Lipid Biosynthesis Pathways Research Articles

Renal Oncocytoma on 1-11C acetate Positron Emission Tomography: Case Report and Literature Review

Renal oncocytomas are uncommon tumors of the renal collecting duct. Although generally benign, these tumors pose a diagnostic and therapeutic dilemma in that they can not be differentiated noninvasively from renal cell carcinomas. We report a 67-year-old man who underwent a clinical 1-11C acetate positron emission tomography (PET) scan for evaluation of possible metastatic prostate carcinoma. The study demonstrated a nodule at the inferior pole of the right kidney with more uptake than the remainder of the kidney. Correlation was made with MRI, which demonstrated that the nodule was solid, and enhanced after contrast agent administration. Upon resection, this nodule was determined to be an oncocytoma. To our knowledge, this marks the first report of the 1-11C acetate PET scan appearance of a renal oncocytoma Possible mechanisms for increased uptake include dysfunctional, but up-regulated oxidative phosphorylation or uptake through lipid biosynthesis pathways.

Altered metabolic responses to intermittent hypoxia in mice with partial deficiency of hypoxia-inducible factor-1α

We have previously shown that exposure of C57BL/6J mice to intermittent hypoxia (IH) leads to 1) hypertriglyceridemia due to upregulation of pathways of lipid biosynthesis, including sterol regulatory element binding protein (SREBP)-1 and stearoyl CoA desaturase (SCD)-1; and 2) hypercholesterolemia due to impaired cholesterol uptake. The goal of the present study was to examine whether hypoxia-inducible factor (HIF)-1 is implicated in changes in lipid metabolism induced by IH. Lean HIF-1alpha (Hif1a)(+/-) mice, which are heterozygous for a null allele at the locus encoding the HIF-1alpha subunit, and their wild-type (WT) Hif1a(+/+) littermates were exposed to IH or control conditions for 5 days. IH increased fasting blood glucose, serum total cholesterol, and high-density lipoprotein-cholesterol, phospholipids, triglycerides (TG), and leptin in mice of both genotypes, whereas serum insulin and interleukin-6 were elevated only in WT mice. The impact of IH on serum TG levels in WT mice was significantly greater than that in Hif1a(+/-) mice (95 +/- 9 vs. 66 +/- 6 mg/dl, P < 0.05), whereas cholesterol and glucose levels were affected independently of genotype. Under hypoxic conditions, mRNA and protein levels of SREBP cleavage-activating protein (SCAP) and SCD-1 and protein levels of nuclear isoform of SREBP-1 in the liver were induced to significantly higher levels in WT mice than in Hif1a(+/-) mice. We conclude that 1) the effect of IH on serum TG levels is mediated through HIF-1, 2) HIF-1 may impact on posttranscriptional regulation of SREBP-1, and 3) the effect of IH on serum cholesterol levels was not altered by partial HIF-1alpha deficiency.

Temporal Correlation of Pathology and DNA Damage With Gene Expression in a Choline-Deficient Model of Rat Liver Injury *

Hepatocellular carcinoma (HCC) is the terminal event in chronic liver diseases with repeated cycles of cellular injury and regeneration. Although much is known about the cellular pathogenesis and etiological agents leading to HCC, the molecular events are not well understood. The choline-deficient (CD) model of rodent HCC involves the consecutive emergence of a fatty liver, apoptosis, compensatory proliferation, fibrosis, and cirrhosis that is markedly similar to the sequence of events typified by human HCC. Moreover, oxidative stress is thought to play a pivotal role in the progression of the disease. Here, we hypothesize that gene expression profiling can temporally mirror the histopathology and oxidative DNA damage observed with this model. We show that clusters of highly co-regulated genes representing distinct cellular pathways for lipid biosynthesis and metabolism, apoptosis, cell proliferation, and tissue remodeling temporally correlate with the well-defined sequential emergence of pathological alterations in the progression of liver disease. Additionally, an oxidative stress signature was observed that was corroborated in a time-dependent manner with increases in oxidized purines and abasic sites in DNA. Collectively, expression patterns were strongly driven by pathology, demonstrating that patterns of gene expression in advanced stages of liver disease are primarily driven by histopathological changes and to a much lesser degree by the original etiological agent. In conclusion, gene expression profiling coupled with the CD model of HCC provides a unique opportunity to unveil the molecular events associated with various stages of liver injury and carcinogenesis and to distinguish between causal and consecutive changes.

Chronic intermittent hypoxia upregulates genes of lipid biosynthesis in obese mice

Obstructive sleep apnea (OSA), a condition tightly linked to obesity, leads to chronic intermittent hypoxia (CIH) during sleep. There is emerging evidence that OSA is independently associated with insulin resistance and fatty liver disease, suggesting that OSA may affect hepatic lipid metabolism. To test this hypothesis, leptin-deficient obese (ob/ob) mice were exposed to CIH during the light phase (9 AM-9 PM) for 12 wk. Liver lipid content and gene expression profile in the liver (Affymetrix 430 GeneChip with real-time PCR validation) were determined on completion of the exposure. CIH caused a 30% increase in triglyceride and phospholipid liver content (P < 0.05), whereas liver cholesterol content was unchanged. Gene expression analysis showed that CIH upregulated multiple genes controlling 1) cholesterol and fatty acid biosynthesis [malic enzyme and acetyl coenzyme A (CoA) synthetase], 2) predominantly fatty acid biosynthesis (acetyl-CoA carboxylase and stearoyl-CoA desaturases 1 and 2), and 3) triglyceride and phospholipid biosynthesis (mitochondrial glycerol-3-phosphate acyltransferase). A majority of overexpressed genes were transcriptionally regulated by sterol regulatory element-binding protein (SREBP) 1, a master regulator of lipogenesis. A 2.8-fold increase in SREBP-1 gene expression in CIH was confirmed by real-time PCR (P = 0.001). Expression of major genes of cholesterol biosynthesis, SREBP-2 and 3-hydroxy-3-methylglutaryl-CoA reductase, was unchanged. In conclusion, we have shown that CIH may exacerbate preexisting fatty liver of obesity via upregulation of the pathways of lipid biosynthesis in the liver.

Lysophosphatidic Acid (LPA) Protects Primary Chronic Lymphocytic Leukemia Cells from Apoptosis through LPA Receptor Activation of the Anti-apoptotic Protein AKT/PKB

Lysophosphatidic acid (LPA) protects epithelial and fibroblast cell lines from apoptosis. In B-cells, LPA acts as a growth factor promoting cell proliferation. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+ B-lymphocytes primarily through a block in apoptosis. The mechanisms underlying this defect are not fully understood. We investigated whether LPA could be a survival factor in CLL cells. Herein, we demonstrate that LPA protects B-cell lines BJAB and I-83 and primary CLL cells but not normal B-cells from fludarabine- and etoposide-induced apoptosis. Furthermore, LPA prevented spontaneous apoptosis in primary CLL cells. The LPA1 expression was found to be increased in primary CLL cells compared with normal B-cells correlating with LPA prevention of apoptosis. Treatment of primary CLL cells with the LPA receptor antagonist, diacylglycerol pyrophosphate, reverses the protective effect of LPA against apoptosis, and down-regulation of the LPA1 by siRNA blocked LPA-mediated protection against spontaneous apoptosis in primary CLL cells. The protective effect of LPA was inhibited by blocking activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. These results indicate that LPA is a survival factor in B-cell lines and primary CLL cells but not normal B-cells. Thus, drugs targeting the LPA receptors might be an effective therapy against B-cell-derived malignancies such as CLL.

Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling.

Trends in increased tuberculosis infection and a fatality rate of approximately 23% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis (Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.

The sulfolipids 2'-O-acyl-sulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1.

The biosynthesis of thylakoid lipids in eukaryotic photosynthetic organisms often involves enzymes in the endoplasmic reticulum (ER) and the chloroplast envelopes. Two pathways of thylakoid lipid biosynthesis, the ER and the plastid pathways, are present in parallel in many species, including Arabidopsis, but in other plants, e.g. grasses, only the ER pathway is active. The unicellular alga Chlamydomonas reinhardtii diverges from plants like Arabidopsis in a different way because its membranes do not contain phosphatidylcholine, and most thylakoid lipids are derived from the plastid pathway. Here, we describe an acylated derivative of sulfolipid, 2'-O-acyl-sulfoquinovosyldiacylglycerol (ASQD), which is present in C. reinhardtii. Although the fatty acids of sulfoquinovosyldiacylglycerol (SQDG) were mostly saturated, ASQD molecular species carried predominantly unsaturated fatty acids. Moreover, directly attached to the head group of ASQD was preferentially an 18-carbon fatty acid with four double bonds. High-throughput robotic screening led to the isolation of a plasmid disruption mutant of C. reinhardtii, designated Deltasqd1, which lacks ASQD as well as SQDG. In this mutant, the SQD1 ortholog was completely deleted and replaced by plasmid sequences. It is proposed that ASQD arises from the sugar nucleotide pathway of sulfolipid biosynthesis by acylation of the 2'-hydroxyl of the sulfoquinovosyl head group. At the physiological level, the mutant showed increased sensitivity to a diuron herbicide and reduced growth under phosphate limitation, suggesting a role for SQDG and/or ASQD in photosynthesis as conducted by C. reinhardtii, particularly under phosphate-limited conditions.

Heterogeneity of Genome and Proteome Content in Bacteria, Archaea, and Eukaryotes

Our analysis compares bacteria, archaea, and eukaryota with respect to a wide assortment of genome and proteome properties. These properties include ribosomal protein gene distributions, chaperone protein contrasts, major variation of transcription/translation factors, gene encoding pathways of energy metabolism, and predicted protein expression levels. Significant differences within and between the three domains of life include protein lengths, information processing procedures, many metabolic and lipid biosynthesis pathways, cellular controls, and regulatory proteins. Differences among genomes are influenced by lifestyle, habitat, physiology, energy sources, and other factors.

Dermatophyte lipids-Composition and regulation of phospholipids.

This review presents the extensive work carried out on lipid components of dermatophytes, their biosynthesis, turnover and regulation. It emerges from the work done so far that the pathways of lipid biosynthesis/ degradation and the lipid composition in dermatophytes are similar to those in yeasts and other fungi. Second messengers (Ca(2+), cAMP) were demonstrated to have a regulatory role in phospholipid metabolism and they mainly act by stimulating Ca(2+)/CaM or cAMP dependent protein kinase(s). Both these kinases were purified and characterized inMicrosporum gypseum. Further work is being carried out to elucidate the molecular mechanism of regulation of phospholipid metabolism by these second messengers.

Cell Adhesion to Fibronectin Regulates Membrane Lipid Biosynthesis through 5′-AMP-activated Protein Kinase

We have shown that attachment to a fibronectin substrate stimulates two pathways of lipid biosynthesis in cultured human fibroblasts. Detachment of these cells (mechanically, with trypsin, or by RGDS peptides) caused a significant decrease in their 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and in their incorporation of [3H]acetate into fatty acids. This inhibition was substantially reversed by the reattachment of cells to fibronectin substrates, but not to poly-L-lysine substrates or to fibronectin in solution. Inhibiting phosphoprotein phosphatase activity with okadaic acid blocked the recovery of both biosynthetic activities. Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5'-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP. For example, in our system, sodium azide and 2-deoxy-D-glucose increased the ratio of cellular AMP to ATP and caused a decrease in lipid biosynthesis. We then verified the prediction that detachment of cells from substrates also caused an increase in the AMP/ATP ratio. We therefore conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix.

Lipid biosynthesis pathways as chemotherapeutic targets in kinetoplastid parasites

Inhibitors of sterol and phospholipid biosynthesis in kinetoplastid parasites such as Trypanosoma cruzi, the causative agent of Chagas' disease, and different species of Leishmania have potent and selective activity as chemotherapeutic agents in vitro and in vivo. Recent work with the sterol C14 alpha-demethylase inhibitor D0870, a bis triazole derivative, showed that this compound is capable of inducing radical parasitological cure in murine models of both acute and chronic Chagas' disease. Other inhibitors of this type, such as SCH 56592, have also shown curative, rather than suppressive, activity against T. cruzi in these models. Leishmania species have different susceptibilities to sterol biosynthesis inhibitors, both in vitro and in vivo. Leishmania braziliensis promastigotes, naturally resistant to C14 alpha-demethylase inhibitors such as ketoconazole and D0870, were susceptible to these drugs when used in combination with the squalene epoxidase inhibitor terbinafine. Inhibitors of delta 24(25) sterol methyl transferase have been shown to act as potent antiproliferative agents against Trypanosoma cruzi, both in vitro and in vivo. New inhibitors of this type which show enhanced activity and novel mechanisms of action have been synthesized. Recent work has also demonstrated that this type of enzyme inhibitors can block sterol biosynthesis and cell proliferation in Pneumocystis carinii, a fungal pathogen which had previously been found resistant to other sterol biosynthesis inhibitors. Ajoene, an antiplatelet compound derived from garlic, was shown to have potent antiproliferative activity against epimastigotes and amastigotes of Trypanosoma cruzi in vitro; this activity was associated with a significant alteration of the phospholipid composition of the cells with no significant effects on the sterol content. In addition, alkyllsophospholipids such as ilmofosine, miltefosine and edelfosine have been shown to block the proliferation of T. cruzi and Leishmania and alter both the phospholipid and sterol composition. These results indicate the potential of lipid biosynthesis inhibitors as useful therapeutic agents in the treatment of leishmaniasis and Chagas' disease.

Molecular Cloning of the Gene Encoding Stearoyl-Acyl Carrier Protein Desaturase in Brassica juncea

Metabolic engineering of the pathways of lipid biosynthesis has generated transgenic oilseed crops with enhanced levels of specialty fatty acids of Industrial value. Stearic acid, a 18:0 saturated fatty acid, is one such important fatty acid. Stearoylacyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis and converts stearoyl-ACP to oleoyl-ACP. We have cloned the complete coding region of the gene for this enzyme in Brassica juncea. Based on the sequence information of the gene in B. napus, 27-mer forward and reverse primers were designed each of which incorporated a Sal I restriciton site at the end. The primers were used to fish out the desaturase gene from B. juncea genome by polymerase chain reaction (PCR). The PCR product conformed to the average size of the coding region of the gene in B. napus. The PCR product was cloned in the pGem-T vector. The cloning was reconfirmed by restriction enzyme analysis and by PCR of the recombinant plasmid. The potential use of this gene in molecular farming of designer oilseed brassicas is discussed.

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase.

Cloning and characterization of a yeast cytochrome b5-encoding gene which suppresses ketoconazole hypersensitivity in a NADPH- P-450 reductase-deficient strain

Cytochrome P-450 (Cyp) 51 or lanosterol-C14-demethylase is the main target for antifungal compounds of the triazole family like ketoconazole (Kz). Disruption of the associated NADPH- P-450 reductase-encoding gene ( YRED) is not lethal, but decreases by about 20-fold the Kz resistance (Kz R of wild-type (wt) Saccharomyces cerevisiae. Transformation of a YRED-disrupted strain by a yeast genomic library based on a multicopy vector allowed us to identify a suppressor of Kz hypersensitivity. Deletion analysis of the 5-kb cloned fragment indicated that yeast cytochrome b 5-encoding gene ( CYB5), which encodes a 120-amino-acid (aa) protein, is required and sufficient for the suppressor effect. The encoded polypeptide shares about 30% aa identity with mammalian cytochromes b 5 (Cyb5). CYB5 disruption and tetrad analysis demonstrate that yeast Cyb5 is not required for growth in a Yred + strain. Determination of the microsomal content of b-type cytochromes by differential spectra indicated the presence of a strongly decreased or null Cyb5 level in the disrupted strain. This confirms that we have cloned the gene encoding the major microsomal form of Cyb5 which appears not to be essential. Minor Cyb5 isoforms could also be present in yeast or other redox proteins could substitute for the pleiotropic roles of Cyb5 in the sterol and lipid biosynthesis pathways.

Development of an RFLP linkage map in diploid peanut species

An RFLP linkage map of peanut has been developed for use in genetic studies and breeding programs aimed at improving the cultivated species (Arachis hypogaea L.). An F2 population derived from the interspecific hybridization of two related diploid species in the sectionArachis (A. stenosperma ×A. cardenasii) was used to construct the map. Both random genomic and cDNA clones were used to develop the framework of the map. In addition, three cDNA clones representing genes coding for enzymes involved in the lipid biosynthesis pathway have been mapped in peanut. Of the 100 genomic and 300 cDNA clones evaluated, 15 and 190, respectively, revealed polymorphisms among the parents of our mapping population. Unfortunately, a large number of these produced complex banding patterns that could not be mapped. Of the 132 markers analyzed for segregation, 117 are distributed among 11 linkage groups, while 15 have not yet been associated with any other marker. A total map distance of approximately 1063 cM has been covered to-date.

A Mutant of Arabidopsis Deficient in Desaturation of Palmitic Acid in Leaf Lipids

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by elevated amounts of palmitic acid and a decreased amount of unsaturated 16-carbon fatty acids as a consequence of a single nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids suggested that the mutant is deficient in the activity of a chloroplast omega9 fatty acid desaturase which normally introduces a double bond in 16-carbon acyl chains esterified to monogalactosyldiacylglycerol (MGD). The mutant exhibited an increased ratio of 18- to 16-carbon fatty acids in MGD due to a change in the relative contribution of the prokaryotic and eukaryotic pathways of lipid biosynthesis. This appears to be a regulated response to the loss of chloroplast omega9 desaturase and presumably reflects a requirement for polyunsaturated fatty acids for the normal assembly of chloroplast membranes. The reduction in mass of prokaryotic MGD species involved both a reduction in synthesis of MGD by the prokaryotic pathway and increased turnover of MGD molecular species which contain 16:0.

Biosynthesis of pulmonary glycoconjugates—V. Biosynthesis of oligosaccharide inner core region of [formula omitted] in lung microsomes

1. 1. Lung microsomes are able to synthesize dolichyl-mannosyl-phosphate, which stimulates the biosynthesis of a product (P 1), soluble in chloroform/methanol/water (10:10:3, v/v). 2. 2. In presence of pulmonary dolichyl-mannosyl-phosphate, liver microsomes synthesized 3 mannosylated products: X 1 product recovered in chloroform/methanol extract, X 2 and X 3 products soluble in chloroform/methanol/water (10:10:3, v/v). 3. 3. Mild acid hydrolysis of P 1 product released one mannosylated oligosaccharide which remained at the origin in our solvent system, since two mannosylated oligosaccharides, released from products synthesized in liver, moved very slowly. 4. 4. Addition of N,N′- diacetyl [ 14C]chitobiosyl-pyrophosphoryl-dolichol to lung microsomes incubated with GDP-[ 3H]mannose stimulated the synthesis of a [ 3H, 14C]product extracted with chloroform/methanol (2:1, v/v). Mild acid hydrolysis of this product released [ 3H, 14C]oligosaccharide which moved very slowly on paper chromatography, in our solvent system. 5. 5. These results demonstrate that lung tissue can synthesize the oligosaccharide inner core region of N- glycoprotein with the sequence (Man) x-(GlcNac) 2. But pulmonary dolichol pool is probably involved in other lipid biosynthesis pathways.

Synthesis and Composition of Surface Lipids of Human Skin

The composition and biosynthesis of lipids in human skin have long been of considerable interest, both because of the highly unusual structures these lipids contain and because of the possible involvement of the lipids in disease processes of the skin. Despite the exceptionally complex nature of surface lipid, its composition is now known in detail. In addition, since efficient procedures are now available for analyzing the class composition of skin lipid and the individual members of these classes, the changes in composition that may occur under specialized circumstances can now be studied in large groups of subjects. Because of the unusual compounds in skin lipids. the pathways of lipogenesis that have been elucidated in other tissues cannot confidently be accepted for skin. Therefore, direct examination of lipogenesis in skin has been desirable, but the numerous studies that have been conducted have been only moderately successful in delineating pathways of lipid biosynthesis. Moreover, because of the extreme variation in surface lipid composition between species, studies of sebaceous lipogenesis in other animals have only limited application to man. Howe\'er, useful inferences regarding the biosynthesis have been based on observations of the composition of human surface I ipid collected under specific conditions. This review emphasizes the results of investigations of the composition of surface fipids from human skin from which deductions concerning their origin and biosynthesis can be made. Some of the more direct examinations of lipogenesis based on in vitro studies in skin are discussed by Wheatley (this issue).

Effect of Dietary Fat and Cholesterol on the in vitro Incorporation of Acetate-14C into Hen Liver and Ovarian Lipids

A study was made of the incorporation of acetate-1-14C into the lipid fractions of liver slices and ovarian tissues from hens fed a basal diet and diets containing either safflower oil (30%), animal fat (30%), cholesterol (1%), or cholesterol (1%) and safflower oil (29%). When safflower oil was fed, acetate, incorporation into total liver lipids decreased. The distribution of 14C (ratio of 14C in a particular fraction to the 14C in total lipids) increased in the cholesterol and phospholipid fractions, decreased in triglycerides, and remained the same in diglycerides and cholesterol esters. When animal fat was fed, the distribution of 14C increased in the cholesterol fraction and decreased in triglycerides, but total lipid synthesis was depressed further than it was when safflower oil was fed. When cholesterol was fed, acetate incorporation into liver cholesterol decreased, while incorporation into triglyceride increased slightly. When both safflower oil and cholesterol were included in the diet, synthetic control by cholesterol predominated. Regulation of the pathways of lipid biosynthesis from acetate was observed in ovarian tissue only when the diet containing both cholesterol and safflower oil was fed. The implications of these results on the synthesis of egg yolk lipids are discussed.