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Mutations in Dyrk1b are associated with metabolic syndrome and nonalcoholic fatty liver disease in humans. Our investigations showed that DYRK1B levels are increased in the liver of patients with nonalcoholic steatohepatitis (NASH) and in mice fed with a high-fat, high-sucrose diet. Increasing Dyrk1b levels in the mouse liver enhanced de novo lipogenesis (DNL), fatty acid uptake, and triacylglycerol secretion and caused NASH and hyperlipidemia. Conversely, knockdown of Dyrk1b was protective against high-calorie-induced hepatic steatosis and fibrosis and hyperlipidemia. Mechanistically, Dyrk1b increased DNL by activating mTORC2 in a kinase-independent fashion. Accordingly, the Dyrk1b-induced NASH was fully rescued when mTORC2 was genetically disrupted. The elevated DNL was associated with increased plasma membrane sn-1,2-diacylglyerol levels and increased PKC[epsilon]-mediated IRK T1150 phosphorylation, which resulted in impaired activation of hepatic insulin signaling and reduced hepatic glycogen storage. These findings provide insights into the mechanisms that underlie Dyrk1binduced hepatic lipogenesis and hepatic insulin resistance and identify Dyrk1b as a therapeutic target for NASH and insulin resistance in the liver., Introduction Nonalcoholic fatty liver disease (NAFLD) is a rapidly growing disorder affecting nearly 25% of the adult population worldwide and is a major risk factor for nonalcoholic steatohepatitis (NASH), atherosclerosis, [...]

The crystal structure of the MraY enzyme from Aquifex aeolicus in complex with the naturally occurring nucleoside inhibitor muraymycin D2 (MD2) reveals that MraY undergoes a large conformational rearrangement near the active site after the binding of MD2, leading to the generation of a nucleoside-binding pocket and a peptide-binding site. Structure of an antibiotic target Peptidoglycan biosynthesis is a well-established target for antibiotics. The first step in this process is catalysed by MraY, for which many naturally occurring inhibitors exist, but the design of new compounds to target this enzyme has been hampered by a lack of structural insight into the mode of inhibition. Here, Seok-Yong Lee and colleagues solve the crystal structure of MraY from the hyperthermophilic bacterium Aquifex aeolicus in complex with the naturally occurring nucleoside inhibitor, muraymycin D2 (MD2). The structure shows that MraY undergoes large conformational rearrangements near the active site following MD2 binding, leading to the generation of a nucleoside-binding pocket and a peptide-binding site. Antibiotic-resistant bacterial infection is a serious threat to public health. Peptidoglycan biosynthesis is a well-established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) catalyses the first and an essential membrane step of peptidoglycan biosynthesis. It is considered a very promising target for the development of new antibiotics, as many naturally occurring nucleoside inhibitors with antibacterial activity target this enzyme.sup.1,2,3,4. However, antibiotics targeting MraY have not been developed for clinical use, mainly owing to a lack of structural insight into inhibition of this enzyme. Here we present the crystal structure of MraY from Aquifex aeolicus (MraY.sub.AA) in complex with its naturally occurring inhibitor, muraymycin D2 (MD2). We show that after binding MD2, MraY.sub.AA undergoes remarkably large conformational rearrangements near the active site, which lead to the formation of a nucleoside-binding pocket and a peptide-binding site. MD2 binds the nucleoside-binding pocket like a two-pronged plug inserting into a socket. Further interactions it makes in the adjacent peptide-binding site anchor MD2 to and enhance its affinity for MraY.sub.AA. Surprisingly, MD2 does not interact with three acidic residues or the Mg.sup.2+ cofactor required for catalysis, suggesting that MD2 binds to MraY.sub.AA in a manner that overlaps with, but is distinct from, its natural substrate, UDP-MurNAc-pentapeptide. We have determined the principles of MD2 binding to MraY.sub.AA, including how it avoids the need for pyrophosphate and sugar moieties, which are essential features for substrate binding. The conformational plasticity of MraY could be the reason that it is the target of many structurally distinct inhibitors. These findings can inform the design of new inhibitors targeting MraY as well as its paralogues, WecA and TarO., Author(s): Ben C. Chung [sup.1] , Ellene H. Mashalidis [sup.1] , Tetsuya Tanino [sup.2] , Mijung Kim [sup.3] , Akira Matsuda [sup.2] , Jiyong Hong [sup.3] , Satoshi Ichikawa [sup.2] [...]

Cellular lipid metabolism has been linked to Immune responses; however, the precise mechanisms by which de novo fatty add synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1-dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2- deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro-IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis., Introduction Sepsis remains the leading cause of death in the medical intensive care unit (MICU) (1). Although inflammatory responses are important for host defense against invading microbes, excessive inflammation in [...]

The obesity epidemic has led to an increased incidence of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP-activated protein kinase (Ampk) regulates energy homeostasis and is activated by [...]

A suite of characteristics is often used to assess egg quality as these properties potentially play important roles in progeny survival and growth. Our objective was to assess egg characteristics including lipid biocomposition of an iteroparous, batch-spawning teleost of wild origin. Maternal allocation to egg number was generally dome-shaped (5 of 8 females) and egg size declined over the breeding season for eight breeding pairs of wild Atlantic cod (Gadus morhua)(n = 43 batches). Egg lipid composition ranged considerably among females and between egg batches within females (e.g., phospholipids 40-86 %; polar 47-87 % and neutral lipids 15-52 % of total lipids; polyunsaturated fatty acids 16-50 % of total fatty acids). Principal component analyses revealed significant interrelationships among maternal traits, batch sequence and fecundity, and egg size and composition. Seasonal trends with regard to lipid deposition were variable; three females showed consistent declines in lipid parameters (µg [egg.sup.-1]) with both batch number and egg diameter, one female showed consistent increase and the four remaining females showed no trend. The three females that exhibited seasonal declines in egg lipid content were characterized as having high fertilization success (>75 %). Our findings highlight the variability in lipid allocation to eggs of batch spawners of wild origin and characterize the composition of endogenous reserves available during embryogenesis and yolk sac larval stages., Introduction In fishes, egg quality is a term coined to describe the overall health of an egg and the impact it has on fertility, embryo survival, hatching success, and subsequent [...]

The failure of pancreatic βcells to adapt to an increasing demand for insulin is the major mechanism by which patients progress from insulin resistance to type 2 diabetes (T2D) and is thought to be related to dysfunctional lipid homeostasis within those cells. In multiple animal models of diabetes, females demonstrate relative protection from βcell failure. We previously found that the hormone 17β-estradiol (E2) in part mediates this benefit. Here, we show that treating male Zucker diabetic fatty (ZDF) rats with E2 suppressed synthesis and accumulation of fatty acids and glycerolipids in islets and protected against βcell failure. The antilipogenic actions of E2 were recapitulated by pharmacological activation of estrogen receptor α (ERα) or ERβin a rat βcell line and in cultured ZDF rat, mouse, and human islets. Pancreas-specific null deletion of ERαin mice [(PERα.sup.-/-]) prevented reduction of lipid synthesis by E2 via a direct action in islets, and [PERα.sup.-/-] mice were predisposed to islet lipid accumulation and βcell dysfunction in response to feeding with a high-fat diet. ER activation inhibited βcell lipid synthesis by suppressing the expression (and activity) of fatty acid synthase via a nonclassical pathway dependent on activated Stat3. Accordingly, pancreas-specific deletion of Stat3 in mice curtailed ER-mediated suppression of lipid synthesis. These data suggest that extranuclear ERs may be promising therapeutic targets to prevent βcell failure in T2D., Introduction Type 2 diabetes (T2D) occurs when pancreatic βcells fail to compensate for the increased insulin demand in the context of obesity-associated insulin resistance. Thus, developing novel therapeutic strategies to [...]

Mass spectrometry imaging of tissue-lipid transfers without MALDI matrix is demonstrated. Commercially available nanostructured surfaces (nano-assisted laser desorption-ionization or NALDI) are used as substrates for imprinting of tissue sections. The lithographic transfers are then washed and the two-dimensional distribution of the lipids is imaged by laser desorption-ionization mass spectrometry. The NALDI imaging of lipid transfers is compared with standard MALDI imaging of matrix-coated tissue sections. The obtained images are of the same quality, and no spatial information is lost due to the imprinting process. NALDI imaging is faster due to the absence of the time-consuming matrix deposition step, and the NALDI mass spectra are less complex and easier to interpret than standard MALDI. In this particular application example, NALDI mass spectrometry is able to identify the same lipid species as MALDI mass spectrometry and provides better distinction between kidney and adrenal gland tissues based on the lipid analysis. 10.1021/ac100661h

Evolutionary loss of traits can result from negative selection on a specific phenotype, or if the trait is selectively neutral, because the phenotype associated with the trait has become redundant. Even essential traits may be lost, however, if the resulting phenotypic deficiencies can be compensated for by the environment or a symbiotic partner. Here we demonstrate that loss of an essential metabolic trait in parasitic wasps has evolved through environmental compensation. We tested 24 species for the ability to synthesize lipids de novo and collected additional data from the literature. We found the majority of adult parasitoid species to be incapable of synthesizing lipids, and phylogenetic analyses showed that the evolution of lack of lipogenesis is concurrent with that of parasitism in insects. Exploitive host manipulation, in which the host is forced to synthesize lipids to the benefit of the parasitoid, presumably facilitates loss of lipogenesis through environmental compensation. Lipogenesis re-evolved in a small number of parasitoid species, particularly host generalists. The wide range of host species in which generalists are able to develop may impede effective host manipulation and could have resulted in regaining of lipogenic ability in generalist parasitoids. As trait loss through environmental compensation is unnoticed at the phenotypic level, it may be more common than currently anticipated, especially in species involved in intricate symbiotic relationships with other species. coevolution | environmental compensation | fat reserves | host manipulation | parasitoids doi/10.1073/pnas.1001744107

Higher concentrations of circulating lipids (cholesterol and triglycerides) and their decreased catabolism pose a major risk in the development of atherosclerosis and coronary heart disease (CHD). Although statins are widely used for treatment of hyperlipidemia, side effects associated with their use have prompted the search for a safer alternative for treating hyperlipidemia. The present study investigated the effect of water-soluble compounds in Abana (WSCA), a polyherbal drug formulation traditionally used in India for the treatment of hyperlipidemia, on lipid metabolism in Hep[G.sub.2] cells. WSCA reduced cholesterol and triglyceride content in the cells and their supernatant. WSCA inhibited the incorporation of [2-[sup.14]C]acetate into cellular cholesterol and fatty acids, suggesting the inhibition of lipid synthesis. In addition, WSCA inhibited HMG-CoA reductase, a key metabolic enzyme involved in the biosynthesis of cholesterol. WSCA also increased cholesterol and fatty acid secretion into the cell supernatant, suggesting the enhanced removal of cholesterol and fatty acids. Furthermore, WSCA showed decreased linoleic acid (18:2) and arachidonic acid (20:4) content in Hep[G.sub.2] cells. The present study is the first to show that WSCA simultaneously inhibited cellular cholesterol biosynthesis and increased cholesterol secretion into the cell supernatant in Hep[G.sub.2] cells. Key words: Abana, cholesterol, triglyceride, fatty acid, HMG-CoA, cholesterol efflux. L'augmentation des concentrations de lipides circulants (cholesterol et triglycerides) et la diminution de leur catabolisme representent un risque majeur de deeveloppement d'atheerosclerose et de maladie coronarienne. Les statines sont couramment utilisees dans le traitement de l'hyperlipidemie, mais leur effet deletere sur la sante a encourage; la recherche d'un composant de substitution d'origine vegeetale stir pour traiter cette affection. La presente etude a eu pour objectif d'examiner l'effet de composes hydrosolubles d'abana (CHSA), une preparation a base de plantes meedicinales, traditionnellement utiliseee en Inde dans le traitement de l'hyperlipidemie, sur le metabolisme des cellules Hep[G.sub.2]. Les resultats font etat de l'effet des CHSA sur la reduction de la teneur en cholesterol et en triglycerides dans les cellules et leur surnageant. Les CHSA ont inhibe l'incorporation de [2-[sup.14]C]acetate dans le cholesterol et les acides gras cellulaires, donnant a penser a une inhibition de la synthase des lipides. Ils ont aussi inhibe la HMG-CoA reductase, une enzyme meetabolique cle impliquee dans la biosynthese du cholesterol. Ils ont de plus augmente la secretion du cholesterol et des acides gras dans le surnageant cellulaire, laissant croire a une elimination accrue du cholesterol et des acides gras. Enfin, ils ont montre une diminution de la teneur en acide linoleeique (18:2) et en acide arachidonique (20:4) dans les cellules Hep[G.sub.2]. Cette etude est la premiere du genre a montrer que les CHSA a la fois inhibent la biosynthese du cholesterol cellulaire et augmentent la secretion du cholesterol dans le surnageant cellulaire dans les cellules Hep[G.sub.2]. Mots-cles: Abana, cholesterol, triglyceride, acide gras, HMG-CoA, cholesterol, efflux. [Traduit par la Redaction], Introduction Plants have been used by humans for the treatment of various ailments since time immemorial, and screening of herbal drugs continues to offer opportunities for new drug discovery. The [...]

Mailhot G, Ravid Z, Barchi S, Moreau A, Rabasa-Lhoret R, Levy E. CFTR knockdown stimulates lipid synthesis and transport in intestinal Caco-2/15 cells. Am J Physiol Gastrointest Liver Physiol 297: G1239-G1249, 2009. First published October 1, 2009; doi: 10.1152/ajpgi.00206.2009.--Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel highly expressed in epithelial ceils of the gastrointestinal tract. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease characterized by pancreatic insufficiency, fat malabsorption, and steatorrhea. Despite the administration of pancreatic enzymes to normalize malabsorption, CF patients still experienced lipid fecal loss, nutritional deficiencies, and abnormalities in serum lipid profile, suggesting the presence of intrinsic defects in the intestinal handling of nutrients. The objective of the present study was to assess the impact of CFTR gene knockdown on intracellular lipid metabolism of the intestinal Caco-2/15 cell line. Partial CFTR gene inactivation led to cellular lipid accretion of phospholipids, triglycerides, and cholesteryl esters. Likewise, secretion of these lipid fractions was significantly increased following CFTR gene manipulation. As expected from these findings, the output of triglyceride-rich lipoproteins showed the same increasing pattern. Investigation of the mechanisms underlying these changes revealed that CFTR knockdown resulted in raised levels of apolipoproteins in cells and media and microsomal transfer protein activity, two important factors for the efficient assembly and secretion of lipoproteins. Similarly, scrutiny of the enzymatic monoacylglycerol acyltransferase and diacylglycerol acyltransferase, which exhibit dynamic function in triacylglycerol resynthesis and chylomicron formation in enterocytes, revealed a significant augmentation in their activity. Conversely, cholesterol uptake mediated by Niemann-Pick C1 like 1, Scavenger Receptor Class B Type I, and ATP-binding cassette G8 remains unaffected by genetic modification of CFTR. Collectively, these results highlight the role played by CFTR in intestinal handling of lipids and may suggest that factors other than defective CFTR are responsible for the abnormal intracellular events leading to fat mal-absorption in CF patients. intestinal fat absorption; lipids; lipoproteins; apolipoproteins; cystic fibrosis doi: 10.1152/ajpgi.00206.2009

Cheng L, Hart X, Shi Y. A regulatory role of LPCAT1 in the synthesis of inflammatory lipids, PAF and LPC, in the retina of diabetic mice. Am J Physiol Endocrinol Metab 297: E1276-E1282, 2009. First published September 22, 2009; doi: 10.1152/ajpendo.00475.2009.--Platelet-activating factor (PAF) and lysophosphatidylcholine (LPC) are potent inflammatory lipids. Elevated levels of PAF and LPC are associated with the onset of diabetic retinopathy and neurodegeneration. However, the molecular mechanisms underlying such defects remain elusive. LPCAT1 is a newly reported lysophospholipid acyl-transferase implicated in the anti-inflammatory response by its role in conversion of LPC to PC. Intriguingly, the LPCATI enzyme also catalyzes the synthesis of PAF from lyso-PAF with use of acetyl-CoA as a substrate. The present studies investigated regulatory roles of LPCAT1 in the synthesis of inflammatory lipids during the onset of diabetes. Our work shows that LPCAT1 plays an important role in the inactivation of PAF by catalyzing the synthesis of alkyl-PC, an inactivated form of PAF with use of acyl-CoA and lyso-PAF as substrates. In support of a role of LPCAT1 in anti-inflammatory responses in diabetic retinopathy, LPCATI is most abundantly expressed in the retina. Moreover, LPCATI mRNA levels and acyltransferase activity toward lyso-PAF and LPC were significantly downregulated in retina and brain tissues in response to the onset of diabetes in lns[2.sup.Akita] and db/db mice, mouse models of type 1 and type 2 diabetes, respectively. Conversely, treatment of db/db mice with rosiglitazone, an antidiabetes compound, significantly upregulated LPCAT1 mRNA levels concurrently with increased acyltransferase activity in the retina and brain. Collectively, these findings identified a novel regulatory role of LPCAT1 in catalyzing the inactivation of inflammatory lipids in the retina of diabetic mice. lysophosphatidylcholine; lysophosphatidylcholine acyltransferase; diabetic retinopathy; platelet-activating factor doi: 10.1152/ajpendo.00475.2009

AcetyI-CoA produced in mitochondria from carbohydrate or amino acid catabolism needs to reach the cytosol to initiate de novo synthesis of fatty acids. All eukaryotes analyzed so far use a citrate/malate shuttle to transfer acetyl group equivalents from the mitochondrial matrix to the cytosol. Here we investigate how this acetyl group transfer occurs in the procyclic life cycle stage of Trypanosoma brucei, a protozoan parasite responsible of human sleeping sickness and economically important livestock diseases. Deletion of the potential citrate lyase gene, a critical cytosolic enzyme of the citrate/malate shuttle, has no effect on de novo biosynthesis of fatty acids from [sup.14]C-labeled glucose, indicating that another route is used for acetyl group transfer. Because acetate is produced from acetyI-CoA in the mitochondrion of this parasite, we considered genes encoding cytosolic enzymes producing acetyl-CoA from acetate. We identified an acetyI-CoA synthetase gene encoding a cytosolic enzyme (AceCS), which is essential for cell viability. Repression of AceCS by inducible RNAi results in a 20-fold reduction of [sup.14]C-incorporation from radiolabeled glucose or acetate into de novo synthesized fatty acids. Thus, we demonstrate that the essential cytosolic enzyme AceCS of T. brucei is responsible for activation of acetate into acetyl-CoA to feed de novo biosynthesis of lipids. To date, Trypanosoma is the only known eukaryotic organism that uses acetate instead of citrate to transfer acetyl groups over the mitochondrial membrane for cytosolic lipid synthesis. acetyl-CoA synthetase | citrate/malate shuttle | de novo lipid biosynthesis | mitochondrial acetate

Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased ~10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4-6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released lAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins. Caco-2 cells; lipid absorption; small intestine; enzyme-labeled fluorescence-97

The storage of fatty acids and fatty alcohols in the form of neutral lipids such as triacylglycerol (TAG), cholesteryl ester (CE), and wax ester (WE) serves to provide reservoirs for membrane formation and maintenance, lipoprotein trafficking, lipid detoxification, evaporation barriers, and fuel in times of stress or nutrient deprivation. This ancient process likely originated in actinomycetes and has persisted in eukaryotes, albeit by different molecular mechanisms. A surfeit of neutral lipids is strongly, perhaps causally, related to several human diseases such as diabetes mellitus, obesity, atherosclerosis and nonalcoholic fatty liver disease. Therefore, understanding the metabolic pathways of neutral lipid synthesis and the roles of the enzymes involved may facilitate the development of new therapeutic interventions for these syndromes. acyltransferase; triacylglycerol; wax ester; wax diester; cholesteryl ester

A key and highly regulated enzyme that is required for the biosynthesis of monounsaturated fatty acids is stearoyl-CoA desaturase (SCD), which catalyzes the [D.sup.9]-cis desaturation of a range of fatty acyl-CoA substrates. The preferred substrates are palmitoyl- and stearoyl-CoA, which are converted into palmitoleoyl- and oleoyl-CoA respectively. Oleate is the most abundant monounsaturated fatty acid in dietary fat and is therefore readily available. Studies of mice that have a naturally occurring mutation in the SCD-1 gene isoform as well as a mouse model with a targeted disruption of the SCD gene (SCD-[1.sup.-/-]) have revealed the role of de novo synthesized oleate and thus the physiological importance of SCD-1 expression. SCD-1 deficiency results in reduced body adiposity, increased insulin sensitivity, and resistance to diet-induced obesity. The expression of several genes of lipid oxidation are upregulated, whereas lipid synthesis genes are downregulated. SCD-1 was also found to be a component of the novel metabolic response to the hormone leptin. Therefore, SCD-1 appears to be an important metabolic control point, and inhibition of its expression could be of benefit for the treatment of obesity, diabetes, and other metabolic diseases. In this article, we summarize the recent and timely advances concerning the important role of SCD in the biochemistry and physiology of lipid metabolism.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.03.048 Byline: Robin Mathew (1)(5), Cristina M. Karp (3)(5), Brian Beaudoin (2)(3), Nhan Vuong (3), Guanghua Chen (2), Hsin-Yi Chen (3), Kevin Bray (3), Anupama Reddy (6), Gyan Bhanot (3)(5)(7), Celine Gelinas (1)(2), Robert S. DiPaola (4)(5), Vassiliki Karantza-Wadsworth (4)(5), Eileen White (1)(2)(3)(5) Keywords: CELLBIO; HUMDISEASE; SIGNALING Abstract: Allelic loss of the essential autophagy gene beclin1 occurs in human cancers and renders mice tumor-prone suggesting that autophagy is a tumor-suppression mechanism. While tumor cells utilize autophagy to survive metabolic stress, autophagy also mitigates the resulting cellular damage that may limit tumorigenesis. In response to stress, autophagy-defective tumor cells preferentially accumulated p62/SQSTM1 (p62), endoplasmic reticulum (ER) chaperones, damaged mitochondria, reactive oxygen species (ROS), and genome damage. Moreover, suppressing ROS or p62 accumulation prevented damage resulting from autophagy defects indicating that failure to regulate p62 caused oxidative stress. Importantly, sustained p62 expression resulting from autophagy defects was sufficient to alter NF-[kappa]B regulation and gene expression and to promote tumorigenesis. Thus, defective autophagy is a mechanism for p62 upregulation commonly observed in human tumors that contributes directly to tumorigenesis likely by perturbing the signal transduction adaptor function of p62-controlling pathways critical for oncogenesis. Author Affiliation: (1) University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA (2) Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854, USA (3) Department of Molecular Biology and Biochemistry, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA (4) Division of Medical Oncology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA (5) The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA (6) RUTCOR, Rutgers University, Piscataway, NJ 08854, USA (7) Department of Physics, Rutgers University, Piscataway, NJ 08854, USA Article History: Received 16 August 2008; Revised 23 December 2008; Accepted 23 March 2009 Article Note: (miscellaneous) Published: June 11, 2009

We investigated the mechanisms whereby peroxisome proliferator-activated receptor-[gamma], (PPAR[gamma]) agonism affects glucose and lipid metabolism in brown adipose tissue (BAT) by studying the impact of PPAR[gamma] activation on BAT glucose uptake and metabolism, lipogenesis, and mRNA levels plus activities of enzymes involved in triacylglycerol (TAG) synthesis. Interscapular BAT of rats treated or not with rosiglitazone (15 mg x [kg.sup.-1] x [day.sup.-1], 7 days) was evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (PAP or lipin-1), and diacylglycerol acyltransferase (DGAT). Rosiglitazone increased BAT mass without affecting whole tissue glucose uptake. BAT glycogen content (-80%), its synthesis from glucose (-50%), and mRNA levels of UDP-glucose pyrophosphorylase (-40%), which generates UDP-linked glucose for glycogen synthesis, were all reduced by rosiglita-zone. In contrast, BAT TAG-glycerol synthesis in vivo and glucose incorporation into TAG-glycerol in vitro were stimulated by the agonist along with the activities and mRNA levels of glycerol 3-phosphate-generating phosphoenolpyruvate carboxykinase and glycerokinase. Furthermore, rosiglitazone markedly increased the activities of GPAT and DGAT but not those of lipin-l-mediated PAP-l, enzymes involved in the sequential acylation of glycerol 3-phosphate and TAG synthesis. Because an adequate supply of fatty acids is essential for BAT nonshivering thermogenesis, the enhanced ability of BAT to synthesize TAG under PPAR[gamma] activation may constitute an important mechanism by which lipid substrates are stored in preparation for an eventual thermogenic activation. glycogen; glycerokinase; phosphoenolpyruvate carboxykinase; glycerolphosphate acyltransferase; lipin; diacylglycerol acyltransferase; peroxisome proliferator-activated receptor-[gamma]

Our aim was to study the effects of increased dietary intake of [alpha]-Iinolenic acid (ALA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on serum lipids and LDL fatty acid compositions. To this end, a controlled parallel study was conducted in 74 healthy normolipidemic men and women aged 19-43 y. Participants were randomly assigned to 1 of 3 interventions and consumed a total intake of 4.4 g/d ALA (ALA group), 2.2 g/d EPA (EPA group), and 2.3 g/d DHA (DHA group) for 6 wk. Fatty acid ethyl esters were incorporated into margarines, which replaced the participant's normal spread. The ALA, EPA, or DHA intake led to a significant enrichment of the LDL with the respective (n-3) fatty acid. In addition, LDL EPA contents in the ALA group increased by 36% (P< 0.05) with no changes in LDL DHA. The EPA intervention led to an additional enrichment with DHA (24%; P< 0.001), whereas the DHA intervention further increased the amount of EPA (249%; P< 0.001). ALA, EPA, or DHA intake did not affect fasting serum concentrations of total and LDL cholesterol, but fasting serum triacylglycerol concentrations significantly decreased in the EPA (-0.14 mmol/L) and DHA (-0.30 mmol/L) interventions and also in the ALA intervention (-0.17 mmol/L). DHA intake significantly increased serum HDL cholesterol, whereas no changes were found with ALA or EPA intake. In conclusion, the present data support the hypothesis that isolated dietary ALA, EPA, and DHA intakes lead to differential enrichment in LDL due to interconversion. Moderate amounts of ALA, EPA, and DHA are effective in improving lipid profiles of normolipidemic humans.

Milk fat depression (MFD) is a naturally occurring condition in dairy cows where milk fat synthesis is inhibited by intermediates of ruminal biohydrogenation. One of these bioactive fatty acids (FA), trans-10, cis-12 conjugated linoleic acid (CLA), decreases milk fat synthesis through transcriptional downregulation of genes involved in mammary lipid synthesis. Energy partitioning during MFD is not well characterized because of the complexity of observing energy metabolism in ruminant animals. To investigate energy partitioning during MFD, adipose tissue biopsies were taken from 4 cows arranged in a switchback design. Treatments were control and 4-d abomasal infusion of trans-10, cis-12 CLA (7.5 g/d). CLA decreased milk fat yield by 38 % and milk fat content by 34%, but yields of milk and other milk components were unchanged. In contrast to reported changes in mammary tissue, adipose tissue expression of lipid synthesis enzymes, including lipoprotein lipase, FA synthase, stearoyl-CoA desaturase, and FA binding protein 4, was increased. Expression of regulators of lipid synthesis, including sterol-response element binding protein 1, thyroid hormone responsive spot 14, and PPAR-[gamma], also increased in adipose tissue. Thus, a CLA dose resulting in near maximal inhibition of mammary lipid synthesis resulted in increased expression of lipid synthesis-related genes in adipose tissue. A meta-analysis of intake response during CLA infusion was conducted to extend the investigation of energy metabolism during MFD. Voluntary intake decreased (P< 0.001) by 1.5 kg/d during CLA-induced MFD in the 14 studies analyzed, but the reduction in intake only partially accounts for the energy spared from reduced milk fat synthesis. Results are consistent with energy spared from the reduction in milk fat synthesis being partitioned toward adipose tissue fat stores during short-term MFD.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.01.024 Byline: Jan-Michael Kugler, Jarred Chicoine, Paul Lasko Keywords: Ribonucleoprotein particles; Endoplasmic reticulum; Trafficking; Epidermal growth factor signaling; Pattern formation Abstract: Bicaudal-C (Bic-C) is a multiple KH-domain RNA-binding protein required for Drosophila oogenesis and, maternally, for embryonic patterning. In early oogenesis, Bic-C negatively regulates target mRNAs, including Bic-C, by recruiting the CCR4 deadenylase through a direct association with its NOT3 subunit. Here, we identify a novel function for Bic-C in secretion of the TGF-[alpha] homolog Gurken (Grk). In Bic-C mutant egg chambers, Grk is sequestered within actin-coated structures during mid-oogenesis. As a consequence, Egfr signalling is not efficiently activated in the dorsal-anterior follicle cells. This phenotype is strikingly similar to that of trailer hitch (tral) mutants. Consistent with the idea that Bic-C and Tral act together in Grk secretion, Bic-C co-localizes with Tral within cytoplasmic granules, and can be co-purified with multiple protein components of a Tral mRNP complex. Taken together, our results implicate translational regulation by Bic-C and Tral in the secretory pathway. Author Affiliation: Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, Canada H3A 1B1 Article History: Received 2 June 2008; Revised 5 January 2009; Accepted 19 January 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.12.025 Byline: Kota Saito (1), Mei Chen (3), Fred Bard (4), Shenghong Chen (5), Huilin Zhou (5), David Woodley (3), Roman Polischuk (6), Randy Schekman (7), Vivek Malhotra (1)(2) Keywords: CELLBIO; SIGNALING Abstract: A genome-wide screen revealed previously unidentified components required for transport and Golgi organization (TANGO). We now provide evidence that one of these proteins, TANGO1, is an integral membrane protein localized to endoplasmic reticulum (ER) exit sites, with a luminal SH3 domain and a cytoplasmic proline-rich domain (PRD). Knockdown of TANGO1 inhibits export of bulky collagen VII from the ER. The SH3 domain of TANGO1 binds to collagen VII; the PRD binds to the COPII coat subunits, Sec23/24. In this scenario, PRD binding to Sec23/24 subunits could stall COPII carrier biogenesis to permit the luminal domain of TANGO1 to guide SH3-bound cargo into a growing carrier. All cells except those of hematopoietic origin express TANGO1. We propose that TANGO1 exports other cargoes in cells that do not secrete collagen VII. However, TANGO1 does not enter the budding carrier, which represents a unique mechanism to load cargo into COPII carriers. Author Affiliation: (1) Department of Cell and Developmental Biology, CRG-Centre de Regulacio Genomica, Dr. Aiguader, 88 08003 Barcelona, Spain (2) Institutcio Catalana de Recerca I Estudis Avancats, Dr. Aiguader, 88 08003 Barcelona, Spain (3) Department of Dermatology, USC/Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA (4) Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673 Proteos, Singapore (5) Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA (6) Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy (7) Department of Molecular and Cellular Biology, HHMI, University of California, Berkeley, Berkeley, CA 94720, USA Article History: Received 20 June 2008; Revised 8 October 2008; Accepted 12 December 2008 Article Note: (miscellaneous) Published: March 5, 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.02.014 Byline: Chan Young Park (1), Paul J. Hoover (2), Franklin M. Mullins (2)(3), Priti Bachhawat (2)(4), Elizabeth D. Covington (2), Stefan Raunser (5), Thomas Walz (5)(6), K. Christopher Garcia (2)(4), Ricardo E. Dolmetsch (1), Richard S. Lewis (2) Keywords: CELLBIO Abstract: Store-operated Ca.sup.2+ channels activated by the depletion of Ca.sup.2+ from the endoplasmic reticulum (ER) are a major Ca.sup.2+ entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca.sup.2+ sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca.sup.2+ entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions. Author Affiliation: (1) Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA (2) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA (3) Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA (4) Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA (5) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA (6) Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA Article History: Received 24 September 2008; Revised 6 January 2009; Accepted 9 February 2009 Article Note: (miscellaneous) Published online: February 26, 2009

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided [approximately equal to] 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches. fatty acid elongation | S. cerevisiae | shotgun lipidomics

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.11.047 Byline: Catalina Ruiz-Canada (1), Daniel J. Kelleher (1), Reid Gilmore (1) Keywords: CELLBIO; PROTEINS Abstract: Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation. Author Affiliation: (1) Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School, Worcester, MA 01605, USA Article History: Received 23 June 2008; Revised 17 October 2008; Accepted 25 November 2008 Article Note: (miscellaneous) Published: January 22, 2009

The molecular details of how lipids influence final topological organization of membrane proteins are not well understood. Here, we present evidence that final topology is influenced by lipid--protein interactions most likely outside of the translocon. The N-terminal half of Escherichia coli lactose permease (LacY) is inverted with respect to the C-terminal half and the membrane bilayer when assembled in mutants lacking phosphatidylethanolamine and containing only negatively charged phospholipids. We demonstrate that inversion is dependent on interactions between the net charge of the cytoplasmic surface of the N-terminal bundle and the negative charge density of the membrane bilayer surface. A transmembrane domain, acting as a molecular hinge between the two halves of the protein, must also exit from the membrane for inversion to occur. Phosphatidylethanolamine dampens the translocation potential of negative residues in favor of the cytoplasmic retention potential of positive residues, thus explaining the dominance of positive over negative amino acids as co- or post-translational topological determinants.

It is well known that fatty acid biosynthesis in leaves is closely related to photosynthesis; although a residual synthesis is also detected at night. But less known is the effect of the day-night cycle in fatty acid biosynthesis in heterotrophic tissues such as developing seeds. Through the use of radioactive precursors, we detected variations in the metabolism of fatty acids and lipids during the day--night period in sunflower (Helianthus annuus L.) seeds. These oscillations have been observed both in the fatty acids bound to triacylglycerols and in polar lipids, indicating a fine regulation of the lipids biosynthetic activities in sunflower developing seeds. Additionally, in the triacylglycerol fraction, periodic variations were observed in the oleic and linoleic acids content as a consequence of qualitative variations in the biosynthetic machinery during the day--night period related to temperature changes. Analyzing the rate of oleic acid desaturation, we observed a significant increase in activity in the middle of the night, indicative of the importance of nocturnal desaturation. Similar variations were also observed in the stearoyl-acyl carrier protein (ACP) desaturase activity. This study has shown the variation in the lipid biosynthetic capacity in sunflower developing seeds during the day--night cycle--including control over individual enzymes such as the stearoyl-ACP desaturase--and the influence of diurnal and nocturnal temperatures in the quality (oleic/linoleic ratio) of the oil.

ABSTRACT. To determine whether nitrogen or light exerts the most control for the rates of carbon production of ice algae and phytoplankton under the ice, nitrogen addition ([NO.sub.3] or [NH.sub.4]) [...]

The goal of this study was to determine the magnitude by which acute consumption of fructose in a morning bolus would stimulate lipogenesis (measured by infusion of [sup.13][C.sub.1]-acetate and analysis by GC-MS) immediately and after a subsequent meal. Six healthy subjects [4 men and 2 women; aged (mean [+ or -] SD) 28 [+ or -] 8 y; BMI, 24.3 [+ or -] 2.8 kg/[m.sup.2]; and serum triacylglycerols (TG), 1.03 [+ or -] 0.32 mmol/L] consumed carbohydrate boluses of sugars (85 g each) in a random and blinded order, followed by a standardized lunch 4 h later. Subjects completed a control test of glucose (100:0) and a mixture of 50:50 glucose:fructose and one of 25:75 (wt:wt). Following the morning boluses, serum glucose and insulin after 100:0 were greater than both other treatments (P < 0.05) and this pattern occurred again after lunch. In the morning, fractional lipogenesis was stimulated when subjects ingested fructose and peaked at 15.9 [+ or -] 5.4% after the 50:50 treatment and at 16.9 [+ or -] 5.2% after the 25:75 treatment, values that were greater than after the 100:0 treatment (7.8 [+ or -] 5.7%; P < 0.02). When fructose was consumed, absolute lipogenesis was 2-fold greater than when it was absent (100:0). Postlunch, serum TG were 11-29% greater than 100:0 and TG-rich lipoprotein-TG concentrations were 76-200% greater after 50:50 and 25:75 were consumed (P< 0.05). The data demonstrate that an early stimulation of lipogenesis after fructose, consumed in a mixture of sugars, augments subsequent postprandial lipemia. The postlunch blood TG elevation was only partially due to carry-over from the morning. Acute intake of fructose stimulates lipogenesis and may create a metabolic milieu that enhances subsequent esterification of fatty acids flowing to the liver to elevate TG synthesis postprandially.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.02.049 Byline: Asvin K.K. Lakkaraju (1), Camille Mary (1), Anne Scherrer (1), Arthur E. Johnson (2), Katharina Strub (1) Keywords: CELLBIO; PROTEINS Abstract: SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted. Author Affiliation: (1) Departement de biologie cellulaire, Universite de Geneve, Sciences III, 1211 Geneva, Switzerland (2) Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA Article History: Received 18 October 2007; Revised 8 January 2008; Accepted 14 February 2008 Article Note: (miscellaneous) Published: May 1, 2008

Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the [beta]-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of [beta]-hydroxydecanoyl-[beta]-hydroxydecanoate from two molecules of [beta]-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily [beta]-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for [beta]-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

In contrast to other cereals, typical barley cultivars have caryopses with adhering hulls at maturity, known as covered (hulled) barley. However, a few barley cultivars are a free-threshing variant called naked (hulless) barley. The covered/naked caryopsis is controlled by a single locus (nud) on chromosome arm 7HL. On the basis of positional cloning, we concluded that an ethylene response factor (ERF) family transcription factor gene controls the covered/naked caryopsis phenotype. This conclusion was validated by (i) fixation of the 17-kb deletion harboring the ERF gene among all 100 naked cultivars studied; (ii) two x-ray-induced nud alleles with a DNA lesion at a different site, each affecting the putative functional motif; and (iii) gene expression strictly localized to the testa. Available results indicate the monophyletic origin of naked barley. The Nud gene has homology to the Arabidopsis WIN1/SHN1 transcription factor gene, whose deduced function is control of a lipid biosynthesis pathway. Staining with a lipophilic dye (Sudan black B) detected a lipid layer on the pericarp epidermis only in covered barley. We infer that, in covered barley, the contact of the caryopsis surface, overlaid with lipids to the inner side of the hull, generates organ adhesion. caryopsis | domestication epidermis | ethylene response factor | grass

The synthesis of a sulfolipid-I (SL-I) analogue bearing unnatural lipid substituents is described. The route developed for the model compound is extended for the synthesis of native SL-I as well as additional analogues for use examining the functions of SL-I.

The nonclassical biological activities of vitamin E are investigated. It is observed that vitamin E is involved in novel transcriptional activities that cause a pronounced inhibition of cholesterol biosynthesis.

The ability to store fat in the form of cytoplasmic triglyceride droplets is conserved from Saccharomyces cerevisiae to humans. Although much is known regarding the composition and catabolism of lipid droplets, the molecular components necessary for the biogenesis of lipid droplets have remained obscure. Here we report the characterization of a conserved gene family important for lipid droplet formation named fat-inducing transcript (FIT). FIT1 and FIT2 are endoplasmic reticulum resident membrane proteins that induce lipid droplet accumulation in cell culture and when expressed in mouse liver, shRNA silencing of FIT2 in 3T3-LI adipocytes prevents accumulation of lipid droplets, and depletion of FIT2 in zebrafish blocks diet-induced accumulation of lipid droplets in the intestine and liver, highlighting an important role for FIT2 in lipid droplet formation in vivo. Together these studies identify and characterize a conserved gene family that is important in the fundamental process of storing fat. adipocytes | diabetes | FIT | obesity | triglyceride

The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) that are annotated as lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes, olsA was previously shown to be an O-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of c-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining plsC316 and plsC3498 genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the Escherichia coli plsC, neither plsC316 nor plsC3498 was essential in R. capsulatus. In contrast, no plsC316 olsA double mutant could be isolated, indicating that an intact copy of either olsA or plsC316 was required for R. capsulatus growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no c-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an E. coli plsC(Ts) mutant of either R. capsulatus plsC316 or olsA gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that plsC316 encodes the main AGPAT required for glycerophospholipid synthesis in R. capsulatus, while olsA acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of R. capsulatus, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.

The liver produces plasma sex hormone-binding globulin (SHBG), which transports sex steroids and regulates their access to tissues. In overweight children and adults, low plasma SHBG levels are a biomarker of the metabolic syndrome and its associated pathologies. Here, we showed in transgenic mice and HepG2 hepatoblastoma cells that monosaccharides (glucose and fructose) reduce human SHBG production by hepatocytes. This occurred via a downregulation of hepatocyte nuclear factor-4[alpha] (HNF-4[alpha]) and replacement of HNF-4[alpha] by the chicken OVA upstream promoter-transcription factor 1 at a cis-element within the human SHBG promoter, coincident with repression of its transcriptional activity. The dose-dependent reduction of HNF-4[alpha] levels in HepG2 cells after treatment with glucose or fructose occurred in concert with parallel increases in cellular palmitate levels and could be mimicked by treatment with palmitoyl-CoA. Moreover, inhibition of lipogenesis prevented monosaccharide-induced downregulation of HNF-4[alpha] and reduced SHBG expression in HepG2 cells. Thus, monosaccharide-induced lipogenesis reduced hepatic HNF-4[alpha] levels, which in turn attenuated SHBG expression. This provides a biological explanation for why SHBG is a sensitive biomarker of the metabolic syndrome and the metabolic disturbances associated with increased fructose consumption., Introduction Body mass index is a major determinant of sex hormone-binding globulin (SHBG) concentrations in the blood of men and women (1-3). Low serum SHBG levels in overweight individuals are [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.10.025 Byline: R. Luke Wiseman (1)(2), Evan T. Powers (1)(2), Joel N. Buxbaum (3), Jeffery W. Kelly (1)(2), William E. Balch (4)(5) Keywords: CELLBIO; PROTEINS; HUMDISEASE Abstract: To provide an integrated view of endoplasmic reticulum (ER) function in protein export, we have described the interdependence of protein folding energetics and the adaptable biology of cellular protein folding and transport through the exocytic pathway. A simplified treatment of the protein homeostasis network and a formalism for how this network of competing pathways interprets protein folding kinetics and thermodynamics provides a framework for understanding cellular protein trafficking. We illustrate how folding and misfolding energetics, in concert with the adjustable biological capacities of the folding, degradation, and export pathways, collectively dictate an adaptable standard for protein export from the ER. A model of folding for export (FoldEx) establishes that no single feature dictates folding and transport efficiency. Instead, a network view provides insight into the basis for cellular diversity, disease origins, and protein homeostasis, and predicts strategies for restoring protein homeostasis in protein-misfolding diseases. Author Affiliation: (1) Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (2) The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (3) Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (4) Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (5) The Institute for Childhood and Neglected Diseases, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Article History: Received 9 March 2007; Revised 31 July 2007; Accepted 10 October 2007 Article Note: (miscellaneous) Published: November 15, 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.10.018 Byline: David Reczek (1), Michael Schwake (2), Jenny Schroder (2), Heather Hughes (1), Judith Blanz (2), Xiaoying Jin (1), William Brondyk (1), Scott Van Patten (1), Tim Edmunds (1), Paul Saftig (2) Keywords: CELLBIO; HUMDISEASE Abstract: [beta]-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of [beta]-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. [beta]-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of [beta]-glucocerebrosidase was secreted. Missorting of [beta]-glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of [beta]-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a [beta]-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for [beta]-glucocerebrosidase. Author Affiliation: (1) Genzyme Corporation, 1 Mountain Road, Framingham, MA 01701, USA (2) Biochemical Institute, Christians-Albrechts University Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany Article History: Received 15 May 2007; Revised 5 September 2007; Accepted 8 October 2007 Article Note: (miscellaneous) Published: November 15, 2007

The biogenesis of thylakoid membranes, an indispensable event for the photoautotrophic growth of plants, requires a significant increase in the level of the unique thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG), which constitutes the bulk of membrane lipids in chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. Here we report the identification and characterization of an Arabidopsis mutant showing a complete defect in MGDG synthase 1. The mutant seeds germinated as small albinos only in the presence of sucrose. The seedlings lacked galactolipids and had disrupted photosynthetic membranes, leading to the complete impairment of photosynthetic ability and photoautotrophic growth. Moreover, invagination of the inner envelope, which is not seen in mature WT chloroplasts, was observed in the mutant, supporting an old hypothesis that envelope invagination is a major event in early chloroplast biogenesis. In addition to the defective seedling phenotype, embryo development was arrested in the mutant, although seeds with impaired embryos could germinate heterotrophically. These results demonstrate the importance of galactolipids not only in photosynthetic growth but also in embryogenesis. glycosyltransferase | thylakoid membrane | monogalactosyldiacylglycerol | monogalactosyldiacylglycerol synthase

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the reversible transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein to the glucosamine 3-OH group of UDP-GlcNAc. Escherichia coli LpxA is highly selective for R-3-hydroxymyristate. The crystal structure of the E. coli LpxA homotrimer, determined previously in the absence of lipid substrates or products, revealed that LpxA contains an unusual, left-handed parallel [beta]-helix fold. We have now solved the crystal structures of E. coli LpxA with the bound product UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc at a resolution of 1.74 [Anstrom] and with bound UDP-3-O-(R-3-hydroxydecanoyl)GlcNAc at 1.85 [Angstrom]. The structures of these complexes are consistent with the catalytic mechanism deduced by mutagenesis and with a recent 3.0-[Angtrom] structure of LpxA with bound UDP-GlcNAc. Our structures show how LpxA selects for 14-carbon R-3-hydroxyacyl chains and reveal two modes of UDP binding. UDP-3-O-(R-3-hydroxymyristoyl)-acetylglucosamine | acyl carrier protein LpxA | Escherichia coli | x-ray crystallography

The accumulation of low-density lipoprotein (LDL) is recognized as one of the main contributors in atherogenesis. Mathematical models have been constructed to simulate mass transport in large arteries and the consequent lipid accumulation in the arterial wall. The objective of this study was to investigate the influences of wall shear stress and transmural pressure on LDL accumulation in the arterial wall by a multilayered, coupled lumen-wall model. The model employs the Navier-Stokes equations and Darcy's Law for fluid dynamics, convection-diffusion-reaction equations for mass balance, and Kedem-Katchalsky equations for interracial coupling. To determine physiologically realistic model parameters, an optimization approach that searches optimal parameters based on experimental data was developed. Two sets of model parameters corresponding to different transmural pressures were found by the optimization approach using experimental data in the literature. Furthermore, a shear-dependent hydraulic conductivity relation reported previously was adopted. The integrated multilayered model was applied to an axisymmetric stenosis simulating an idealized, mildly stenosed coronary artery. The results show that low wall shear stress leads to focal LDL accumulation by weakening the convective clearance effect of transmural flow, whereas high transmural pressure, associated with hypertension, leads to global elevation of LDL concentration in the arterial wall by facilitating the passage of LDL through wall layers. low-desity lipoprotein transport; lipid accumulation; atherosclerosis; hypertension doi:10.1152/ajpheart.01281.2006

Two experiments were conducted with lactating Angus x Gelbvieh beef cows to determine the effects of postpartum lipid supplementation, BCS at parturition, and day of lactation on fatty acid profiles in plasma, adipose tissue, and milk. In Exp. 1, 36 primiparous cows (488 [+ or -] 10 kg of initial BW; 5.5 [+ or -] 0.02 initial BCS) were given ad libitum access to hay and assigned randomly to a low-fat (control) supplement or supplements with cracked, high-linoleate safflower seeds (linoleate) or cracked, high-oleate safflower seeds (oleate) from d 3 to 90 of lactation. Diets were formulated to be isonitrogenous and isocaloric; safflower seed diets provided 5% of DMI as fat. Plasma and milk samples were collected on d 30, 60, and 90 of lactation. Adipose tissue biopsies were collected near the tailhead region of cows on d 45 and 90 of lactation. In Exp. 2, 3-yr-old cows achieving a BCS of 4 [+ or -] 0.07 (479 [+ or -] 36 kg of BW) or 6 [+ or -] 0.07 (580 [+ or -] 53 kg of BW) at parturition were used in a 2-yr experiment (n = 36/yr). Beginning 3 d postpartum through d 61 of lactation, cows were fed diets similar to those of Exp. 1. Adipose tissue and milk samples were collected on d 30 and 60, and plasma was collected on d 31 and 61 of lactation. Responses to postpartum dietary treatment were comparable in both experiments. Cows fed linoleate and oleate had greater (P [less than or equal to] 0.001) total fatty acid concentrations in plasma than cows fed control. Except for 15:1, milk fatty acids with Key words: adipose tissue, beef cow, body condition score, fatty acid, lipid supplementation, milk

The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.09.043 Byline: Xiaodong Wang (1), John Venable (1), Paul LaPointe (1), Darren M. Hutt (1), Atanas V. Koulov (1), Judith Coppinger (1), Cemal Gurkan (1), Wendy Kellner (1), Jeanne Matteson (1), Helen Plutner (1), John R. Riordan (5), Jeffery W. Kelly (2)(3), John R. Yates (1), William E. Balch (1)(4) Abstract: The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, [DELTA]F508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of [DELTA]F508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the 'chaperome') is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease. Author Affiliation: (1) Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (2) Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (3) The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (4) The Institute for Childhood and Neglected Disease, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (5) Department of Biochemistry and Biophysics, 5011 Thurston-Bowles Building, University of North Carolina, Chapel Hill, NC, 27510, USA Article History: Received 8 April 2006; Revised 8 July 2006; Accepted 11 September 2006 Article Note: (miscellaneous) Published: November 16, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.08.004 Byline: Ramanujan S. Hegde (1), Harris D. Bernstein (2) Abstract: Most secreted and many membrane proteins contain cleavable N-terminal signal sequences that mediate their targeting to and translocation across the endoplasmic reticulum or bacterial cytoplasmic membrane. Recent studies have identified many exceptions to the widely held view that signal sequences are simple, degenerate and interchangeable. Growing evidence indicates that signal sequences contain information that specifies the choice of targeting pathway, the efficiency of translocation, the timing of cleavage and even postcleavage functions. As a consequence, signal sequences can have important roles in modulating protein biogenesis. Based on a synthesis of studies in numerous experimental systems, we propose that substrate-specific sequence elements embedded in a conserved domain structure impart unique and physiologically important functionality to signal sequences. Author Affiliation: (1) Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA (2) Genetics and Biochemistry Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.06.059 Byline: Huiping Tu (1), Omar Nelson (1), Arseny Bezprozvanny (1), Zhengnan Wang (1), Sheu-Fen Lee (2), Yi-Heng Hao (2), Lutgarde Serneels (3), Bart De Strooper (3), Gang Yu (2), Ilya Bezprozvanny (1) Abstract: Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for [approximately equal to]40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca.sup.2+) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for [approximately equal to]80% of passive Ca.sup.2+ leak from the endoplasmic reticulum. Deficient Ca.sup.2+ signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca.sup.2+ leak function of presenilins is independent of their [gamma]-secretase activity. Our data suggest a Ca.sup.2+ signaling function for presenilins and provide support for the 'Ca.sup.2+ hypothesis of AD.' Author Affiliation: (1) Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, TX, 75390, USA (2) Center for Basic Neuroscience, UT Southwestern Medical Center at Dallas, Dallas, TX, 75390, USA (3) Neuronal Cell Biology and Gene Transfer, Center for Human Genetics, Flanders Interuniversity Institute for Biotechnology (VIB4) and KU Leuven, 3000 Leuven, Belgium Article History: Received 1 June 2005; Revised 9 April 2006; Accepted 30 June 2006 Article Note: (miscellaneous) Published: September 7, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.06.045 Byline: Soo Hee Lee (1), Jennifer L. Stephens (1), Kimberly S. Paul (1), Paul T. Englund (1) Abstract: All eukaryotic and prokaryotic organisms are thought to synthesize fatty acids using a type I or type II synthase. In addition, eukaryotes extend pre-existing long chain fatty acids using microsomal elongases (ELOs). We have found that Trypanosoma brucei, a eukaryotic human parasite that causes sleeping sickness, uses three elongases instead of type I or type II synthases for the synthesis of nearly all its fatty acids. Trypanosomes encounter diverse environments during their life cycle with different fatty acid requirements. The tsetse vector form requires synthesis of stearate (C18), whereas the bloodstream form needs myristate (C14). We find that trypanosome fatty acid synthesis is modular, with ELO1 converting C4 to C10, ELO2 extending C10 to C14, and ELO3 elongating C14 to C18. In blood, ELO3 downregulation favors myristate synthesis, whereas low concentrations of exogenous fatty acids in cultured parasites cause upregulation of the entire pathway, allowing the parasite to adapt to different environments. Author Affiliation: (1) Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, MD 21205, USA Article History: Received 17 April 2006; Revised 15 May 2006; Accepted 12 June 2006 Article Note: (miscellaneous) Published: August 24, 2006