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1.  Thematic review series: Adipocyte Biology. Adipose tissue function and plasticity orchestrate nutritional adaptation 
Journal of lipid research  2007;48(6):1253-1262.
This review focuses on adipose tissue biology and introduces the concept of adipose tissue plasticity and expandability as key determinants of obesity-associated metabolic dysregulation. This concept is fundamental to our understanding of adipose tissue as a dynamic organ at the center of nutritional adaptation. Here, we summarize the current knowledge of the mechanisms by which adipose tissue can affect peripheral energy homeostasis, particularly in the context of overnutrition. Two mechanisms emerge that provide a molecular understanding for obesity-associated insulin resistance. These are a) the dysregulation of adipose tissue expandability and b) the abnormal production of adipokines. This knowledge has the potential to pave the way for novel therapeutic concepts and strategies for managing and/or correcting complications associated with obesity and the metabolic syndrome.
PMCID: PMC4303760  PMID: 17374880
obesity; adipokines; lipotoxicity; insulin resistance; Metabolic syndrome
2.  Three-fold effect of lovastatin treatment on low density lipoprotein metabolism in subjects with hyperlipidemia: increase in receptor activity, decrease in apoB production, and decrease in particle affinity for the receptor. Results from a novel triple-tracer approach 
Journal of lipid research  1998;39(4):913-924.
To differentiate effects of lovastatin on low density lipoprotein (LDL) receptor activity from effects on LDL metabolic properties, LDL apolipoprotein B (apoB) turnover was studied in eight hyperlipidemic subjects during baseline and lovastatin treatment, in the latter case with LDL tracers isolated during both baseline (C-LDL) and drug treatment (Rx-LDL) conditions. Lovastatin (40 mg/day) significantly lowered total plasma and LDL cholesterol levels (27% and 25%, respectively) as well as plasma triglyceride levels (30%). Using contemporaneous tracers (C-LDL before and Rx-LDL during treatment), lovastatin caused a modest increase in LDL fractional catabolic rate (FCR) (0.410 ± 0.113 vs. 0.339 ± 0.108 pools/day, P < 0.04 by paired t). The increase in LDL tracer FCR was higher when C-LDL tracer isolated during the untreated period was injected during lovastatin treatment (0.496 ± 0.177 vs. 0.339 ± 0.108 pools/day, P < 0.02). These in vivo studies in humans were confirmed by injecting LDL tracers from two patients into five guinea pigs. The C-LDL tracer was cleared consistently faster than the Rx-LDL tracer (0.082 ± 0.018 vs. 0.057 ± 0.015 pools/h, P < 0.001). The results demonstrate three important outcomes of lovastatin treatment in these subjects: LDL receptor activity increased by 49% (P < 0.02); LDL apoB production rate decreased by 17% (P < 0.03), and LDL particle in vivo affinity for the LDL receptor decreased by 15% (P < 0.01). The decrease in LDL particle affinity partially negated the expected effect of increased LDL receptors on LDL clearance. The present study provides an explanation for earlier observations by several investigators using contemporaneous tracers that treatment with HMGCoA reductase inhibitors resulted in only modest increases in low density lipoprotein functional catabolic rate.
PMCID: PMC3988111  PMID: 9555954
HMG-CoA reductase inhibitors; LDL receptor; apolipoprotein B; lipoprotein metabolism; tracer kinetics; lipid-lowering therapy
3.  A polymorphism in the gene encoding procolipase produces a colipase, Arg92Cys, with decreased function against long-chain triglycerides 
Journal of lipid research  2007;48(11):2478-2484.
Type 2 diabetes mellitus is a multifactorial and polygenic disorder with increasing prevalence. Recently, a polymorphism in the gene encoding procolipase, a cysteine for arginine substitution at position 92, was associated with type 2 diabetes in two human populations. Because procolipase plays a critical role in dietary fat metabolism, polymorphisms that affect the function of procolipase could influence the development of type 2 diabetes. We hypothesized that the Arg92Cys polymorphism has functional consequences. To test our hypothesis, we expressed recombinant cysteine 92 (Cys92) procolipase in a yeast expression system and compared the function and stability of purified Cys92 with that of the more common arginine 92 (Arg92) procolipase. Cys92 fully restored the activity of bile-salt inhibited lipase with short- and medium-chain triglycerides but only had 50% of Arg92 function with long-chain triglycerides. After storage at 4°C, Cys92 lost the ability to restore pancreatic triglyceride lipase activity with medium- and long-chain triglycerides. The loss of function correlated with the inability of Cys92 to anchor lipase on an emulsion surface and oxidation of the cysteine. No detectable degradation or intramolecular disulfide formation occurred in Cys92 after storage. Our findings demonstrate that the Arg92Cys polymorphism decreases the function of Cys92 colipase. This change may contribute to the development of type 2 diabetes.
PMCID: PMC3684974  PMID: 17715423
lipase; type 2 diabetes; digestion; recombinant protein; mutagenesis
4.  LDL phospholipid hydrolysis produces modified electronegative particles with an unfolded apoB-100 protein 
Journal of Lipid Research  2004;46(1):115-122.
Electronegative low density lipoprotein (LDL− formation that structurally resembles LDL− isolated from plasma was evaluated after LDL treatment with snake venom phospholipase A2 (PLA2). PLA2 treatment of LDL increased its electrophoretic mobility in proportion to the amount of LDL− formed without evidence of lipid peroxidation. These changes dose-dependently correlated with the degree of phospholipid hydrolysis. Strong immunoreactivity of LDL− subfraction from plasma and PLA2-treated LDL (PLA2-LDL) to amyloid oligomer-specific antibody was observed. Higher β-strand structural content and unfolding proportionate to the loss of α-helical structure of apolipoprotein B-100 (apoB-100) of LDL− isolated from both native and PLA2-LDLs was demonstrated by circular dichroism (CD) spectropolarimetry. These structural changes resembled the characteristics of some oxidatively modified LDLs and soluble oligomeric aggregates of amyloidogenic proteins. PLA2-LDL was also more susceptible to nitration by peroxynitrite, likely because of exposure of otherwise inaccessible hydrophilic and hydrophobic domains arising from apoB-100 unfolding. This was also demonstrated for plasma LDL−. In contrast, PLA2-LDL was more resistant to copper-mediated oxidation that was reversed upon the addition of small amounts of unsaturated fatty acids.
PMCID: PMC3383313  PMID: 15489541
apolipoprotein B-100; atherogenic low density lipoprotein; lipid peroxidation; oxidation; nitrotyrosine; unsaturated fatty acids; β-strand structures; protein structure; secretory phospholipase A2
5.  On determining the extent of side-pool synthesis in a three-pool model for whole body cholesterol kinetics 
Journal of Lipid Research  1981;22(8):1174-1180.
Whole body cholesterol turnover is well described by a three-pool model. This model has eight unknown parameters: three masses, three synthesis rates, and two inter-compartmental exchange rates. Only six parameters can be estimated by fitting the model to the plasma specific radioactivity-time curve which results from the intravenous injection of labeled cholesterol. Additional information is obtained if a precursor of cholesterol, labeled with a different isotope, is also injected. Equations are derived to enable the calculation of all eight model parameters from the two sum-of-exponentials equations that are fitted to the two tracer curves. The characteristics of a satisfactory precursor are discussed.
PMCID: PMC3277742  PMID: 7320627
compartmental analysis; mathematical model; exchangeable cholesterol; cholesterol precursor; parameter estimation; pool models; identifiability; double-isotope study
6.  Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments 
Journal of Lipid Research  2006;47(12):2738-2753.
Lipoprotein kinetic parameters are determined from mass spectrometry data after administering mass isotopes of amino acids, which label proteins endogenously. The standard procedure is to model the isotopic content of the labeled precursor amino acid and of proteins of interest as tracer-to-tracee ratio (TTR). It is shown here that even though the administered tracer alters amino acid mass and turnover, apolipoprotein synthesis is unaltered and hence the apolipoprotein system is in a steady state, with the total (labeled plus unlabeled) masses and fluxes remaining constant. The correct model formulation for apolipoprotein kinetics is shown to be in terms of tracer enrichment, not of TTR. The needed mathematical equations are derived. A theoretical error analysis is carried out to calculate the magnitude of error in published results using TTR modeling. It is shown that TTR modeling leads to a consistent underestimation of the fractional synthetic rate. In constant-infusion studies, the bias error percent is shown to equal approximately the plateau enrichment, generally <10%. It is shown that, in bolus studies, the underestimation error can be larger. Thus, for mass isotope studies with endogenous tracers, apolipoproteins are in a steady state and the data should be fitted by modeling enrichments.
PMCID: PMC3276318  PMID: 16951401
amino acid metabolism; fractional synthetic rate; mass isotopes; tracer kinetics; tracer-to-tracee ratio; precursor-product relationship
7.  Nonuniform radiolabeling of VLDL apolipoprotein B: implications for the analysis of studies of the kinetics of the metabolism of lipoproteins containing apolipoprotein B 
Journal of Lipid Research  1990;31(6):1031-1042.
Radiolabeling of whole lipoproteins or individual apolipoproteins has been an essential tool for the determination of the kinetics of apolipoprotein metabolism in vivo. Mathematical analysis of specific radioactivity (SA) or total radioactivity data has demonstrated the existence of significant complexity in the plasma decay curves of several apolipoproteins. Results obtained during development of methods to study the metabolism of apolipoprotein B (apoB) in very low density lipoprotein (VLDL) subclasses isolated according to flotation (Sf) rates from whole radiolabeled (d < 1.006 g/ml) VLDL suggested nonuniform radiolabeling of apoB in the three Sf subclasses being studied. We therefore determined apoB SA in VLDL Sf subclasses in ten hypertriglyceridemic and five normal subjects. After radioiodination of apoB in whole VLDL, different apoB SA were found in Sf 400–100, Sf 100–60, and Sf 60–20. The pattern of labeling was quite variable among subjects. On average, apoB SA in the VLDL tracer was greatest in Sf 400–100, and least in Sf 60–20. Nonuniform labeling could also be demonstrated in five studies in which samples were obtained 3 min after intravenous injection of the tracer into subjects with a wide range of plasma triglycerides. Nonuniform labeling of apoB in whole VLDL was also demonstrated in two of the subjects by isolating subclasses of their VLDL that did and did not bind to an anti-apolipoprotein E immunoaffinity column. These results indicate that the usual assumption of homogeneous labeling of apoB may be erroneous. We have derived a simple mathematical formula to study the consequences of this assumption in estimating kinetic parameters. It is shown that an erroneous assumption of homogeneous tracer labeling may significantly underestimate or overestimate the true production rate, even in a simple two-pool model. Identification of labeling characteristics and incorporation of this information into the mathematical analysis of the plasma radioactivity data can improve the accuracy of the analysis as well as the sensitivity of compartmental models generated by such data.
PMCID: PMC3275143  PMID: 2373953
lipoproteins; kinetic analysis; compartmental models
8.  Metabolism of apoB lipoproteins of intestinal and hepatic origin during constant feeding of small amounts of fat 
Journal of lipid research  2006;47(8):1771-1779.
We aimed to identify mechanisms by which apolipoprotein B-48 (apoB-48) could have an atherogenic role by simultaneously studying the metabolism of postprandial apoB-48 and apoB-100 lipoproteins. The kinetics of apoB-48 and apoB-100, each in four density subfractions of VLDL and intermediate density lipoprotein (IDL), were studied by stable isotope labeling in a constantly fed state with half-hourly administration of almond oil in five postmenopausal women. A non-steady-state, multicompartmental model was used. Despite a much lower production rate, VLDL and IDL apoB-48 shared a similar secretion pattern with apoB-100: both were directly secreted into all fractions with similar percentage mass distributions. Fractional catabolic rates (FCRs) of apoB-48 and apoB-100 were similar in VLDL and IDL. We identified a fast turnover compartment of light VLDL that had a residence time of <30 min for apoB-48 and apoB-100. Finally, a high secretion rate of apoB-48 was associated with a slow FCR of VLDL and IDL apoB-100. In conclusion, the intestine secretes a spectrum of apoB lipoproteins, similar to what the liver secretes, albeit with a much lower secretion rate. Once in plasma, intestinal and hepatic triglyceride-rich lipoproteins have similar rates of clearance and participate interactively in similar metabolic pathways, with high apoB-48 production inhibiting the clearance of apoB-100.
PMCID: PMC3219222  PMID: 16685082
kinetics; stable isotopes; triglyceride-rich lipoproteins; apolipoprotein B-48; apolipoprotein B-100
9.  Agpat6—a Novel Lipid Biosynthetic Gene Required for Triacylglycerol Production in Mammary Epithelium 
Journal of lipid research  2006;47(4):734-744.
In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, Agpat6, with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6’s closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6−/−) mice. Agpat6−/− mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6−/− mice was a defect in lactation. Pups nursed by Agpat6−/− mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6−/− lactating mammary glands were underdeveloped, and there was a dramatic decrease in size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6−/− mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the endoplasmic reticulum, AGPAT6, which is crucial for the production of milk fat by the mammary gland.
PMCID: PMC3196597  PMID: 16449762
LPAAT; acyltransferase; transacylase; milk fat
10.  Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome 
Journal of lipid research  2007;49(2):410-419.
Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). FALDH is an enzyme component of fatty alcohol:NAD oxidoreductase (FAO), which is necessary for fatty alcohol metabolism. To better understand the biochemical basis for the cutaneous symptoms in this disease, we investigated lipid metabolism in cultured keratinocytes from SLS patients. Enzyme activities of FALDH and FAO in SLS cells were <10% of normal. SLS keratinocytes accumulated 45-fold more fatty alcohol (hexadecanol, octadecanol, and octadecenol) than normal, whereas wax esters and 1-O-alkyl-2,3-diacylglycerols were increased by 5.6-fold and 7.5-fold, respectively. SLS keratinocytes showed a reduced incorporation of radioactive octadecanol into fatty acid (24% of normal) and triglyceride (13% of normal), but incorporation into wax esters and 1-O-alkyl-2,3-diacylglycerol was increased by 2.5-fold and 2.8-fold, respectively. Our results indicate that FALDH deficiency in SLS keratinocytes causes the accumulation and diversion of fatty alcohol into alternative biosynthetic pathways. The striking lipid abnormalities in cultured SLS keratinocytes are distinct from those seen in fibroblasts and may be related to the stratum corneum dysfunction and ichthyosis in SLS.
PMCID: PMC3057183  PMID: 17971613
aldehyde dehydrogenase; alkylglycerol; epidermis; ether glycerolipid; fatty aldehyde; ichthyosis; mental retardation; plasmalogen; stratum corneum; wax ester
11.  Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation 
Journal of lipid research  2007;48(11):2443-2452.
Oxidative stress and inflammation are fundamental for the onset of aging and appear to be causatively linked. Previously, we reported that hepatocytes from aged rats, compared with young rats, are hyperresponsive to interleukin-1β (IL-1β) stimulation and exhibit more potent c-Jun N-terminal kinase (JNK) activation and attenuated interleukin-1 receptor-associated kinase-1 (IRAK-1) degradation. An age-related increase in the activity of neutral sphingomyelinase-2 (NSMase-2), a plasma membrane enzyme, was found to be responsible for the IL-1β hyperresponsiveness. The results reported here show that increased NSMase activity during aging is caused by a 60–70% decrease in hepatocyte GSH levels. GSH, at concentrations typically found in hepatocytes from young animals, inhibits NSMase activity in a biphasic dose-dependent manner. Inhibition of GSH synthesis in young hepatocytes activates NSMase, causing increased JNK activation and IRAK-1 stabilization in response to IL-1β, mimicking the hyperresponsiveness typical for aged hepatocytes. Vice versa, increased GSH content in hepatocytes from aged animals by treatment with N-acetylcysteine inhibits NSMase activity and restores normal IL-1β response. Importantly, the GSH decline, NSMase activation, and IL-1β hyperresponsiveness are not observed in aged, calorie-restricted rats. In summary, this report demonstrates that depletion of cellular GSH during aging plays an important role in regulating the hepatic response to IL-1β by inducing NSMase-2 activity.— Rutkute, K., R. H. Asmis, and M. N. Nikolova-Karakashian. Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation.
PMCID: PMC3010975  PMID: 17693623
ceramide; calorie restriction; interleukin-1 receptor-associatedkinase-1; c-JunN-terminalkinase; reduced glutathione
12.  Agpat6 deficiency causes subdermal lipodystrophy and resistance to obesityS 
Journal of lipid research  2006;47(4):745-754.
Triglyceride synthesis in most mammalian tissues involves the sequential addition of fatty acids to a glycerol backbone, with unique enzymes required to catalyze each acylation step. Acylation at the sn-2 position requires 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) activity. To date, seven Agpat genes have been identified based on activity and/or sequence similarity, but their physiological functions have not been well established. We have generated a mouse model deficient in AGPAT6, which is normally expressed at high levels in brown adipose tissue (BAT), white adipose tissue (WAT), and liver. Agpat6-deficient mice exhibit a 25% reduction in body weight and resistance to both diet-induced and genetically induced obesity. The reduced body weight is associated with increased energy expenditure, reduced triglyceride accumulation in BAT and WAT, reduced white adipocyte size, and lack of adipose tissue in the subdermal region. In addition, the fatty acid composition of triacylglycerol, diacylglycerol, and phospholipid is altered, with proportionally greater polyunsaturated fatty acids at the expense of monounsaturated fatty acids. Thus, Agpat6 plays a unique role in determining triglyceride content and composition in adipose tissue and liver that cannot be compensated by other members of the Agpat family.
PMCID: PMC2901549  PMID: 16436371
acyltransferase; gene-trap; adipose tissue; energy expenditure; 1-acylglycerol-3-phosphate O-acyltransferase
13.  Cellular palmitoylation and trafficking of lipidated peptides 
Journal of lipid research  2007;48(8):1873-1884.
Many important signaling proteins require the posttranslational addition of fatty acid chains for their proper subcellular localization and function. One such modification is the addition of palmitoyl moieties by enzymes known as palmitoyl acyltransferases (PATs). Substrates for PATs include C-terminally farnesylated proteins, such as H- and N-Ras, as well as N-terminally myristoylated proteins, such as many Src-related tyrosine kinases. The molecular and biochemical characterization of PATs has been hindered by difficulties in developing effective methods for the analysis of PAT activity. In this study, we describe the use of cell-permeable, fluorescently labeled lipidated peptides that mimic the PAT recognition domains of farnesylated and myristoylated proteins. These PAT substrate mimetics are accumulated by SKOV3 cells in a saturable and time-dependent manner. Although both peptides are rapidly palmitoylated, the SKOV3 cells have a greater capacity to palmitoylate the myristoylated peptide than the farnesylated peptide. Confocal microscopy indicated that the palmitoylated peptides colocalized with Golgi and plasma membrane markers, whereas the corresponding nonpalmitoylatable peptides accumulated in the Golgi but did not traffic to the plasma membrane. Overall, these studies indicate that the lipidated peptides provide useful cellular probes for quantitative and compartmentalization studies of protein palmitoylation in intact cells.
PMCID: PMC2895159  PMID: 17525474
palmitoyl acyltransferase; Ras; lipidation; subcellular trafficking
14.  Formation of 7-dehydrocholesterol-containing membrane rafts in vitro and in vivo, with relevance to the Smith-Lemli-Opitz syndrome 
Journal of lipid research  2003;45(2):347-355.
Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease typified by 7-dehydrocholesterol (7DHC) accumulation and depletion of cholesterol. Because cholesterol is a primary component of detergent-resistant membrane domains (“rafts”), we examined the compatibility of 7DHC with raft formation. Liposomes containing bovine brain phosphatidylcholine, sphingomyelin, cerebrosides, and either cholesterol, 7DHC, or coprostanol (the latter being incompatible with raft formation) were prepared. 7DHC was indistinguishable from cholesterol in its ability to become incorporated into membrane rafts, as judged by physical and chemical criteria, whereas coprostanol did not form rafts. The in vivo compatibility of 7DHC with raft formation was evaluated in brains of rats treated with trans-1,4-bis(2-dichlorobenzylamino-ethyl)cyclohexane dihydrochloride (AY9944), which mimics the SLOS biochemical defect. 7DHC/cholesterol ratios in rafts and whole brains from AY9944-treated rats were similar, indicating comparable efficiency of 7DHC and cholesterol incorporation into brain rafts. In contrast, dolichol (a nonsterol isoprenoid incompatible with raft formation) was greatly depleted in brain rafts relative to whole brain. Although brain raft fractions prepared from AY9944-treated and control rats yielded similar sterol-protein ratios, their gel electrophoresis profiles exhibited multiple differences, suggesting that altered raft sterol composition perturbs raft protein content. These results are discussed in the context of the SLOS phenotype, particularly with regard to the associated central nervous system defects.
PMCID: PMC2851617  PMID: 14594996
lipid domains; proteomics; brain; rat
15.  Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay 
Journal of lipid research  2007;48(6):1422-1427.
An assay was set up for glyceryl ether monooxygenase activity in tissue samples using the novel substrate 1-O- pyrenedecyl-sn glycerol and high performance liquid chromatographic analysis of reaction mixtures with fluorescence detection, allowing robust detection of enzymatic activity in microgram amounts of tissue homogenates. The activity partially purified from rat liver strictly depended on presence of a tetrahydropteridine. Tetrahydrobiopterin-dependent glyceryl ether monooxygenase activity was observed in all rat tissues tested except female heart, with highest activities in liver, intestine and cerebellum. Activity was not uniformly distributed in brain, but was higher in cerebellum than in striatum or cortex. These data demonstrate that tetrahydrobiopterin-dependent glyceryl ether monooxygenase is not only found in liver and the gastrointestinal tract, but also in brain and other organs of the rat and provides an additional goal for tetrahydrobiopterin biosynthesis in these organs.
PMCID: PMC2851153  PMID: 17303893
tetrahydrobiopterin; pteridine; ether lipid; alkylglycerol; chemical synthesis; activity assay
16.  Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism 
Journal of lipid research  2008;49(4):697-714.
Many of the ichthyoses are associated with inherited disorders of lipid metabolism. These disorders have provided unique models to dissect physiologic processes in normal epidermis and the pathophysiology of more common scaling conditions. In most of these disorders, a permeability barrier abnormality “drives” pathophysiology through stimulation of epidermal hyperplasia. Among primary abnormalities of nonpolar lipid metabolism, triglyceride accumulation in neutral lipid storage disease as a result of a lipase mutation provokes a barrier abnormality via lamellar/nonlamellar phase separation within the extracellular matrix of the stratum corneum (SC). Similar mechanisms account for the barrier abnormalities (and subsequent ichthyosis) in inherited disorders of polar lipid metabolism. For example, in recessive X-linked ichthyosis (RXLI), cholesterol sulfate (CSO4) accumulation also produces a permeability barrier defect through lamellar/nonlamellar phase separation. However, in RXLI, the desquamation abnormality is in part attributable to the plurifunctional roles of CSO4 as a regulator of both epidermal differentiation and corneodesmosome degradation. Phase separation also occurs in type II Gaucher disease (GD; from accumulation of glucosylceramides as a result of to β-glucocerebrosidase deficiency). Finally, failure to assemble both lipids and desquamatory enzymes into nascent epidermal lamellar bodies (LBs) accounts for both the permeability barrier and desquamation abnormalities in Harlequin ichthyosis (HI). The barrier abnormality provokes the clinical phenotype in these disorders not only by stimulating epidermal proliferation, but also by inducing inflammation.
PMCID: PMC2844331  PMID: 18245815
ATP binding cassette transporter 12; arachidonate lipoxygenase; barrier function; epidermal lipids; harlequin ichthyosis; neutral lipid storage disease; recessive X-linked ichthyosis; stratum corneum; transepidermal water loss
17.  Polyunsaturated phospholipids promote the oxidation and fragmentation of γ-hydroxyalkenals: formation and reactions of oxidatively truncated ether phospholipidsS 
Journal of lipid research  2007;49(4):832-846.
Low density lipoprotein contains traces of biologically active platelet-activating factor (PAF)-like ether phosphatidylcholines (PCs). These oxidatively truncated alkylacylphosphatidylcholines (OxPAFs) are presumably formed through the oxidative truncation of 1-alkyl-2-polyunsaturated fatty acyl PCs. We now report that a diverse structural variety of OxPAFs are generated in small unilamellar vesicles (SUVs) uponmyeloperoxidase (MPO)-promoted autoxidation of ether PCs that incorporate linoleoyl, arachidonyl, or docosahexaenoyl groups at the sn-2 position. Total syntheses are reported that confirm the identities of the new OxPAFs and will facilitate the evaluation of their biologically important chemistry and activities. Especially noteworthy is the formation of OxPAFs containing γ-hydroxyalkenal functionality. Analogous oxidatively truncated diacylphosphatidylcholines are biologically important because they and their more oxidized derivatives are strong ligands for the scavenger receptor CD36. Furthermore, their covalent adduction with proteins can interfere with protein function or generate biologically active carboxyalkylpyrrole derivatives. We now find a profound influence of membrane composition on the stability of OxPAFs. In the presence of a polyunsaturated diacyl PC, the linoleic acid ester of 2-lysophosphatidylcholine, MPO induces the oxidation of aldehydes to carboxylic acids and the further oxidative truncation of γ-hydroxyalkenals. Remarkably, these reactions do not occur readily with MPO in SUVs composed entirely of saturated diacyl-PCs. A mechanistic rationale is presented that can account for this dichotomy.
PMCID: PMC2836539  PMID: 18165704
myeloperoxidase; platelet-activating factor; liquid chromatography-tandem mass spectrometry; low density lipoprotein
18.  Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6−/− mice 
Journal of lipid research  2008;49(4):823.
Elucidation of the metabolic pathways of triacylglycerol (TAG) synthesis is critical to the understanding of chronic metabolic disorders such as obesity, cardiovascular disease, and diabetes. sn-Glycerol-3-phosphate acyltransferase (GPAT) and sn-1-acylglycerol-3-phosphate acyltransferase (AGPAT) catalyze the first and second steps in de novo TAG synthesis. AGPAT6 is one of eight AGPAT isoforms identified through sequence homology, but the enzyme activity for AGPAT6 has not been confirmed. We found that in liver and brown adipose tissue from Agpat6-deficient (Agpat6−/−) mice, N-ethylmaleimide (NEM)-sensitive GPAT specific activity was 65% lower than in tissues from wild-type mice, but AGPAT specific activity was similar. Overexpression of Agpat6 in Cos-7 cells increased an NEM-sensitive GPAT specific activity, but AGPAT specific activity was not increased. Agpat6 and Gpat1 overexpression in Cos-7 cells increased the incorporation of [14C]oleate into diacylglycerol (DAG) or into DAG and TAG, respectively, suggesting that the lysophosphatidic acid, phosphatidic acid, and DAG intermediates initiated by each of these isoforms lie in different cellular pools. Together, these data show that “Agpat6−/− mice” are actually deficient in a novel NEM-sensitive GPAT, GPAT4, and indicate that the alterations in lipid metabolism in adipose tissue, liver, and mammary epithelium of these mice are attributable to the absence of GPAT4
PMCID: PMC2819352  PMID: 18192653
triacylglycerol; phospholipid; lipodystrophy; acyl-coenzyme A; steatosis; sn-l-acylglycerol-3-phosphate O-acyltransferase-deficient mice
19.  Genetic determinants of obesity-related lipid traits 
Journal of lipid research  2004;45(4):610-615.
In our ongoing effort to identify genes influencing the biological pathways that underlie the metabolic disturbances associated with obesity, we performed genome-wide scanning in 2,209 individuals distributed over 507 Caucasian families to localize quantitative trait loci (QTLs), which affect variation of plasma lipids. Pedigree-based analysis using a quantitative trait variance component linkage method that localized a QTL on chromosome 7q35-q36, which linked to variation in levels of plasma triglyceride [TG, logarithm of odds (LOD) score = 3.7] and was suggestive of linkage to LDL-cholesterol (LDL-C, LOD = 2.2). Covariates of the TG linkage included waist circumference, fasting insulin, and insulin:glucose, but not body mass index or hip circumference. Plasma HDL-cholesterol (HDL-C) levels were suggestively linked to a second QTL on chromosome 12p12.3 (LOD = 2.6). Five other QTLs with lower LOD scores were identified for plasma levels of LDL-C, HDL-C, and total cholesterol. These newly identified loci likely harbor genetic elements that influence traits underlying lipid adversities associated with obesity.
PMCID: PMC2771634  PMID: 14754912
linkage analysis; triglycerides; obesity; lipid profiles; high density lipoprotein cholesterol
20.  Acyl-coenzyme A 
Journal of lipid research  2005;46(9):1933-1943.
7-Ketocholesterol (7KC) is a cytotoxic component of oxidized low density lipoproteins (OxLDLs) and induces apoptosis in macrophages by a mechanism involving the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we examined the role of ACAT in 7KC-induced and OxLDL-induced apoptosis in murine macrophages. An ACAT inhibitor, Sandoz 58-035, suppressed 7KC-induced apoptosis in P388D1 cells and both 7KC-induced and OxLDL-induced apoptosis in mouse peritoneal macrophages (MPMs). Furthermore, compared with wild-type MPMs, ACAT-1-deficient MPMs demonstrated significant resistance to both 7KC-induced and OxLDL-induced apoptosis. Macrophages treated with 7KC accumulated ACAT-derived [14C]cholesteryl and [3H]7-ketocholesteryl esters. Tandem LC-MS revealed that the 7KC esters contained primarily saturated and monounsaturated fatty acids. An inhibitor of cPLA2, arachidonyl trifluoromethyl ketone, prevented the accumulation of 7KC esters and inhibited 7KC-induced apoptosis in P388D1 cells. The decrease in 7KC ester accumulation produced by the inhibition of cPLA2 was reversed by supplementing with either oleic or arachidonic acid (AA); however, only AA supplementation restored the induction of apoptosis by 7KC. These results suggest that 7KC not only initiates the apoptosis pathway by activating cPLA2, as we have reported previously, but also participates in the downstream signaling pathway when esterified by ACAT to form 7KC-arachidonate.
PMCID: PMC2768430  PMID: 15995174
7-ketocholesterol; oxysterol esterification; low density lipoproteins
21.  Lipoprotein lipase and hepatic lipase 
Journal of lipid research  2003;44(8):1552-1558.
HDL subspecies Lp(A-I) and Lp(A-I,A-II) have different anti-atherogenic potentials. To determine the role of lipoprotein lipase (LPL) and hepatic lipase (HL) in regulating these particles, we measured these enzyme activities in 28 healthy subjects with well-controlled Type 1 diabetes, and studied their relationship with Lp(A-I) and Lp(A-I,A-II). LPL was positively correlated with the apolipoprotein A-I (apoA-I), cholesterol, and phospholipid mass in total Lp(A-I), and with the apoA-I in large Lp(A-I) (r ≥ 0.58, P ≥ 0.001). HL was negatively correlated with all the above Lp(A-I) parameters plus Lp(A-I) triglyceride (r ≥ -0.53, P ≤ 0.003). No correlation was detected between LPL and Lp(A-I,A-II). However, HL was inversely correlated with total Lp(A-I,A-II) phospholipid, and with large Lp(A-I,A-II) (r ≥ 0.50, P ≤ 0.006). Similar studies were performed with phospholipid transfer protein (PLTP). Only total Lp(A-I) triglyceride in women (not men) (r = 0.71, P = 0.009) was significantly correlated with PLTP activity. These observations indicate that LPL and HL play major roles in determining the level and composition of plasma Lp(A-I), particularly large Lp(A-I), but not with Lp(A-I,A-II) level. Furthermore, select correlations of LPL and/or HL with the apoA-I, cholesterol, and triglyceride of Lp(A-I) but not Lp(A-I,A-II) imply that the apoA-I and lipid of Lp(A-I) and Lp(A-I,A-II) are not fully equilibrated.
PMCID: PMC2768368  PMID: 12777470
phospholipid transfer protein; high density lipoprotein size profile; Type 1 diabetes
22.  Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity 
Journal of lipid research  2006;47(9):2028-2041.
Fatty acid elongases and desaturases play an important role in hepatic and whole body lipid composition. We examined the role that key transcription factors played in the control of hepatic elongase and desaturase expression. Studies with peroxisome proliferator-activated receptor α (PPARα)-deficient mice establish that PPARα was required for WY14643-mediated induction of fatty acid elongase-5 (Elovl-5), Elovl-6, and all three desaturases [Δ5 desaturase (Δ5D), Δ6D, and Δ9D]. Increased nuclear sterol-regulatory element binding protein-1 (SREBP-1) correlated with enhanced expression of Elovl-6, Δ5D, Δ6D, and Δ9D. Only Δ9D was also regulated independently by liver X receptor (LXR) agonist. Glucose induction of L-type pyruvate kinase, Δ9D, and Elovl-6 expression required the carbohydrate-regulatory element binding protein/MAX-like factor X (ChREBP/MLX) heterodimer. Suppression of Elovl-6 and Δ9D expression in livers of streptozotocin-induced diabetic rats and high fat-fed glucose-intolerant mice correlated with low levels of nuclear SREBP-1. In leptin-deficient obese mice (Lepob/ob), increased SREBP-1 and MLX nuclear content correlated with the induction of Elovl-5, Elovl-6, and Δ9D expression and the massive accumulation of monoun-saturated fatty acids (18:1,n-7 and 18:1,n-9) in neutral lipids. Diabetes- and obesity-induced changes in hepatic lipid composition correlated with changes in elongase and desaturase expression. In conclusion, these studies establish a role for PPARα, LXR, SREBP-1, ChREBP, and MLX in the control of hepatic fatty acid elongase and desaturase expression and lipid composition.
PMCID: PMC2764365  PMID: 16790840
peroxisome proliferator-activated receptor α; sterol-regulatory element binding protein-1; carbohydrate-regulatory element binding protein; MAX-like factor X; liver X receptor
23.  Docosahexaneoic acid (22:6,n-3) regulates rat hepatocyte SREBP-1 nuclear abundance by Erk- and 26S proteasome-dependent pathways 
Journal of lipid research  2005;47(1):181-192.
Insulin induces and dietary n-3 PUFAs suppress hepatic de novo lipogenesis by controlling sterol-regulatory element binding protein-1 nuclear abundance (nSREBP-1). Our goal was to define the mechanisms involved in this regulatory process. Insulin treatment of rat primary hepatocytes rapidly augments nSREBP-1 and mRNASREBP-1c while suppressing mRNAInsig-2 but not mRNAInsig-1. These events are preceded by rapid but transient increases in Akt and Erk phosphorylation. Removal of insulin from hepatocytes leads to a rapid decline in nSREBP-1 [half-time (T1/2) ~ 10 h] that is abrogated by inhibitors of 26S proteasomal degradation. 22:6,n-3, the major n-3 PUFA accumulating in livers of fish oil-fed rats, suppresses hepatocyte levels of nSREBP-1, mRNASREBP-1c, and mRNAInsig-2 but modestly and transiently induces mRNAInsig-1. More importantly, 22:6,n-3 accelerates the disappearance of hepatocyte nSREBP-1 (T1/2 ~ 4 h) through a 26S proteasome-dependent process. 22:6,n-3 has minimal effects on microsomal SREBP-1 and sterol-regulatory element binding protein cleavage-activating protein or nuclear SREBP-2. 22:6,n-3 transiently inhibits insulin-induced Akt phosphorylation but induces Erk phosphorylation. Inhibitors of Erk phosphorylation, but not overexpressed constitutively active Akt, rapidly attenuate 22:6,n-3 suppression of nSREBP-1. Thus, 22:6,n-3 suppresses hepatocyte nSREBP-1 through 26S proteasome- and Erk-dependent pathways. These studies reveal a novel mechanism for n-3 PUFA regulation of hepatocyte nSREBP-1 and lipid metabolism.—Botolin, D., Y. Wang, B. Christian, and D. B. Jump. Docosahexaneoic acid (22:6,n-3) regulates rat hepatocyte SREBP-1 nuclear abundance by Erk- and 26S proteasome-dependent pathways.
PMCID: PMC2764363  PMID: 16222032
sterol regulatory element binding protein-1; Insig-1; Insig-2
24.  Lipoprotein lipase- and hepatic triglyceride lipase-promoted very low density lipoprotein degradation proceeds via an apolipoprotein E-dependent mechanism 
Journal of lipid research  2000;41(11):1858-1871.
Apolipoprotein E (apoE) is the primary recognition signal on triglyceride-rich lipoproteins responsible for interacting with low density lipoprotein (LDL) receptors and LDL receptor-related protein (LRP). It has been shown that lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) promote receptor-mediated uptake and degradation of very low density lipoproteins (VLDL) and remnant particles, possibly by directly binding to lipoprotein receptors. In this study we have investigated the requirement for apoE in lipase-stimulated VLDL degradation. We compared binding and degradation of normal and apoE-depleted human VLDL and apoE knockout mouse VLDL in human foreskin fibroblasts. Surface binding at 37°C of apoE knockout VLDL was greater than that of normal VLDL by 3-and 40-fold, respectively, in the presence of LPL and HTGL. In spite of the greater stimulation of surface binding, lipase-stimulated degradation of apoE knockout mouse VLDL was significantly lower than that of normal VLDL (30, 30, and 80%, respectively, for control, LPL, and HTGL treatments). In the presence of LPL and HTGL, surface binding of apoE-depleted human VLDL was, respectively, 40 and 200% of normal VLDL whereas degradation was, respectively, 25 and 50% of normal VLDL. LPL and HTGL stimulated degradation of normal VLDL in a dose-dependent manner and by a LDL receptor-mediated pathway. Maximum stimulation (4-fold) was seen in the presence LPL (1 µg/ml) or HTGL (3 µg/ml) in lovastatin-treated cells. On the other hand, degradation of apoE-depleted VLDL was not significantly increased by the presence of lipases even in lovastatin-treated cells. Surface binding of apoE-depleted VLDL to metabolically inactive cells at 4°C was higher in control and HTGL-treated cells, but unchanged in the presence of LPL. Degradation of prebound apoE-depleted VLDL was only 35% as efficient as that of normal VLDL. Surface binding of apoE knockout or apoE-depleted VLDL was to heparin sulfate proteoglycans because it was completely abolished by heparinase treatment. However, apoE appears to be a primary determinant for receptor-mediated VLDL degradation.
PMCID: PMC2759277  PMID: 11060356
heparan sulfate proteoglycans; LDL receptors; fibroblasts; apoE knockout mice
25.  The L-4F mimetic peptide prevents insulin resistance through increased levels of HO-1, pAMPK, and pAKT in obese mice*1 
Journal of Lipid Research  2009;50(7):1293-1304.
We examined mechanisms by which L-4F reduces obesity and diabetes in obese (ob) diabetic mice. We hypothesized that L-4F reduces adiposity via increased pAMPK, pAKT, HO-1, and increased insulin receptor phosphorylation in ob mice. Obese and lean mice were divided into five groups: lean, lean-L-4F-treated, ob, ob-L-4F-treated, and ob-L-4F-LY294002. Food intake, insulin, glucose adipocyte stem cells, pAMPK, pAKT, CB1, and insulin receptor phosphorylation were determined. Subcutaneous (SAT) and visceral adipose tissue (VAT) were determined by MRI and hepatic lipid content by magnetic resonance spectroscopy. SAT and VAT volumes decreased in ob-L-4F-treated animals compared with control. L-4F treatment decreased hepatic lipid content and increased the numbers of small adipocytes (P < 0.05) and phosphorylation of insulin receptors. L-4F decreased CB1 in SAT and VAT and increased pAKT and pAMPK in endothelium. L-4F-mediated improvement in endothelium was prevented by LY294002. Inhibition of pAKT and pAMPK by LY294002 was associated with an increase in glucose levels. Upregulation of HO-1 by L-4F produced adipose remodeling and increased the number of small differentiated adipocytes. The anti-obesity effects of L-4F are manifested by a decrease in visceral fat content with reciprocal increases in adiponectin, pAMPK, pAKT, and phosphorylation of insulin receptors with improved insulin sensitivity.
PMCID: PMC2694329  PMID: 19224872
diabetes; adiponectin; adiposity; apolipoprotein A-I; heme oxygenase-1; insulin receptor; insulin sensitivity; obesity; endothelial dysfunction

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