Endothelial lipase provides an alternative pathway for FFA uptake in lipoprotein lipase-deficient mouse adipose tissue.

Lipoprotein lipase (LPL) is thought to be the only enzyme responsible for the catabolism of triglycerides (TGs) associated with TG-rich lipoproteins in adipose tissue (AT). However, LPL deficiency in humans and induced mutant mice is not associated with decreased fat mass. We investigated whether endothelial lipase (EL), a recently discovered phospholipase, might represent an alternative mechanism for the uptake of phospholipid-derived fatty acids in murine lipoprotein-deficient AT. When LPL was expressed in AT and isolated murine adipocytes, EL mRNA was not detectable. In contrast, mouse AT and isolated adipocytes that lacked LPL expressed large amounts of EL mRNA. The cellular phospholipase activity in LPL-deficient fat pads was increased 4-fold compared with control fat pads and could be inhibited to control levels by a specific EL antibody. Fatty acids produced by EL activity were absorbed by adipocytes and incorporated into the TG moiety of AT. Our results suggest that EL activity in AT and other peripheral tissues might contribute to the tissue uptake of free fatty acids, which could have important implications for the metabolism of plasma lipoproteins.

Dopamine receptor D3 mRNA expression in human lymphocytes is negatively correlated with the personality trait of persistence.

It has been proposed that neurotransmitter receptor expression in peripheral immune cells reflects expression of these receptors in the brain. To test this "peripheral marker hypothesis", we compared mRNA expression of the dopamine receptors D3 (DRD3) and D4 (DRD4) in peripheral blood lymphocytes (PBL) to personality traits assessed with the Temperament and Character Inventory (TCI) in 50 healthy and unmedicated Caucasian individuals. A shared variance of at least 17% (p=0.016) between DRD3 mRNA expression in PBL and the personality trait of persistence was found. As personality traits have been generally assumed polygenic with a single gene accounting for rarely more than 1-2% of observed variance in a trait, this result lends further support to the peripheral marker hypothesis for DRD3 mRNA expression in PBL. It may also suggest a significant role for the DRD3 in the neurobiology of persistence and point to an interesting link between personality and functioning of the immune system.

Reduced dopamine D4 receptor mRNA expression in lymphocytes of long-term abstinent alcohol and heroin addicts.

AIM: It has been repeatedly suggested that dopamine receptor expression in peripheral blood lymphocytes reflects, to some extent, brain status. The aim of the present study was to investigate dopamine receptor expression in peripheral blood lymphocytes of long-term abstinent alcohol and heroin addicts against the background of the hypothesis, that a persisting dysfunction of the dopaminergic system contributes a biological cause to the chronic character of addiction. DESIGN: Dopamine D3 and D4 receptor mRNA expression in peripheral blood lymphocytes was measured by real-time polymerase chain reaction (PCR) in 19 alcohol addicts, abstinent for 6.2 +/- 4.7 months (mean +/- SD), and 20 heroin addicts, abstinent for 6.7 +/- 3.7 months (mean +/- SD), and compared to a control group of 29 age- and sex-matched individuals with no life-time history of substance abuse. FINDINGS: One-way anova showed significant differences in D4 mRNA expression between the groups (P = 0.005): both groups of addicts showed an approximately 50% reduction in D4 receptor mRNA expression in peripheral blood lymphocytes (PBL) compared to controls. No differences were found for D3 mRNA expression between the groups. CONCLUSION: The results of the present study indicate a withdrawal-persisting dopaminergic imbalance in abstinent addicts as measured by a suggested peripheral marker.

Dependence of lipoprotein-lipase-catalyzed triacylglycerol hydrolysis on droplet size of synthetic monodisperse emulsions measured with static light scattering.

Well-defined triolein emulsions of low polydispersity were prepared by shearing a crude emulsion in a modified Couette cell, resulting in radii in the range of 300 to 900 nm. These emulsions were used as synthetic substrates for lipoprotein lipase, a key enzyme for the hydrolysis of serum triacylglycerols. The change in radius with time was studied with on-line static light scattering at 37 degrees C. An optimum radius of about 750 nm was found for this reaction.

Monitoring gene expression: quantitative real-time rt-PCR.

Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.

Enhanced ABCG1 expression increases atherosclerosis in LDLr-KO mice on a western diet.

ABCG1 promotes cholesterol efflux from cells, but ABCG1(-/-) bone marrow transplant into ApoE(-/-) and LDLr(-/-) mice reduces atherosclerosis. To further investigate the role of ABCG1 in atherosclerosis, ABCG1 transgenic mice were crossed with LDLr-KO mice and placed on a high-fat western diet. Increased expression of ABCG1 mRNA was detected in liver (1.8-fold) and macrophages (2.7-fold), and cholesterol efflux from macrophages to HDL was also increased (1.4-fold) in ABCG1xLDLr-KO vs. LDLr-KO mice. No major differences were observed in total plasma lipids. However, cholesterol in the IDL-LDL size range was increased by approximately 50% in ABCG1xLDLr-KO mice compared to LDLr-KO mice. Atherosclerosis increased by 39% (10.1+/-0.8 vs 6.1+/-0.9% lesion area, p=0.02), as measured by en face analysis, and by 53% (221+/-98 vs 104+/-58x10(3)microm(2), p =0.01), as measured by cross-sectional analysis in ABCG1xLDLr-KO mice. Plasma levels for MCP-1 (1.5-fold) and TNF-alpha (1.2-fold) were also increased in ABCG1xLDLr-KO mice. In summary, these findings suggest that enhanced expression of ABCG1 increases atherosclerosis in LDLr-KO mice, despite its role in promoting cholesterol efflux from cells.

ABCA1 overexpression in the liver of LDLr-KO mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis.

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of (125)I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7alpha1, CYP27alpha1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions.

Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue.

It has been observed previously that hormone-sensitive lipase-deficient (HSL-ko) mice have reduced white adipose tissue (WAT) stores compared to control mice. These findings contradict the expectation that the decreased lipolytic activity in WAT of HSL-ko mice would cause accumulation of triglycerides (TGs) in that tissue. Here we demonstrate that the cellular TG synthesis in HSL-deficient WAT is markedly reduced due to downregulation of the enzymatic activities of glycerophosphate acyltransferase, dihydroxyacetonphosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase. Fatty acid de novo synthesis is also decreased due to reduced cellular glucose uptake, reduced glucose incorporation into adipose tissue lipids, and reduced activities of acetyl:CoA carboxylase and fatty acid synthase. Finally, the activities of phosphoenolpyruvate carboxykinase (PEPCK), acyl:CoA synthetase (ACS), and glucose 6-phosphate dehydrogenase, the enzymes that provide glycerol-3-phosphate, acyl-CoA, and NADPH for TG synthesis, respectively, are decreased in HSL-ko mice. The reduced expression of the peroxisome proliferator-activated receptor gamma (PPAR gamma) target genes PEPCK, ACS, and aP2, as well as reduced mRNA levels of PPAR gamma itself, suggest the involvement of this transcription factor in the downregulation of lipogenesis. Taken together, these results establish that in the absence of HSL, the reduced NEFA production is counteracted by a drastic reduction of NEFA reesterification that provides sufficient quantities of NEFA for release into the circulation. These metabolic adaptations result in decreased fat mass in HSL-ko mice.

Defective uptake of triglyceride-associated fatty acids in adipose tissue causes the SREBP-1c-mediated induction of lipogenesis.

Lipoprotein lipase (LPL) is the only known enzyme in the capillary endothelium of peripheral tissues that hydrolizes plasma triglycerides and provides fatty acids (FAs) for their subsequent tissue uptake. Previously, we demonstrated that mice that express LPL exclusively in muscle develop essentially normal fat mass despite the absence of LPL and the deprivation of nutritionally derived FAs in adipose tissue (AT). Using this mouse model, we now investigated the metabolic response to LPL deficiency in AT that enables maintenance of normal AT mass. We show that the rate of FA production was 1.8-fold higher in LPL-deficient AT than in control AT. The levels of mRNA and enzymatic activities of important enzymes involved in FA and triglyceride biosynthesis were induced concomitantly. Increased plasma glucose clearing and (14)C-deoxyglucose uptake into LPL-deficient mouse fat pads indicated that glucose provided the carbon source for lipid synthesis. Leptin expression was decreased in LPL-deficient AT. Finally, the induction of de novo FA synthesis in LPL-deficient AT was associated with increased expression and processing of sterol regulatory element binding protein 1 (SREBP-1), together with an increase in INSIG-1 expression. These results suggest that in the absence of LPL in AT, lipogenesis is activated through increased SREBP-1 expression and processing triggered by decreased availability of nutrition-derived FAs, elevated insulin, and low leptin levels.

Reduced dopamine D3 receptor expression in blood lymphocytes of smokers is negatively correlated with daily number of smoked cigarettes: a peripheral correlate of dopaminergic alterations in smokers.

The mesolimbic dopaminergic system is known to mediate rewarding effects of nicotine administration, and dysfunctions of this system may underlie failure to stop cigarette smoking. Expression of dopamine receptors in peripheral blood lymphocytes (PBLs) has been indicated as a peripheral correlate of brain status. Dopamine receptor D(3) (DRD3) and D(4) (DRD4) mRNA expression in PBLs was measured by real-time polymerase chain reaction in smokers (n=26) and former smokers (n=14), compared with nonsmoking control subjects (n=35). A significant (p=.032, Bonferroni corrected) 30% reduction of DRD3 mRNA expression in PBLs was found in smokers but not former smokers in comparison with controls. DRD3 mRNA expression in PBLs in smokers but not former smokers was negatively correlated with daily number of cigarettes consumed (Pearson correlation coefficient r=-.54, p=.005). These data suggest a selective inhibiting effect of smoking on DRD3 mRNA expression and, with the known involvement of DRD3 in reward mediation, indicates a vicious-cycle explanation for the motivation for continued smoking.

Transgenic mice expressing lipoprotein lipase in adipose tissue. Absence of the proximal 3'-untranslated region causes translational upregulation.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and adipocyte metabolism. Defects in LPL can lead to hypertriglyceridemia and the subsequent development of atherosclerosis. The mechanisms of regulation of this enzyme are complex and may occur at multiple levels of gene expression. Because the 3'-untranslated region (UTR) is involved in LPL translational regulation, transgenic mice were generated with adipose tissue expression of an LPL construct either with or without the proximal 3'-UTR and driven by the aP2 promoter. Both transgenic mouse colonies were viable and expressed the transgene, resulting in a 2-fold increase in LPL activity in white adipose tissue. Neither mouse colony exhibited any obvious phenotype in terms of body weight, plasma lipids, glucose, and non-esterified fatty acid levels. In the mice expressing hLPL with an intact 3'-UTR, hLPL mRNA expression approximately paralleled hLPL activity. However in the mice without the proximal 3'-UTR, hLPL mRNA was low in the setting of large amounts of hLPL protein and LPL activity. In previous studies, the 3'-UTR of LPL was critical for the inhibitory effects of constitutively expressed hormones, such as thyroid hormone and catecholamines. Therefore, these data suggest that the absence of the 3'-UTR results in a translationally unrepressed LPL, resulting in a moderate overexpression of adipose LPL activity.