Intestinal Carnitine Status and Fatty Acid Oxidation in Response to Clofibrate and Medium-Chain Triglyceride Supplementation in Newborn Pigs.

To investigate the role of peroxisome proliferator-activated receptor alpha (PPARα) in carnitine status and intestinal fatty acid oxidation in neonates, a total of 72 suckled newborn piglets were assigned into 8 dietary treatments following a 2 (±0.35% clofibrate) × 4 (diets with: succinate+glycerol (Succ), tri-valerate (TC5), tri-hexanoate (TC6), or tri-2-methylpentanoate (TMPA)) factorial design. All pigs received experimental milk diets with isocaloric energy for 5 days. Carnitine statuses were evaluated, and fatty acid oxidation was measured in vitro using [1-14C]-palmitic acid (1 mM) as a substrate in absence or presence of L659699 (1.6 µM), iodoacetamide (50 µM), and carnitine (1 mM). Clofibrate increased concentrations of free (41%) and/or acyl-carnitine (44% and 15%) in liver and plasma but had no effects in the intestine. The effects on carnitine status were associated with the expression of genes involved in carnitine biosynthesis, absorption, and transportation. TC5 and TMPA stimulated the increased fatty acid oxidation rate induced by clofibrate, while TC6 had no effect on the increased fatty acid oxidation induced by clofibrate (p > 0.05). These results suggest that dietary clofibrate improved carnitine status and increased fatty acid oxidation. Propionyl-CoA, generated from TC5 and TMPA, could stimulate the increased fatty acid oxidation rate induced by clofibrate as anaplerotic carbon sources.

The oncogenic transcription factor ERG represses the transcription of the tumour suppressor gene PTEN in prostate cancer cells.

The oncogene ETS-related gene (ERG) encodes a transcription factor with roles in the regulation of haematopoiesis, angiogenesis, vasculogenesis, inflammation, migration and invasion. The ERG oncogene is activated in >50% of prostate cancer cases, generally through a gene fusion with the androgen-responsive promoter of transmembrane protease serine 2. Phosphatase and tensin homologue (PTEN) is an important tumour suppressor gene that is often inactivated in cancer. ERG overexpression combined with PTEN inactivation or loss is often associated with aggressive prostate cancer. The present study aimed to determine whether or not ERG regulates PTEN transcription directly. ERG was demonstrated to bind to the PTEN promoter and repress its transcription. ERG overexpression reduced endogenous PTEN expression, whereas ERG knockdown increased PTEN expression. The ability of ERG to repress PTEN may contribute to a more cancer-permissive environment.

Stearoyl-CoA Desaturase 1 Is a Key Determinant of Membrane Lipid Composition in 3T3-L1 Adipocytes.

Stearoyl-CoA desaturase 1 (SCD1) is a lipogenic enzyme important for the regulation of membrane lipid homeostasis; dysregulation likely contributes to obesity associated metabolic disturbances. SCD1 catalyses the Δ9 desaturation of 12-19 carbon saturated fatty acids to monounsaturated fatty acids. To understand its influence in cellular lipid composition we investigated the effect of genetic ablation of SCD1 in 3T3-L1 adipocytes on membrane microdomain lipid composition at the species-specific level. Using liquid chromatography/electrospray ionisation-tandem mass spectrometry, we quantified 70 species of ceramide, mono-, di- and trihexosylceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, bis(monoacylglycero)phosphate, phosphatidylinositol and cholesterol in 3T3-L1 adipocytes in which a 90% reduction in scd1 mRNA expression was achieved with siRNA. Cholesterol content was unchanged although decreases in other lipids resulted in cholesterol accounting for a higher proportion of lipid in the membranes. This was associated with decreased membrane lateral diffusion. An increased ratio of 24:0 to 24:1 in ceramide, mono- and dihexosylceramide, and sphingomyelin likely also contributed to this decrease in lateral diffusion. Of particular interest, we observed a decrease in phospholipids containing arachidonic acid. Given the high degree of structural flexibility of this acyl chain this will influence membrane lateral diffusion, and is likely responsible for the transcriptional activation of Lands' cycle enzymes lpcat3 and mboat7. Of relevance these profound changes in the lipidome were not accompanied by dramatic changes in gene expression in mature differentiated adipocytes, suggesting that adaptive homeostatic mechanisms to ensure partial maintenance of the biophysical properties of membranes likely occur at a post-transcriptional level.

Brown Adipose Tissue Thermogenic Capacity Is Regulated by Elovl6.

Although many transcriptional pathways regulating BAT have been identified, the role of lipid biosynthetic enzymes in thermogenesis has been less investigated. Whereas cold exposure causes changes in the fatty acid composition of BAT, the functional consequences of this remains relatively unexplored. In this study, we demonstrate that the enzyme Elongation of Very Long Chain fatty acids 6 (Elovl6) is necessary for the thermogenic action of BAT. Elovl6 is responsible for converting C16 non-essential fatty acids into C18 species. Loss of Elovl6 does not modulate traditional BAT markers; instead, it causes reduced expression of mitochondrial electron transport chain components and lower BAT thermogenic capacity. The reduction in BAT activity appears to be counteracted by increased beiging of scWAT. When beige fat is disabled by thermoneutrality or aging, Elovl6 KO mice gain weight and have increased scWAT mass and impaired carbohydrate metabolism. Overall, our study suggests fatty acid chain length is important for BAT function.

Quantitative analysis of ERG expression and its splice isoforms in formalin-fixed, paraffin-embedded prostate cancer samples: association with seminal vesicle invasion and biochemical recurrence.

OBJECTIVES: The proto-oncogene ETS-related gene (ERG) is consistently overexpressed in prostate cancer. Alternatively spliced isoforms of ERG have variable biological activities; inclusion of exon 11 (72 base pairs [bp]) is associated with aggressiveness and progression of disease. Exon 10 (81 bp) has also been shown to be alternatively spliced. Within this study, we assess whether ERG protein, messenger RNA (mRNA), and ERG splice isoform mRNA expression is altered as prostate cancer progresses. METHODS: Detection of the TMPRSS2-ERG fusion was done using direct methods (reverse transcription polymerase chain reaction [PCR] and fluorescence in situ hybridization) and indirect methods for ERG mRNA and protein expression using quantitative PCR and immunohistochemistry, respectively. A linear equation method was used to quantitatively determine relative proportions of ERG variants (ERG72/Δ72, ERG81/Δ81) for each sample. RESULTS: ERG mRNA and protein expression is increased in patients with advanced prostate cancer, with higher levels of ERG expression significantly associated with seminal vesicle invasion (stage pT3b) and biochemical recurrence. Genes involved in cell migration and invasiveness (matrix metalloproteinase 7, osteopontin, and septin 9) are increased in prostate cancers that overexpress ERG. In addition, there is a clear indication of increased retention of exons 10 and 11 in prostate cancer. CONCLUSIONS: Analysis of ERG and its variants may be valuable in determining prognosis and development of prostate cancer.

Conjugated linoleate reduces prostate cancer viability whereas the effects of oleate and stearate are cell line-dependent.

BACKGROUND: In this study, responses to fatty acid treatments in commonly used prostate cancer cell culture models and variability of gene expression between them were determined. MATERIALS AND METHODS: PC3, DU145, LNCaP, VCaP and PNT2 cells were treated with 100 µM of either oleate, stearate or conjugated linoleate. Cell proliferation and viability were assessed using trypan blue and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay respectively. Gene expression was measured using real-time polymerase chain reaction (PCR). RESULTS: Conjugated linoleic acid reduced cell proliferation and viability in all prostate cancer cell lines, whilst the effects of oleic and stearic acid on proliferation were found to be cell line-dependent. A reduction in gene expression of fatty acid desaturases was observed in prostate cancer cell lines compared to normal prostate cells. CONCLUSION: Differential responses of the cell lines investigated here to fatty acid treatment suggest that multiple prostate cancer cell line models should be used when designing experiments aimed at examining lipid metabolism in prostate cancer.

Adaptive changes of the Insig1/SREBP1/SCD1 set point help adipose tissue to cope with increased storage demands of obesity.

The epidemic of obesity imposes unprecedented challenges on human adipose tissue (WAT) storage capacity that may benefit from adaptive mechanisms to maintain adipocyte functionality. Here, we demonstrate that changes in the regulatory feedback set point control of Insig1/SREBP1 represent an adaptive response that preserves WAT lipid homeostasis in obese and insulin-resistant states. In our experiments, we show that Insig1 mRNA expression decreases in WAT from mice with obesity-associated insulin resistance and from morbidly obese humans and in in vitro models of adipocyte insulin resistance. Insig1 downregulation is part of an adaptive response that promotes the maintenance of SREBP1 maturation and facilitates lipogenesis and availability of appropriate levels of fatty acid unsaturation, partially compensating the antilipogenic effect associated with insulin resistance. We describe for the first time the existence of this adaptive mechanism in WAT, which involves Insig1/SREBP1 and preserves the degree of lipid unsaturation under conditions of obesity-induced insulin resistance. These adaptive mechanisms contribute to maintain lipid desaturation through preferential SCD1 regulation and facilitate fat storage in WAT, despite on-going metabolic stress.

A M-MLV reverse transcriptase with reduced RNaseH activity allows greater sensitivity of gene expression detection in formalin fixed and paraffin embedded prostate cancer samples.

Formalin fixed and paraffin embedded (FFPE) human tissue collections are an invaluable resource for retrospective gene expression studies. However formalin fixation results in chemical modification of RNA and increased RNA degradation. This can affect RNA yield and quality. A critical step when analysing gene expression is the conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase (RT) enzyme. FFPE derived RNA may affect the performance and efficiency of the RT enzyme and cDNA synthesis. We directly compared three commonly used FFPE RNA isolation methods and measured RNA yield, purity and integrity. We also assessed the effectiveness of three commercially available Moloney Murine Leukemia Virus (M-MLV) RTs on cDNA synthesis and gene expression sensitivity when using FFPE RNA as a template. Our results show that gene detection sensitivity is dependent on the isolation method, RT and length of the PCR amplicon (<200bp) when using FFPE RNA. The use of an M-MLV RT enzyme with reduced RNaseH activity gave significantly increased qRT-PCR sensitivity when using FFPE RNA derived from prostate tissue. The choice of RT can also affect perceived changes in target gene expression and thus the same RT should be used when attempting to reproduce results from different studies. This study highlights the need to optimise and evaluate RNA isolation methods and RTs when using FFPE RNA as a template in order to maximise a successful outcome in PCR applications.

Epigallocatechin-3-gallate promotes apoptosis and expression of the caspase 9a splice variant in PC3 prostate cancer cells.

Growing evidence suggests that the flavonoid epigallocatechin-3-gallate (EGCG), notably abundant in green tea, has health-promoting properties. We examined the effect of EGCG on cell survival and apoptosis in the prostate cancer cell line PC3. Cell survival was reduced and apoptosis increased significantly with a low dose of 1 µM EGCG. The ability of the anticancer drug cisplatin to promote apoptosis was enhanced by EGCG. Furthermore, EGCG, both alone and in combination with cisplatin, promoted the expression of the pro-apoptotic splice isoform of caspase 9.

Role of splice variants in the metastatic progression of prostate cancer.

AS (alternative splicing) and its role in disease, especially cancer, has come to forefront in research over the last few years. Alterations in the ratio of splice variants have been widely observed in cancer. Splice variants of cancer-associated genes have functions that can alter cellular phenotype, ultimately altering metastatic potential. As metastases are the cause of approximately 90% of all human cancer deaths, it is crucial to understand how AS is dysregulated in metastatic disease. We highlight some recent studies into the relationship between altered AS of key genes and the initiation of prostate cancer metastasis.

No association between polymorphisms in the INSIG1 gene and the risk of type 2 diabetes and related traits.

BACKGROUND: The insulin-induced gene 1 (INSIG1) encodes a protein that blocks proteolytic activation of sterol regulatory element binding proteins, which are transcription factors that activate genes that regulate cholesterol, fatty acid, and glucose metabolism. OBJECTIVE: We tested for associations between 6 INSIG1 tag single nucleotide polymorphisms (SNPs) (and captured all common variations in INSIG1) and the risk of type 2 diabetes (T2D), obesity, and related traits in 10,567 adults and 1155 adolescents from 5 population-based studies, a T2D case-control study, and a T2D case-series. DESIGN: We genotyped tag SNPs and tested them for associations with the risk of T2D or obesity and with body mass index, waist circumference, systolic and diastolic blood pressure, and concentrations of fasting glucose, 2-h oral-glucose-tolerance test glucose, cholesterol, and triglyceride, with the assumption of an additive effect of the minor allele. Dominant effects were tested for the less-frequent SNPs (minor allele frequency <5%). Summary statistics of each study underwent meta-analysis. RESULTS: Meta-analyses, which included 1655 T2D cases and 2911 control subjects, showed no association between any of the INSIG1 SNPs and T2D (P > 0.08). Furthermore, none of the SNPs showed an association with obesity in 1666 obese and 5737 nonobese individuals (P > 0.17). In agreement, none of the associations between the SNPs and any of the metabolic traits showed convincing associations in the 7562 adults from 4 population-based studies. Although a few nominally significant associations emerged, none of the associations survived multiple-testing correction. We observed no convincing associations with any of the studied traits in 1155 adolescents. CONCLUSION: Although our study was sufficiently powered to identify small effects, the results suggest that common variation in INSIG1 is unlikely to have a major effect on T2D and obesity risk and related traits in white Europeans.

An allostatic control of membrane lipid composition by SREBP1.

The maintenance of membrane lipid composition within strict limits is critical to maintain optimum cellular function. The biophysical properties of the membrane can be influenced among other factors by the saturation/unsaturation of the phospholipid fatty acyl chain. The rate-limiting enzyme in unsaturated fatty acid biosynthesis is the desaturase enzyme which in turn is regulated by the lipid transcription factor sterol regulatory element binding protein (SREBP1). In this review, we collect some evidence suggesting SREBP1 network as an important allostatic regulator necessary to maintain the pool of unsaturated fatty acid lipid species that can be incorporated into biological membranes.

The secreted Salmonella dublin phosphoinositide phosphatase, SopB, localizes to PtdIns(3)P-containing endosomes and perturbs normal endosome to lysosome trafficking.

Invasion and survival in mammalian cells by Salmonella enterica is mediated by bacterial proteins that are delivered to the host cell cytoplasm by type III secretion systems. One of these proteins, SopB/SigD, is a phosphoinositide phosphatase that can hydrolyse a number of substrates in vitro including PtdIns(3,5)P2. These substrates are, however, likely to be restricted in vivo by the localization of SopB, as different phosphoinositides have distinct spatial distributions in mammalian cells. In the present study, we show that heterologously expressed SopB localizes almost exclusively to endosomes containing the lipid PtdIns(3)P, and on which ESCRT (endosomal sorting complexes required for transport) proteins assemble. Furthermore, we present evidence that SopB can inhibit trafficking of activated epidermal growth factor receptor to the lysosome. These results provide further evidence that PtdIns(3,5)P2, a lipid involved in endosomal maturation, may be a relevant in vivo substrate of SopB. We hypothesize that reduction of PtdIns(3,5)P2 levels in cells by the action of SopB may perturb the function of a subset of ESCRT proteins that have previously been shown to bind to this lipid.