-two male Sprague-Dawley rats weighing 100 (sd 2·50) g were divided into four groups, and started on one of four treatments: basal diet, basal diet with betaine administration, high-fat diet and high-fat diet with betaine administration. The results showed that no significant difference of body weight was found among experimental groups. Compared with high-fat diet-fed rats, a betaine supplementation decreased (P< 0·05) hepatic TAG accumulation induced by high-fat diet, which was also supported by hepatic histology results. Additionally, hepatic betaine-homocysteine methyltransferase concentration [corrected] as well as its mRNA abundance and lecithin level were found increased (P< 0·05) by betaine supplementation in both basal diet-fed rats and high-fat diet-fed rats. Betaine administration in high-fat diet-fed rats exhibited a higher (P< 0·05) concentration [corrected] of hepatic carnitine palmitoyltransferase 1 (CPT1) compared with high-fat diet-fed rats. High-fat diet inhibited (P< 0·05) the gene expression of hepatic PPARα and CPT1. However, betaine administration in high-fat diet-fed rats elevated (P< 0·05) the gene expression of PPARα and CPT1. Moreover, concentration, gene and protein expressions of hepatic fibroblast growth factor 21 (FGF21) were increased (P< 0·05) in response to betaine administration in high-fat diet group; meanwhile the gene expression of hepatic AMP-activated protein kinase was increased (P< 0·05) as well. The results suggest that betaine administration enhanced hepatic lipid export and fatty acid oxidation in high-fat diet-fed rats, thus effectively alleviating fat accumulation in the liver.

The pathophysiology of intestinal lipoprotein production.

Intestinal lipoprotein production is a multistep process, essential for the absorption of dietary fats and fat-soluble vitamins. Chylomicron assembly begins in the endoplasmic reticulum with the formation of primordial, phospholipids-rich particles that are then transported to the Golgi for secretion. Several classes of transporters play a role in the selective uptake and/or export of lipids through the villus enterocytes. Once secreted in the lymph stream, triglyceride-rich lipoproteins (TRLs) are metabolized by Lipoprotein lipase (LPL), which catalyzes the hydrolysis of triacylglycerols of very low density lipoproteins (VLDLs) and chylomicrons, thereby delivering free fatty acids to various tissues. Genetic mutations in the genes codifying for these proteins are responsible of different inherited disorders affecting chylomicron metabolism. This review focuses on the molecular pathways that modulate the uptake and the transport of lipoproteins of intestinal origin and it will highlight recent findings on TRLs assembly.

Cholesterol sensing by the ABCG1 lipid transporter: Requirement of a CRAC motif in the final transmembrane domain

The ATP-binding cassette (ABC) transporter, ABCG1, is a lipid exporter involved in removal of cholesterol from cells that has been investigated for its role in foam cells formation and atherosclerosis. The mechanism by which ABC lipid transporters bind and recognise their substrates is currently unknown. In this study, we identify a critical region in the final transmembrane domain of ABCG1, which is essential for its export function and stabilisation by cholesterol, a post-translational regulatory mechanism that we have recently identified as dependent on protein ubiquitination. This transmembrane region contains several Cholesterol Recognition/interaction Amino acid Consensus (CRAC) motifs, and its inverse CARC motifs. Mutational analyses identify one CRAC motif in particular with Y667 at its core, that is especially important for transport activity to HDL as well as stability of the protein in the presence of cholesterol. In addition, we present a model of how cholesterol docks to this CRAC motif in an energetically favourable manner. This study identifies for the first time how ABCG1 can interact with cholesterol via a functional CRAC domain, which provides the first insight into the substrate–transporter interaction of an ABC lipid exporter.

Cytosolic Lipid Trafficking Proteins STARD4 and STARD5 Modulate Hepatic Neutral Lipid Metabolism: Implications for Diabetic Dyslipidaemia and Steatosis

This study explored expression of the START domain family in hyperinsulinaemic, genetically obese rodents, and the functional roles of cytosolic StarD4 and StarD5 proteins in hepatic lipid synthesis and export. Genetic obesity in (fa/fa) Zucker rats repressed StarD4, StarD5, StarD8 and StarD13, and decreased levels of cytosolic StarD4 and StarD5 proteins, compared with lean controls, suggesting links with hepatic storage or secretion of lipids. Stable overexpression of STARD4 in rat McArdle RH7777 hepatoma cells increased lipidation of exogenous ApoA-I compared with empty vector (EV) control, modestly increased secretion of endogenously synthesized cholesterol and reduced incorporation of [14C]oleate into cellular cholesteryl esters. Synthesis and secretion of triacylglycerol did not change in STARD4 overexpressing cells. By contrast, STARD5 overexpression did not alter cholesterol synthesis, secretion, or lipidation of apoA-I, but increased synthesis of triacylglycerol from [3H]glycerol under basal conditions, enhanced incorporation of [14C]oleate into triacylglycerol, and increased expression of Dgat1 and Dgat2 compared with EV. In summary, cytosolic STARD4 impacts predominantly on cholesterol efflux to apoA-I, implying directional transport of cholesterol to the plasma membrane, while STARD5 increases basal triacylglycerol synthesis and enriches this pool with exogenous fatty acids. Cytosolic StarD proteins may be involved in diabetic dyslipidaemia and steatosis.

Glacial cooling as inferred from marine temperature proxies TEXH86 and UK′37

Knowledge of the magnitude of the Last Glacial Maximum (LGM) cooling is a useful constraint for estimating the climate sensitivity used in projecting future climate change. Proxy comparison, especially that between the alkenone-based UK′37 and the archaeal tetraether-based TEX86, has been increasingly applied in paleoceanographic studies as a measure to better constrain proxy-derived temperature estimates. In this study, we compile and compare published multiproxy (UK′37 and TEXH86) records of glacial cooling measured on the same sediment cores. In spite of the diversity in oceanographic and sedimentation settings spanned by the study sites, we find that the TEXH86-derived mean tropical LGM cooling is approximately twice as strong as that suggested by the UK′37 and MARGO estimates. The extent of proxy discrepancy varies with the application of various regional calibrations, but the mean TEXH86-inferred cooling remains stronger than that inferred from UK′37. To understand the discrepancy between proxies, we examine the seasonal and water column structure of LGM cooling simulated by state-of-the-art climate models. We find that the dissimilar magnitudes of proxy-derived glacial cooling cannot be fully explained by proxies reflecting temperature of different seasons or different water depths, if the recording season and depth are assumed to stay constant through time. A hypothetical shift in recording season and/or depth between the Holocene and the LGM could in theory cause the proxy discrepancy, but this hypothesis cannot be constrained due to a lack of information on lipid production and export in the water column during the LGM. Alternatively, the systematic proxy discrepancy, which persists across diverse oceanographic settings, may imply that the commonly applied proxy calibrations for reconstructing past temperatures are fundamentally biased. As evidenced by the improved consistency between UK′37 and TEXH86-based estimates of LGM cooling after we applied a global subsurface (0–200 m) temperature calibration for TEXH86, it is plausible that the TEXH86 signal originates from deeper in the water column than typically assumed for the proxy calibration.

A systems biology approach utilizing a mouse diversity panel identifies genetic differences influencing isoniazid-induced microvesicular steatosis.

Isoniazid (INH), the mainstay therapeutic for tuberculosis infection, has been associated with rare but serious hepatotoxicity in the clinic. However, the mechanisms underlying inter-individual variability in the response to this drug have remained elusive. A genetically diverse mouse population model in combination with a systems biology approach was utilized to identify transcriptional changes, INH-responsive metabolites, and gene variants that contribute to the liver response in genetically sensitive individuals. Sensitive mouse strains developed severe microvesicular steatosis compared with corresponding vehicle control mice following 3 days of oral treatment with INH. Genes involved in mitochondrial dysfunction were enriched among liver transcripts altered with INH treatment. Those associated with INH treatment and susceptibility to INH-induced steatosis in the liver included apolipoprotein A-IV, lysosomal-associated membrane protein 1, and choline phosphotransferase 1. These alterations were accompanied by metabolomic changes including reduced levels of glutathione and the choline metabolites betaine and phosphocholine, suggesting that oxidative stress and reduced lipid export may additionally contribute to INH-induced steatosis. Finally, genome-wide association mapping revealed that polymorphisms in perilipin 2 were linked to increased triglyceride levels following INH treatment, implicating a role for inter-individual differences in lipid packaging in the susceptibility to INH-induced steatosis. Taken together, our data suggest that INH-induced steatosis is caused by not one, but multiple events involving lipid retention in the livers of genetically sensitive individuals. This work also highlights the value of using a mouse diversity panel to investigate drug-induced responses across a diverse population.

Intracellular cholesterol transporters and modulation of hepatic lipid metabolism: Implications for diabetic dyslipidaemia and steatosis

Aims/hypothesesTo examine hepatic expression of cholesterol-trafficking proteins, mitochondrial StarD1 and endosomal StarD3, and their relationship with dyslipidaemia and steatosis in Zucker (fa/fa) genetically obese rats, and to explore their functional role in lipid metabolism in rat McArdle RH-7777 hepatoma cells. MethodsExpression of StarD1 and StarD3 in rat liver and hepatoma samples were determined by Q-PCR and/or immunoblotting; lipid mass by colorimetric assays; radiolabelled precursors were utilised to measure lipid synthesis and secretion, and lipidation of exogenous apolipoprotein A-I. ResultsHepatic expression of StarD3 protein was repressed by genetic obesity in (fa/fa) Zucker rats, compared with lean (Fa/?) controls, suggesting a link with storage or export of lipids from the liver. Overexpression of StarD1 and StarD3, and knockdown of StarD3, in rat hepatoma cells, revealed differential effects on lipid metabolism. Overexpression of StarD1 increased utilisation of exogenous (preformed) fatty acids for triacylglycerol synthesis and secretion, but impacted minimally on cholesterol homeostasis. By contrast, overexpression of StarD3 increased lipidation of exogenous apoA-I, and facilitated de novo biosynthetic pathways for neutral lipids, potentiating triacylglycerol accumulation but possibly offering protection against lipotoxicity. Finally, StarD3 overexpression altered expression of genes which impact variously on hepatic insulin resistance, inducing Ppargcla, Cyp2e1, Nr1h4, G6pc and Irs1, and repressing expression of Scl2a1, Igfbp1, Casp3 and Serpine 1. Conclusions/interpretationTargeting StarD3 may increase circulating levels of HDL and protect the liver against lipotoxicity; loss of hepatic expression of this protein, induced by genetic obesity, may contribute to the pathogenesis of dyslipidaemia and steatosis.

Crystal Structure of the Transcriptional Regulator Rv0678 of Mycobacterium tuberculosis

Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.

Abstract 418: Bifunctional Role of Autophagy in Intracellular ApoB Degradation and Lipid Droplet Mobilization

Introduction: As apolipoprotein B (apoB) is translated and translocated into the ER, lipids from cytoplasmic lipid droplets (LDs) are added to promote folding and to initiate very-low-density-lipoprotein (VLDL) assembly. However, without sufficient lipid availability, apoB is misfolded and subject to proteasomal degradation. Evidence now shows that apoB can also be degraded through autophagy under certain conditions, and that LDs are also subject to autophagic degradation, a process referred to as lipophagy. We postulate that apoB autophagy and LD lipophagy integrate to regulate hepatic lipid export and VLDL production. Methods/Results: Studies were conducted in vitro using the human hepatoma cell line, HepG2 and ex vivo using primary Syrian Golden hamster hepatocytes. Cells were fat loaded with/without 0.4 mM OA for 4 hours and simultaneously treated with an autophagy inhibitor 3-methyadenine (3-MA; 100uM) or an autophagy inducer, Torin 1 (250nM). HepG2 cells were transfected with 10 nM of atg12 siRNA, an essential autophagy related gene, for a total of 72 hours. In freshly isolated primary hamster hepatocytes, inhibition of autophagosome formation, through treatment with 3-MA, significantly increased cellular levels of newly synthesized apoB, without a significant increase in apoB secreted into the media. Interestingly, treating these cells with Torin 1 to promote autophagy also significantly increased apoB recovery. However, modulation of autophagy activity also affected the average number of LDs per cell, indicating that lipophagy activity had also been modified, potentially affecting VLDL formation. Conversely, while inhibition and induction of autophagy in HepG2 cells, a human hepatoma cell line, reduced and increased apoB co-localization with autophagosomes respectively, siRNA knockdown of atg12 as well as 3-MA treatment decreased apoB recovery. However, this did not appear to be due to reduction in LD breakdown through autophagy. The data obtained with primary hamster hepatocytes suggest that autophagy may play a dual role in VLDL assembly in vivo by regulating both degradation of apoB and lipidation of VLDL particles through mobilization of lipid from LDs. Defects in these pathways can induce hepatic LD accumulation and steatosis.

Hepatocyte-specificPtpn6deletion promotes hepatic lipid accretion, but reduces NAFLD in diet-induced obesity: Potential role of PPARγ

Hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)) are protected from hepatic insulin resistance evoked by high-fat diet (HFD) feeding for 8 weeks. Unexpectedly, we report herein that Ptpn6(H-KO) mice fed an HFD for up to 16 weeks are still protected from insulin resistance, but are more prone to hepatic steatosis, as compared with their HFD-fed Ptpn6(f/f) counterparts. The livers from HFD-fed Ptpn6(H-KO) mice displayed 1) augmented lipogenesis, marked by increased expression of several hepatic genes involved in fatty acid biosynthesis, 2) elevated postprandial fatty acid uptake, and 3) significantly reduced lipid export with enhanced degradation of apolipoprotein B (ApoB). Despite more extensive hepatic steatosis, the inflammatory profile of the HFD-fed Ptpn6(H-KO) liver was similar (8 weeks) or even improved (16 weeks) as compared to their HFD-fed Ptpn6(f/f) littermates, along with reduced hepatocellular damage as revealed by serum levels of hepatic enzymes. Interestingly, comparative microarray analysis revealed a significant up-regulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene expression, confirmed by quantitative polymerase chain reaction. Elevated PPARγ nuclear activity also was observed and found to be directly regulated by Shp1 in a cell-autonomous manner. These findings highlight a novel role for hepatocyte Shp1 in the regulation of PPARγ and hepatic lipid metabolism. Shp1 deficiency prevents the development of severe hepatic inflammation and hepatocellular damage in steatotic livers, presenting hepatocyte Shp1 as a potential novel mediator of nonalcoholic fatty liver diseases in obesity.