Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling.

The folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, as non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by using molecular chaperones to safeguard folding cotranslationally2. However, although most of the cellular proteome forms oligomeric assemblies3, little is known about the final step of folding: the assembly of polypeptides into complexes. In prokaryotes, a proof-of-concept study showed that the assembly of heterodimeric luciferase is an organized cotranslational process that is facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences-such as the rarity of polycistronic mRNAs and different chaperone constellations-raise the question of whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of the assembly of protein complexes in eukaryotes using ribosome profiling. We determined the in vivo interactions of the nascent subunits from twelve hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find nine complexes assemble cotranslationally; the three complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit, thereby counteracting its propensity for aggregation. The onset of cotranslational subunit association coincides directly with the full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains and then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for the assembly of hetero-oligomers in yeast and indicates that translation, folding and the assembly of protein complexes are integrated processes in eukaryotes.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

BACKGROUND: Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. RESULTS: The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L- 1, which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. CONCLUSIONS: This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

NADPH accumulation is responsible for apoptosis in breast cancer cells induced by fatty acid synthase inhibition.

Fatty acid synthase (FAS), as a key enzyme involved in de novo lipogenesis, is highly expressed in many cancers. FAS inhibition induces cell death in vivo and in vitro, rendering FAS as an attractive target for cancer therapy, but the defined mechanism is still not well understood. Herein, we confirmed that FAS was highly expressed in breast cancers and FAS inhibition by its inhibitors or knockdown induced apoptosis in breast cancer cells. Our results showed that a significantly high level of reactive oxygen species was induced but not responsible for apoptosis in breast cancer cells by FAS inhibition. Instead, NADPH accumulation resulting from FAS inhibition was found to stimulate NADPH oxidase to generate reactive oxygen species and highly associated with apoptosis induction. Suppression of NADPH oxidase almost totally blocked reactive oxygen species generation while significantly potentiated the in vitro and in vivo killing of breast cancers by FAS inhibition. Taken together, these data suggest that FAS plays a critical role in maintaining cellular redox homeostasis and its inhibition leads to NADPH accumulation-mediated apoptosis. Our finding may provide new insights into cancer metabolism and aid in designing effective anticancer treatments.

Increased HAGLR expression promotes non-small cell lung cancer proliferation and invasion via enhanced de novo lipogenesis.

Lung cancers are broadly classified into small cell lung cancer and non-small cell lung cancer, with non-small cell lung cancer one of the leading causes of cancer-associated deaths worldwide. Presently, the mechanisms underlying lung tumorigenesis remain incompletely understood. Accumulating evidence indicates that abnormal expression of long non-coding RNAs is associated with tumorigenesis in multiple cancers, including lung cancer. HAGLR messenger RNA of non-small cell lung cancer tissues was significantly higher. Moreover, high levels of HAGLR expression were associated with non-small cell lung cancer tumor lymph node metastasis status, stage, and poor overall survival. Inhibition of HAGLR in non-small cell lung cancer cells suppressed cell proliferation and invasion. RNA interference-mediated downregulation of HAGLR also decreased levels of fatty acid synthase, with fatty acid synthase levels positively correlated with HAGLR expression in non-small cell lung cancer specimens. In addition, the cellular free fatty acid content of cancer cells was decreased following HAGLR knockdown. HAGLR depletion significantly inhibited the growth of non-small cell lung cancer cells in vivo. Furthermore, the expression levels of p21 and matrix metallopeptidase-9 (MMP-9) were dysregulated when HAGLR expression was suppressed. Our results suggest that HAGLR is an important regulator of non-small cell lung cancer cell proliferation and invasion, perhaps by regulating fatty acid synthase. Therefore, targeting HAGLR may be a possible therapeutic strategy for non-small cell lung cancer.

Hepatic Peroxisome Proliferator-Activated Receptor Gamma Signaling Contributes to Alcohol-Induced Hepatic Steatosis and Inflammation in Mice.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) signaling has been shown to regulate lipogenesis and lipid accumulation. Previous studies have shown that hepatic PPARγ is up-regulated in steatotic liver of both animal and human. However, the effects of hepatic PPARγ signaling on alcoholic liver disease (ALD) remain elusive. METHODS: To determine the role of hepatic PPARγ signaling on ALD, wild-type (WT) and hepatocyte-specific PPARγ knockdown (PPARγ∆Hep) mice were fed a modified Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 8 weeks to induce ALD. Blood parameters, hepatic steatosis, and inflammation were measured after 8-week alcohol feeding. RESULTS: Alcohol feeding to WT mice resulted in liver damage (alanine aminotransferase [ALT], 94.68 ± 17.05 U/L; aspartate aminotransferase [AST], 55.87 ± 11.29 U/L), which was significantly alleviated by hepatic PPARγ knockdown (ALT, 57.36 ± 14.98 U/L; AST, 38.06 ± 3.35 U/L). Alcohol feeding led to marked lipid accumulation and up-regulation of lipogenic genes including fatty acid transport protein 1 (FATP1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), lipin1 (LIPIN1), diacylglycerol acyltransferase 1 (DGAT1), and diacylglycerol acyltransferase 2 (DGAT2) in the livers of WT mice. Knockdown of hepatic PPARγ significantly alleviated alcohol-induced lipid accumulation and abolished the up-regulation of FASN, DGAT1, and DGAT2. Silencing of PPARγ in FL83B cells significantly decreased ethanol (EtOH)-, linoleic acid-, and EtOH plus linoleic acid-induced lipid accumulation. Knockdown of hepatic PPARγ also significantly reduced alcohol-induced inflammatory chemokine (monocyte chemotactic protein 1 [MCP1], keratinocyte-derived chemokine [KC], interferon gamma-induced protein 10 [IP-10]) and inflammatory infiltration (lymphocyte antigen 6 complex, locus G [Ly6G], and F4/80). CONCLUSIONS: The results suggest that hepatic PPARγ signaling contributes to alcohol-induced liver injury by promoting hepatic steatosis and inflammation.

Circulating Fatty Acid Synthase in pregnant women: Relationship to blood pressure, maternal metabolism and newborn parameters.

The enzyme FASN (fatty acid synthase) is potentially related with hypertension and metabolic dysfunction. FASN is highly expressed in the human placenta. We aimed to investigate the relationship circulating FASN has with blood pressure, maternal metabolism and newborn parameters in healthy pregnant women. Circulating FASN was assessed in 115 asymptomatic pregnant women in the second trimester of gestation along with C-peptide, fasting glucose and insulin, post-load glucose lipids, HMW-adiponectin and blood pressure (the latter was assessed in each trimester of gestation). At birth, newborns and placentas were weighed. FASN expression was also able to be assessed in 80 placentas. Higher circulating FASN was associated with lower systolic blood pressure (SBP), with a more favourable metabolic phenotype (lower fasting glucose and insulin, post load glucose, HbAc1, HOMA-IR and C-peptide), and with lower placental and birth weight (all p < 0.05 to p < 0.001). Placental FASN expression related positively to circulating FASN (p < 0.005) and negatively to placental weight (p < 0.05). Our observations suggest a physiological role of placental FASN in human pregnancy. Future studies will clarify whether circulating FASN of placental origin does actually regulate placental and fetal growth, and (thereby) has a favourable influence on the pregnant mother's insulin sensitivity and blood pressure.

Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis.

UNLABELLED: Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE: Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.

Antitumor effects of a drug combination targeting glycolysis, glutaminolysis and de novo synthesis of fatty acids.

There is a strong rationale for targeting the metabolic alterations of cancer cells. The most studied of these are the higher rates of glycolysis, glutaminolysis and de novo synthesis of fatty acids (FAs). Despite the availability of pharmacological inhibitors of these pathways, no preclinical studies targeting them simultaneously have been performed. In the present study it was determined whether three key enzymes for glycolysis, glutaminolysis and de novo synthesis of FAs, hexokinase-2, glutaminase and fatty acid synthase, respectively, were overexpressed as compared to primary fibroblasts. In addition, we showed that at clinically relevant concentrations lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat, known inhibitors of the mentioned enzymes, exerted a cell viability inhibitory effect. Genetic downregulation of the three enzymes also reduced cell viability. The three drugs were highly synergistic when administered as a triple combination. Of note, the cytotoxicity of the triple combination was low in primary fibroblasts and was well tolerated when administered into healthy BALB/c mice. The results suggest the feasibility and potential clinical utility of the triple metabolic targeting which merits to be further studied by using either repositioned old drugs or newer, more selective inhibitors.

To complete its replication cycle, a shrimp virus changes the population of long chain fatty acids during infection via the PI3K-Akt-mTOR-HIF1α pathway.

White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD), is a serious and aggressive shrimp viral pathogen with a worldwide distribution. At the genome replication stage (12 hpi), WSSV induces a metabolic rerouting known as the invertebrate Warburg effect, which boosts the availability of energy and biosynthetic building blocks in the host cell. Here we show that unlike the lipogenesis that is seen in cancer cells that are undergoing the Warburg effect, at 12 hpi, all of the long chain fatty acids (LCFAs) were significantly decreased in the stomach cells of WSSV-infected shrimp. By means of this non-selective WSSV-induced lipolysis, the LCFAs were apparently diverted into ß-oxidation and used to replenish the TCA cycle. Conversely, at 24 hpi, when the Warburg effect had ceased, most of the LCFAs were significantly up-regulated and the composition was also significantly altered. In crayfish these changes were in a direction that appeared to favor the formation of WSSV virion particles. We also found that, at 24 hpi, but not at 12 hpi, the PI3K-Akt-mTOR-HIF1α pathway induced the expression of fatty acid synthase (FAS), an enzyme which catalyzes the conversion of acetyl-CoA into LCFAs. WSSV virion formation was impaired in the presence of the FAS inhibitor C75, although viral gene and viral DNA levels were unaffected. WSSV therefore appears to use the PI3K-Akt-mTOR pathway to induce lipid biosynthesis at 24 hpi in order to support viral morphogenesis.

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production.

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.

Fatty acid synthase-positive hepatocytes and subsequent steatosis in rat livers by irinotecan.

Using a rat model, we investigated factors contributing to the pathogenesis of irinotecan-associated fatty liver disease. Male Sprague-Dawley rats were administered 200 mg/kg irinotecan by intraperitoneal injection on days 1-4, but not on days 5-7. This schedule was repeated 3 times. Rats were sacrificed 4, 18 and 25 days after the last injection, and liver steatosis was evaluated by hematoxylin and eosin (H&E) staining, microarray analysis and immunohistochemistry. Panacinar intrahepatocyte vacuoles were absent on days 4 and 25, but present on day 18, and this alteration was more prominent around the bile ducts than the central veins. Microarray analysis showed that the expression of genes involved in the synthesis of cholesterol and fatty acids was upregulated on day 4. Immunohistochemistry detected fatty acid synthase (Fasn)-strongly positive hepatocytes as well as the activation of liver progenitor cells on day 4, whereas intracellular vacuoles were evident in carbonic anhydrase 3 (CA3)-positive hepatocytes on day 18. Thus, irinotecan-induced liver steatosis was preceded by Fasn-strongly-positive hepatocytes and liver progenitor cell activation. The magnitude of the decrease in the number of Fasn-strongly positive hepatocytes between days 4 and 18 was similar to that of the increase in the number of CA3-positive hepatocytes accompanying vacuoles.

Morphotype transition and sexual reproduction are genetically associated in a ubiquitous environmental pathogen.

Sexual reproduction in an environmental pathogen helps maximize its lineage fitness to changing environment and the host. For the fungal pathogen Cryptococcus neoformans, sexual reproduction is proposed to have yielded hyper virulent and drug resistant variants. The life cycle of this pathogen commences with mating, followed by the yeast-hypha transition and hyphal growth, and it concludes with fruiting body differentiation and sporulation. How these sequential differentiation events are orchestrated to ensure developmental continuality is enigmatic. Here we revealed the genetic network of the yeast-to-hypha transition in Cryptococcus by analyzing transcriptomes of populations with a homogeneous morphotype generated by an engineered strain. Among this network, we found that a Pumilio-family protein Pum1 and the matricellular signal Cfl1 represent two major parallel circuits directing the yeast-hypha transition. Interestingly, only Pum1 coordinates the sequential morphogenesis events during a-α bisexual and α unisexual reproduction. Pum1 initiates the yeast-to-hypha transition, partially through a novel filament-specific secretory protein Fas1; Pum1 is also required to sustain hyphal growth after the morphological switch. Furthermore, Pum1 directs subsequent differentiation of aerial hyphae into fruiting bodies in both laboratory and clinical isolates. Pum1 exerts its control on sexual reproduction partly through regulating the temporal expression of Dmc1, the meiosis-specific recombinase. Therefore, Pum1 serves a pivotal role in bridging post-mating morphological differentiation events with sexual reproduction in Cryptococcus. Our findings in Cryptococcus illustrate how an environmental pathogen can ensure the completion of its life cycle to safeguard its long-term lineage success.

Fatty acid synthase overexpression in adult testicular germ cell tumors: potential role in the progression of non-seminomatous germ cell tumors.

Overexpression of fatty acid synthase (FASN), which is a key enzyme responsible for the endogenous synthesis of fatty acids, and its association with multistep progression have been demonstrated in various human malignant tumors. We aimed to clarify the potential role of FASN overexpression in the development and progression of adult testicular germ cell tumors (TGCTs). From the primary sites of a cohort of 113 TGCT cases, we obtained 221 histological components: 53 intratubular germ cell neoplasias, unclassified (IGCNUs), 84 seminomas, 32 embryonal carcinomas, seven choriocarcinomas, 21 yolk sac tumors, and 24 teratomas. Samples were analyzed for overexpression of FASN by immunohistochemistry. Intensities of immunoreactivity and the fraction of positive cells were classified into each four categories (intensity, 0 to 3; fraction, 0-10 % = 1, 11-50 % = 2, 51-80 % = 3, and >80 % = 4). The overall score was determined by multiplication of both scores and overall scores greater than 6 were considered FASN overexpression. On a component basis, FASN overexpression was detected in 8 % of seminomas but not in IGCNUs (0 %) and was detected frequently in non-seminomatous germ cell tumors (NSGCTs) (88 % of embryonal carcinomas, all choriocarcinomas, 81 % of yolk sac tumors, and 54 % of teratomas). There were no cases of a mixed tumor (i.e., a tumor with multiple histological components) that overexpressed FASN in seminoma components but not in co-existing NSGCT components, suggesting sequential progression. Our immunohistochemical data suggest that FASN overexpression occurs as a late event during the progression from IGCNUs/seminomas to NSGCTs.

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.

As the serious effects of global climate change become apparent and access to fossil fuels becomes more limited, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. In recent years, microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fatty acid-derived biofuels and chemicals from simple sugars. Specifically, we overexpressed all three fatty acid biosynthesis genes, namely acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1) and fatty acid synthase 2 (FAS2), in S. cerevisiae. When coupled to triacylglycerol (TAG) production, the engineered strain accumulated lipid to more than 17% of its dry cell weight, a four-fold improvement over the control strain. Understanding that TAG cannot be used directly as fuels, we also engineered S. cerevisiae to produce drop-in fuels and chemicals. Altering the terminal "converting enzyme" in the engineered strain led to the production of free fatty acids at a titer of approximately 400 mg/L, fatty alcohols at approximately 100mg/L and fatty acid ethyl esters (biodiesel) at approximately 5 mg/L directly from simple sugars. We envision that our approach will provide a scalable, controllable and economic route to this important class of chemicals.

[Expression, purification and characterization of a novel fatty acid synthase from Rhodosporidium toruloides].

Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids. It is one of the most important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21 (DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.

Adipogenic effect of calcium sensing receptor activation.

We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein ß. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.

Cirsium brevicaule A. GRAY leaf inhibits adipogenesis in 3T3-L1 cells and C57BL/6 mice.

BACKGROUND: Various flavonoids obtained from the genus Cirsium have been reported to exhibit beneficial effects on health. The present study evaluated the antiobesity effects of Cirsium brevicaule A. GRAY leaf (CL) by using 3T3-L1 cells and C57BL/6 mice that were fed a high-fat diet (HFD). METHODS: Dried CL powder was serially extracted with solvents of various polarities, and these extracts were tested for antiadipogenic activity using 3T3-L1 adipocytes. Mice were fed experimental HFD supplemented with dried CL powder for 4 wk. Lipid levels and mRNA levels of genes related to lipid metabolism were determined in 3T3-L1 adipocytes and the white adipose tissue (WAT) and liver of mice fed on a HFD. RESULTS: Treatment of 3T3-L1 adipocytes with a hexane extract of CL significantly reduced cellular lipid accumulation and expression of the fatty acid synthase (FASN) gene. Dietary CL reduced the serum levels of non-esterified fatty acids in HFD-fed mice. Significant decreases in subcutaneous WAT weight and associated FASN gene expression were observed in the mice fed the experimental CL diet. Dietary CL also reduced the hepatic lipid and serum levels of a hepatopathic indicator in the HFD-fed mice. A significant reduction in mRNA levels of FASN and HMG-CoA reductase were observed in the livers of the CL-diet group. Dietary CL, on the other hand, increased in the hepatic mRNA levels of genes related to ß-oxidation, namely peroxisome proliferator-activated receptor α, calnitine palmitoyltrasferase 1A, and uncoupling protein 2. Expression of the insulin receptor gene was also significantly increased in the livers of mice-fed the CL diet. CONCLUSIONS: The present study therefore demonstrated that CL suppresses lipid accumulation in the WAT and liver partly through inhibiting mRNA levels of FASN gene and enhancing the lipolysis-related gene expression.

Effects of short-term refeeding on the expression of genes involved in lipid metabolism in chicks (Gallus gallus).

The aim of this study was to analyze the expression patterns of key genes involved in lipid metabolism in response to feeding in chicks. A total of 18 thirteen day-old male chicks were fasted for 12h. The mRNA levels of the genes in the liver and white adipose tissue were analyzed after 0, 2, and 4h of refeeding. The mRNA levels of sterol regulatory element-binding protein (SREBP) 1, liver X receptor α, peroxisome proliferator-activated receptor (PPAR) γ, acetyl-CoA carboxylase α and fatty acid synthase were significantly increased after 2h of refeeding. In contrast, the mRNA levels of PPARα and carnitine palmitoyltransferase 1a were significantly decreased after 2h of refeeding. The mRNA level of acyl-CoA oxidase was significantly decreased after 4h of refeeding. The mRNA levels of cholesterol metabolism-related genes such as SREBP2 and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased after 2h of refeeding. In the white adipose tissue, the mRNA level of PPARγ was significantly increased after 2h of refeeding, whereas the mRNA level of adipose triglyceride lipase was significantly decreased after 4h of refeeding. These results demonstrated that expression of lipid metabolism-related genes is regulated by short-term refeeding in chicks.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ß-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.

Metabolomic and transcriptomic profiling of human K-ras oncogene transgenic rats with pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most debilitating malignancies in humans, and one of the reasons for this is the inability to diagnose this disease early in its development. To search for biomarkers that can be used for early diagnosis of PDAC, we established a rat model of human PDAC in which expression of a human K-ras(G12V) oncogene and induction of PDAC are regulated by the Cre/lox system. In the present study, transgenic rats bearing PDAC and control transgenic rats with normal pancreatic tissues were used for metabolomic analysis of serum and pancreatic tissue by non-targeted and targeted gas chromatography-mass spectrometry and transcriptomic analysis of pancreatic tissue by microarray. Comparison of the metabolic profiles of the serum and pancreatic tissue of PDAC-bearing and control rats identified palmitoleic acid as a metabolite, which was significantly decreased in the serum of PDAC-bearing animals. Transcriptomic analysis indicated that several transcripts involved in anaerobic glycolysis and nucleotide degradation were increased and transcripts involved in the trichloroacetic acid cycle were decreased. Other transcripts that were changed in PDAC-bearing rats were adenosine triphosphate citrate lyase (decreased: fatty acid biosynthesis), fatty acid synthase (increased: fatty acid biosynthesis) and arachidonate 5-lipoxygenase activating protein (increased: arachidonic acid metabolism). Overall, our results suggest that the decreased serum levels of palmitoleic acid in rats with PDAC was likely due to its decrease in pancreatic tissue and that palmitoleic acid should be investigated in human samples to assess its diagnostic significance as a serum biomarker for human PDAC.