高脂饮食诱导肝脏MED1特异性敲除小鼠呈高胆固醇血症

为研究肝脏MED1对脂质代谢的影响,以肝脏MED1特异性敲除（MED1ΔLiv）小鼠为模型,对其进行基因型鉴定,H&E染色观察肝脏组织学变化,免疫组织化学染色检测肝脏MED1蛋白表达|高脂饲料（脂肪含量为60%）饲喂小鼠,并分别在0、1、2 和4 周动态检测血浆胆固醇和甘油三酯及血糖水平. 结果显示,与MED1fl/fl小鼠相比,MED1ΔLiv 小鼠仅肝脏MED1 mRNA表达水平显著降低,其它组织表达无明显变化. 高脂饲喂1 周和2 周,MED1ΔLiv小鼠血浆总胆固醇水平显著升高（P<0.01）|普通或高脂饲料饲喂状态下,与MED1fl/fl小鼠相比,MED1ΔLiv 小鼠血糖水平均显著降低（P<0.05）. 短期给予高脂饲料可诱导MED1ΔLiv 小鼠呈现高胆固醇血症,提示MED1在胆固醇代谢中发挥重要调控作用.     

Direct Comparison of Mice Null for Liver or Intestinal Fatty Acid-binding Proteins Reveals Highly Divergent Phenotypic Responses to High Fat Feeding

The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP^−/− and LFABP^−/− mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP^−/− mice, whereas LFABP^−/− mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP^−/− mice fed high fat diets became obese relative to WT, whereas IFABP^−/− mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP^−/− mice preferentially utilizing lipids, and IFABP^−/− mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP^−/−, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP^−/− mice, result in divergent downstream effects at the systemic level.     

Selection of Reference Genes for qRT-PCR in High Fat Diet-Induced Hepatic Steatosis Mice Model

With the epidemic proportions of obesity worldwide and the concurrent prevalence of hepatic steatosis, there is an urgent need for better understanding the intrinsic mechanism of hepatic steatosis, especially the changes of gene expression underlying the development of hepatic steatosis and its associated abnormal liver function. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible technique of gene expression analysis. However, for accurate and reliable gene expression results, it is vital to have an internal control gene expressed at constant levels under all the experimental conditions being analyzed for. In this study, the authors validated candidate reference genes suitable for qRT-PCR profiling experiments using livers from control mice and high fat diet-induced obese mice. Cross-validation of expression stability of ten selected reference genes using three popular algorithms, GeNorm, NormFinder, and BestKeeper found HPRT1 and GAPDH as most stable reference genes. Thus, HPRT1 and GAPDH are recommended as stable reference genes most suitable for gene expression studies in the development of hepatic steatosis.     

Accelerated Fatty Acid Oxidation in Muscle Averts Fasting-induced Hepatic Steatosis in SJL/J Mice

The accumulation of triglycerides (TG) in the liver, designated hepatic steatosis, is characteristically associated with obesity and insulin resistance, but it can also develop after fasting. Here, we show that fasting-induced hepatic steatosis is under genetic control in inbred mice. After a 24-h fast, C57BL/6J mice and SJL/J mice both lost more than 20% of body weight and ∼60% of total body TG. In C57BL/6J mice, TG accumulated in liver, producing frank steatosis. In striking contrast, SJL/J mice failed to accumulate any hepatic TG even though they lost nearly as much adipose tissue mass as the C57BL/6J mice. Mice from five other inbred strains developed fasting-induced steatosis like the C57BL/6J mice. Measurements of the uptake of free fatty acids (FA) in vivo and in vitro demonstrated that SJL/J mice were protected from steatosis because their heart and skeletal muscle took up and oxidized twice as much FA as compared with C57BL/6J mice. As a result of this muscle diversion, serum-free FA and ketone bodies rose much less after fasting in SJL/J mice as compared with C57BL/6J mice. When livers of SJL/J and C57BL/6J mice were perfused with similar concentrations of FA, the livers took up and esterified similar amounts. We conclude that SJL/J mice express one or more variant genes that lead to enhanced FA uptake and oxidation in muscle, thereby sparing the liver from FA overload in the fasting state.Liver and adipose tissue coordinate metabolic responses to oscillations in nutrient availability (1, 2). In the postprandial state, the liver secretes triglycerides (TG)⁴ into the blood in very low-density lipoproteins (VLDL). In adipose tissue, lipoprotein lipase hydrolyzes the TG, producing fatty acids (FA) and monoglycerides that enter fat cells for reesterification and storage as TG (1). The activity of adipose tissue lipoprotein lipase is enhanced by the postprandial rise in insulin. At the same time, insulin inhibits lipolysis of stored TG in fat cells, assuring that the TG will be retained in the cells (3).Under fasting conditions, insulin falls and the inhibitory effect of insulin on adipose tissue lipolysis is diminished. The released FA enters the blood and is used as an energy source in liver, heart, and skeletal muscle. In the liver, excess FA are either re-esterified into TG for intracellular storage or oxidized and secreted as ketone bodies, which become the main energy source for the brain. In skeletal muscle during fasting, FA are oxidized to CO₂ (1, 2).We (4–6) and others (7) previously reported that livers of mice accumulate large amounts of TG after fasting for 6–24 h. In the current study, we screened 7 strains of inbred mice to study the genetic control of fasting-induced hepatic TG accumulation. Mice from 6 of 7 strains exhibited fasting-induced fatty liver. In the unique mouse strain (SJL/J), hepatic TG failed to accumulate after a 24-h fast even though the SJL/J mice lost amounts of body weight and adipose tissue that were similar to those of the other 6 strains. To trace the mechanism for the difference in hepatic TG accumulation, we conducted extensive comparisons of SJL/J mice and C57BL/6J mice. We provide evidence that mice from both strains release comparable amounts of FA from adipose tissue into blood after fasting. In the SJL/J mice, the bulk of these FA are taken up by muscle and oxidized. In C57BL/6J mice, FA uptake in muscle is comparatively low, and the excess FA are taken up by the liver where they are converted to TG. Thus, genetic control of muscle FA uptake determines the level of hepatic TG accumulation in fasted mice.     

Insulin Resistance Induces Posttranslational Hepatic Sortilin 1 Degradation in Mice

Insulin promotes hepatic apolipoprotein B100 (apoB100) degradation, whereas insulin resistance is a major cause of hepatic apoB100/triglyceride overproduction in type 2 diabetes. The cellular trafficking receptor sortilin 1 (Sort1) was recently identified to transport apoB100 to the lysosome for degradation in the liver and thus regulate plasma cholesterol and triglyceride levels. Genetic variation of SORT1 was strongly associated with cardiovascular disease risk in humans. The major goal of this study is to investigate the effect and molecular mechanism of insulin regulation of Sort1. Results showed that insulin induced Sort1 protein, but not mRNA, in AML12 cells. Treatment of PI3K or AKT inhibitors decreased Sort1 protein, whereas expression of constitutively active AKT induced Sort1 protein in AML12 cells. Consistently, hepatic Sort1 was down-regulated in diabetic mice, which was partially restored after the administration of the insulin sensitizer metformin. LC-MS/MS analysis further revealed that serine phosphorylation of Sort1 protein was required for insulin induction of Sort1 in a casein kinase 2-dependent manner and that inhibition of PI3K signaling or prevention of Sort1 phosphorylation accelerated proteasome-dependent Sort1 degradation. Administration of a PI3K inhibitor to mice decreased hepatic Sort1 protein and increased plasma cholesterol and triglyceride levels. Adenovirus-mediated overexpression of Sort1 in the liver prevented PI3K inhibitor-induced Sort1 down-regulation and decreased plasma triglyceride but had no effect on plasma cholesterol in mice. This study identified Sort1 as a novel target of insulin signaling and suggests that Sort1 may play a role in altered hepatic apoB100 metabolism in insulin-resistant conditions.     

High Fat Diet Feeding Exaggerates Perfluorooctanoic Acid-Induced Liver Injury in Mice via Modulating Multiple Metabolic Pathways

High fat diet (HFD) is closely linked to a variety of health issues including fatty liver. Exposure to perfluorooctanoic acid (PFOA), a synthetic perfluorinated carboxylic acid, also causes liver injury. The present study investigated the possible interactions between high fat diet and PFOA in induction of liver injury. Mice were pair-fed a high-fat diet (HFD) or low fat control with or without PFOA administration at 5 mg/kg/day for 3 weeks. Exposure to PFOA alone caused elevated plasma alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels and increased liver weight along with reduced body weight and adipose tissue mass. HFD alone did not cause liver damage, but exaggerated PFOA-induced hepatotoxicity as indicated by higher plasma ALT and AST levels, and more severe pathological changes including hepatocyte hypertrophy, lipid droplet accumulation and necrosis as well as inflammatory cell infiltration. These additive effects of HFD on PFOA-induced hepatotoxicity correlated with metabolic disturbance in liver and blood as well as up-regulation of hepatic proinflammatory cytokine genes. Metabolomic analysis demonstrated that both serum and hepatic metabolite profiles of PFOA, HFD, or HFD-PFOA group were clearly differentiated from that of controls. PFOA affected more hepatic metabolites than HFD, but HFD showed positive interaction with PFOA on fatty acid metabolites including long chain fatty acids and acylcarnitines. Taken together, dietary high fat potentiates PFOA-induced hepatic lipid accumulation, inflammation and necrotic cell death by disturbing hepatic metabolism and inducing inflammation. This study demonstrated, for the first time, that HFD increases the risk of PFOA in induction of hepatotoxicity.     

Chronic Uridine Administration Induces Fatty Liver and Pre-Diabetic Conditions in Mice

Uridine is a pyrimidine nucleoside that exerts restorative functions in tissues under stress. Short-term co-administration of uridine with multiple unrelated drugs prevents drug-induced liver lipid accumulation. Uridine has the ability to modulate liver metabolism; however, the precise mechanism has not been delineated. In this study, long-term effects of uridine on liver metabolism were examined in both HepG2 cell cultures and C57BL/6J mice. We report that uridine administration was associated with O-GlcNAc modification of FOXO1, increased gluconeogenesis, reduced insulin signaling activity, and reduced expression of a liver-specific fatty acid binding protein FABP1. Long-term uridine feeding induced systemic glucose intolerance and severe liver lipid accumulation in mice. Our findings suggest that the therapeutic potentials of uridine should be designed for short-term acute administration.     

High Fat Feeding in Mice Is Insufficient to Induce Cardiac Dysfunction and Does Not Exacerbate Heart Failure

Preclinical studies of animals with risk factors, and how those risk factors contribute to the development of cardiovascular disease and cardiac dysfunction, are clearly needed. One such approach is to feed mice a diet rich in fat (i.e. 60%). Here, we determined whether a high fat diet was sufficient to induce cardiac dysfunction in mice. We subjected mice to two different high fat diets (lard or milk as fat source) and followed them for over six months and found no significant decrement in cardiac function (via echocardiography), despite robust adiposity and impaired glucose disposal. We next determined whether antecedent and concomitant exposure to high fat diet (lard) altered the murine heart’s response to infarct-induced heart failure; high fat feeding during, or before and during, heart failure did not significantly exacerbate cardiac dysfunction. Given the lack of a robust effect on cardiac dysfunction with high fat feeding, we then examined a commonly used mouse model of overt diabetes, hyperglycemia, and obesity (db/db mice). db/db mice (or STZ treated wild-type mice) subjected to pressure overload exhibited no significant exacerbation of cardiac dysfunction; however, ischemia-reperfusion injury significantly depressed cardiac function in db/db mice compared to their non-diabetic littermates. Thus, we were able to document a negative influence of a risk factor in a relevant cardiovascular disease model; however, this did not involve exposure to a high fat diet. High fat diet, obesity, or hyperglycemia does not necessarily induce cardiac dysfunction in mice. Although many investigators use such diabetes/obesity models to understand cardiac defects related to risk factors, this study, along with those from several other groups, serves as a cautionary note regarding the use of murine models of diabetes and obesity in the context of heart failure.     

Soybean Aphid Infestation Induces Changes in Fatty Acid Metabolism in Soybean

The soybean aphid (Aphis glycines Matsumura) is one of the most important insect pests of soybeans in the North-central region of the US. It has been hypothesized that aphids avoid effective defenses by inhibition of jasmonate-regulated plant responses. Given the role fatty acids play in jasmonate-induced plant defenses, we analyzed the fatty acid profile of soybean leaves and seeds from aphid-infested plants. Aphid infestation reduced levels of polyunsaturated fatty acids in leaves with a concomitant increase in palmitic acid. In seeds, a reduction in polyunsaturated fatty acids was associated with an increase in stearic acid and oleic acid. Soybean plants challenged with the brown stem rot fungus or with soybean cyst nematodes did not present changes in fatty acid levels in leaves or seeds, indicating that the changes induced by aphids are not a general response to pests. One of the polyunsaturated fatty acids, linolenic acid, is the precursor of jasmonate; thus, these changes in fatty acid metabolism may be examples of “metabolic hijacking” by the aphid to avoid the induction of effective defenses. Based on the changes in fatty acid levels observed in seeds and leaves, we hypothesize that aphids potentially induce interference in the fatty acid desaturation pathway, likely reducing FAD2 and FAD6 activity that leads to a reduction in polyunsaturated fatty acids. Our data support the idea that aphids block jasmonate-dependent defenses by reduction of the hormone precursor.     

Mice Lacking Natural Killer T Cells Are More Susceptible to Metabolic Alterations following High Fat Diet Feeding

Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD). Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT) cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d^−/− mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD) feeding. Compared with their WT counterparts, CD1d^−/− mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d^−/− mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.     

N-3 Long-Chain Polyunsaturated Fatty Acid Supplementation Significantly Reduces Liver Oxidative Stress in High Fat Induced Steatosis

Omega-3 (n-3) long-chain polyunsaturated fatty acids (n-3 LCPUFA) are associated with several physiological functions, suggesting that their administration may prevent non transmissible chronic diseases. Therefore, we investigate whether dietary n-3 LCPUFA supplementation triggers an antioxidant response preventing liver steatosis in mice fed a high fat diet (HFD) in relation to n-3 LCPUFA levels. Male C57BL/6J mice received (a) control diet (10% fat, 20% protein, 70% carbohydrate), (b) control diet plus n-3 LCPUFA (108 mg/kg/day eicosapentaenoic acid plus 92 mg/kg/day docosahexaenoic acid), (c) HFD (60% fat, 20% protein, 20% carbohydrate), or (d) HFD plus n-3 LCPUFA for 12 weeks. Parameters of liver steatosis, glutathione status, protein carbonylation, and fatty acid analysis were determined, concomitantly with insulin resistance and serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels. HFD significantly increased total fat and triacylglyceride contents with macrovesicular steatosis, concomitantly with higher fasting serum glucose and insulin levels, HOMA, and serum TNF-α, IL-1β, and IL-6. Reduced and total liver glutathione contents were diminished by HFD, with higher GSSG/GSH ratio and protein carbonylation, n-3 LCPUFA depletion and elevated n-6/n-3 ratio over control values. These changes were either reduced or normalized to control values in animals subjected to HFD and n-3 LCPUFA, with significant increased hepatic total n-3 LCPUFA content and reduced n-6/n-3 ratio being observed after n-3 LCPUFA supplementation alone. So, repletion of liver n-3 LCPUFA levels by n-3 LCPUFA dietary supplementation in HFD obese mice reduces hepatic lipid content, with concomitant antioxidant and anti-inflammatory responses favouring insulin sensitivity.     

A Diet High in Saturated Fat and Sucrose Alters Glucoregulation and Induces Aortic Fatty Streaks in New Zealand White Rabbits

A new and convenient animal model for studying peripheral vascular and coronary artery disease in diabetes was established in this study. Male New Zealand White rabbits weighing approximately 2 kg were divided into 2 groups: a normal control group fed standard laboratory chow and a diabetogenic diet–fed group received a high-fat/high-sucrose diet. The high-fat/high-sucrose diet (contained 10% lard and 37% sucrose) feeding was maintained for 6 months. Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, superoxide dismutase, nitric oxide, nitric oxide synthase, insulin, and glucose were quantitated at monthly or bimonthly intervals. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. The aortic samples were observed by electron microscopy. High plasma triglyceride and glucose concentrations were induced. At the end of 6 months, the aortic fatty streak lesions were present in the animals'' vascular specimens. As far as we know, this is the first report that demonstrates that New Zealand White rabbits can develop obvious aortic fatty streaks by feeding a high-fat/high-sucrose diet. Our results suggest that NewZealand White rabbits fed a high-fat/high-sucrose diet would provide a convenient model for studying peripheral vascular and coronary artery disease in diabetes.     

COH-SR4 Reduces Body Weight,Improves Glycemic Control and Prevents Hepatic Steatosis in High Fat Diet-Induced Obese Mice

Obesity is a chronic metabolic disorder caused by imbalance between energy intake and expenditure, and is one of the principal causative factors in the development of metabolic syndrome, diabetes and cancer. COH-SR4 (“SR4”) is a novel investigational compound that has anti-cancer and anti-adipogenic properties. In this study, the effects of SR4 on metabolic alterations in high fat diet (HFD)-induced obese C57BL/J6 mice were investigated. Oral feeding of SR4 (5 mg/kg body weight.) in HFD mice for 6 weeks significantly reduced body weight, prevented hyperlipidemia and improved glycemic control without affecting food intake. These changes were associated with marked decreases in epididymal fat mass, adipocyte hypertrophy, increased plasma adiponectin and reduced leptin levels. SR4 treatment also decreased liver triglycerides, prevented hepatic steatosis, and normalized liver enzymes. Western blots demonstrated increased AMPK activation in liver and adipose tissues of SR4-treated HFD obese mice, while gene analyses by real time PCR showed COH-SR4 significantly suppressed the mRNA expression of lipogenic genes such as sterol regulatory element binding protein-1c (Srebf1), acetyl-Coenzyme A carboxylase (Acaca), peroxisome proliferator-activated receptor gamma (Pparg), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr), as well as gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) in the liver of obese mice. In vitro, SR4 activates AMPK independent of upstream kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Together, these data suggest that SR4, a novel AMPK activator, may be a promising therapeutic compound for treatment of obesity, fatty liver disease, and related metabolic disorders.     

酶法水解油脂生产脂肪酸的研究

豆油和猪油用解脂假丝酵母脂肪酶水解,酶解产物的酸值分别达到196和194.酶解条件是:豆油酶量100单位/克油,猪油酶量250单位/克油,豆油:水=1:1,猪油:水=0.6:0.4,豆油温度40℃,猪油温度42℃,摇床转速180r/min,水解时间36h,酶解过程中加20%NaOH溶液1%.这样的条件适合脂肪酸生产.     

Arabidopsis ECERIFERUM2 Is a Component of the Fatty Acid Elongation Machinery Required for Fatty Acid Extension to Exceptional Lengths

Primary aerial surfaces of land plants are coated by a lipidic cuticle, which forms a barrier against transpirational water loss and protects the plant from diverse stresses. Four enzymes of a fatty acid elongase complex are required for the synthesis of very-long-chain fatty acid (VLCFA) precursors of cuticular waxes. Fatty acid elongase substrate specificity is determined by a condensing enzyme that catalyzes the first reaction carried out by the complex. In Arabidopsis (Arabidopsis thaliana), characterized condensing enzymes involved in wax synthesis can only elongate VLCFAs up to 28 carbons (C28) in length, despite the predominance of C29 to C31 monomers in Arabidopsis stem wax. This suggests additional proteins are required for elongation beyond C28. The wax-deficient mutant eceriferum2 (cer2) lacks waxes longer than C28, implying that CER2, a putative BAHD acyltransferase, is required for C28 elongation. Here, we characterize the cer2 mutant and demonstrate that green fluorescent protein-tagged CER2 localizes to the endoplasmic reticulum, the site of VLCFA biosynthesis. We use site-directed mutagenesis to show that the classification of CER2 as a BAHD acyltransferase based on sequence homology does not fit with CER2 catalytic activity. Finally, we provide evidence for the function of CER2 in C28 elongation by an assay in yeast (Saccharomyces cerevisiae).Land plants have a lipidic cuticle that seals the outer surface of all of their primary aerial organs. Structurally, the cuticle consists of two components, cutin and cuticular waxes. Together these form a hydrophobic barrier that plays a critical role in plant survival by restricting nonstomatal water loss (Riederer and Schreiber, 2001). Cuticles also protect the plant from biotic and abiotic stresses, profoundly affect plant-insect interactions (Müller, 2006), prevent epidermal fusions (Sieber et al., 2000), and are involved in drought stress signaling (Wang et al., 2011).Cutin is a polymer of mainly midchain- and ω-hydroxy and -epoxy 16 carbon (C16) and C18 fatty acids, which are cross-linked in ester bonds directly or through a glycerol backbone (Pollard et al., 2008). Cuticular waxes are aliphatic monomers that are deposited within the cutin matrix as intracuticular wax, and on top of it as epicuticular wax film and crystals. Wax is a heterogeneous mixture of very-long-chain fatty acids (VLCFAs) and their alkane, aldehyde, alcohol, ketone, and ester derivatives, which typically range from C24 to C32 in length (Samuels et al., 2008). The composition of cuticular wax varies greatly among species and tissues, often providing physical and chemical properties to the plant surface that are advantageous in specific environments.Genetic analyses have revealed that a fatty acid elongase (FAE) complex is responsible for the synthesis of VLCFA wax precursors (Millar et al., 1999; Fiebig et al., 2000; Kunst and Samuels, 2009). FAE complexes are heterotetramers of independently transcribed, monofunctional proteins localized to the endoplasmic reticulum (ER). Together, they catalyze a series of four reactions to elongate long-chain acyl-CoAs or very-long-chain acyl-CoAs by sequential addition of two carbon units. The condensing enzyme, or β-ketoacyl-CoA synthase (KCS), catalyzes the first reaction in this sequence and is both rate limiting and specific for the chain length of acyl-CoA synthesized (Millar and Kunst, 1997). Two very dissimilar families of KCSs have been identified in Arabidopsis (Arabidopsis thaliana): a FAE1-type family homologous to the first such KCS enzyme discovered in association with seed oil biosynthesis (Kunst et al., 1992; James et al., 1995; Lassner et al., 1996), and an ELONGATION DEFECTIVE (ELO)-like family homologous to the yeast (Saccharomyces cerevisiae) ELO family responsible for sphingolipid synthesis (Dunn et al., 2004). To date, no function has been ascribed to Arabidopsis ELOs. Of the 21 FAE1-type KCS enzymes in Arabidopsis (Joubès et al., 2008), 11 have been shown by microarray analysis to be up-regulated in the stem epidermis (Suh et al., 2005). Only one of these, ECERIFERUM6 (CER6/KCS6/CUT1; Millar et al., 1999; Fiebig et al., 2000; Joubès et al., 2008), has a dominant role in the elongation of VLCFAs for cuticular wax synthesis, as CER6 suppression results in a dramatic reduction of all wax monomers longer than C24 (Millar et al., 1999). Heterologous expression of CER6 in yeast has demonstrated that the CER6 condensing enzyme can produce C28 VLCFAs (O. Rowland and L. Kunst, unpublished data). However, CER6 appears to be unable to produce VLCFAs longer than C28 in yeast; this presents a problem as the bulk of Arabidopsis stem wax is made up of C29 alkanes, secondary alcohols, and ketones derived from C30 VLCFAs. Mutant screens have not revealed any other KCS enzymes necessary for VLCFA elongation past C28 in Arabidopsis. Therefore, there may be other proteins unrelated to condensing enzymes that are required for acyl chain extension beyond C28 that remain unknown.The wax-deficient mutant cer2 shows a dramatic reduction in all stem waxes longer than C28 and increased accumulation of waxes C28 or shorter, suggesting that CER2 has a role in the final steps of VLCFA elongation. Surprisingly, the cer2 mutation has been mapped to At4g24510 (Negruk et al., 1996; Xia et al., 1996), a gene homologous to plant BAHD acyltransferases. However, the CER2 protein was reported to localize exclusively to the nucleus (Xia et al., 1997). This does not fit with CER2 annotation as a BAHD acyltransferase, as all characterized BAHD acyltransferases are soluble cytosolic enzymes (D’Auria, 2006).The objective of this work was to more precisely evaluate the role of CER2 in fatty acid elongation using a new CER2 allele, cer2-5 (Columbia-0 [Col-0] ecotype). We provide evidence that CER2 has a metabolic function specific to wax synthesis, and that the CER2 homolog CER2-LIKE1 has an analogous role in leaf wax synthesis. Despite the classification of CER2 as a BAHD acyltransferase based on sequence homology, we demonstrate that CER2 cannot share the catalytic mechanism that has been confirmed for other members of the BAHD family, and provide biochemical support for a function of CER2 in VLCFA elongation by an assay in yeast.     

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa^−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.