Expression Of Lipid-related Genes Research Articles

Long-chain base kinase1 affects freezing tolerance in Arabidopsis thaliana

Long-chain base kinases (LCBKs) phosphorylate sphingolipid-derived long-chain base lipids and participate in the regulation of stress responses in plants. Here, we isolated a novel Arabidopsis thaliana mutant, lcbk1-2, which was extremely sensitive to freezing temperatures with or without cold acclimation. Physiological assays revealed that concentrations of osmolytes (proline and soluble sugars) and the activity of superoxide dismutase were significantly decreased in the lcbk1-2 mutant, compared with wild type. Also, the balance of reactive oxygen species (ROS) was disrupted in the lcbk1-2 mutant with or without cold treatment and, consistent with this, gene expression profiling analysis showed that the expression of cold-responsive ROS-scavenging genes was substantially decreased in the lcbk1-2 mutant. The expression of membrane lipid-related genes, which are linked to freezing tolerance in plants, was also impaired in the lcbk1-2 mutant. Furthermore, transgenic lines overexpressing LCBK1 showed enhanced freezing tolerance with over-accumulation of osmolytes. Collectively, our results suggested that LCBK1 functions as a novel positive regulator of freezing tolerance in Arabidopsis and may participate in the accumulation of osmolytes, the regulation of ROS homeostasis and lipid metabolism.

Regulation of growth, antioxidant capacity, fatty acid profiles, hematological characteristics and expression of lipid related genes by different dietary n-3 highly unsaturated fatty acids in juvenile black seabream (Acanthopagrus schlegelii)

An 8-week feeding trial was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acid (n-3HUFA) on growth performance, antioxidant capacity, fatty acid profiles, hematological characteristics and expression of some lipid related genes of juvenile black seabream (Acanthopagrus schlegelii) (initial weight 3.77±0.00g). Five isonitrogenous and isolipidic experimental diets were formulated with graded levels of n-3 HUFA (0.23, 0.87, 1.29, 1.75 and 2.53% of dry weight, DHA/EPA ratio approximately at 1.0). The results revealed that fish fed the diet containing 1.75% n-3 HUFA had higher weight gain (WG) and specific growth rate (SGR) than those fed the control diet. Fish fed diets containing 1.29% and 1.75% n-3 HUFA had higher feed efficiency (FE) than those fed the other diets. Hepatic and muscular fatty acid profiles reflected that of diets. The content of malondialdehyde (MDA) increased both in the serum and liver of fish fed high n-3 HUFA level diets, and the highest hepatic activity of glutathione peroxidase (GSH-PX) was recorded in fish fed the diet containing 1.29% n-3 HUFA. The expression of accα, g6pd, fas, srebp-1, lpl, atgl, hsl, elovl5 and fads2 was down-regulated in fish fed the diets with high n-3 HUFA levels. However, the expression levels of 6pgd and pparα significantly increased when the dietary contents of n-3 HUFA increased from 0.23% to 1.29% and then decreased when dietary n-3 HUFA levels increased from 1.75% to 2.53%. The highest expression of cpt1a was found in fish fed the diet containing 1.75% n-3 HUFA. The content of cholesterol (CHOL) in serum increased along with n-3 HUFA level. Over all, this study indicated that fish fed moderate dietary n-3 HUFA (1.34–1.80% n-3 HUFA) could enhance growth, feed utilization and antioxidant capacity. Different dietary levels of n-3 HUFA could strongly affect expression levels of some lipid metabolism relevant genes of the juvenile black seabream. This may contribute to our understanding of the mechanisms related to lipid metabolism (anabolism and catabolism) effects of different dietary levels of n-3 HUFA.

Growth performance, tissue composition, and gene expression responses in Atlantic salmon (Salmo salar) fed varying levels of different lipid sources

We investigated the influence of dietary fatty acid (FA) composition on growth performance, muscle tissue FA composition, and lipid-related hepatic gene expression in Atlantic salmon. Currently the commercial feed industry primarily uses terrestrial-based fats and oils as the main lipid component in the diet, with minimal provision of fish oil (FO). Therefore, we compared the growth and tissue FA response of salmon smolts (mean initial weight 256gfish−1) after 16weeks on three different terrestrial-based diets, with inclusion at three increasing levels of substitution with FO. The lipid sources used in this study were: FO, camelina oil (CO), canola oil (CA), and camelina oil with poultry fat (CO+PF). The terrestrial-based lipid sources had different FA signatures, on account of varied proportions of the C18 FA: 18:2n−6, 18:3n−3, or 18:1n−9. Inclusion of terrestrial FA and removal of marine FA (20:5n−3, 22:6n−3) were reflected in the FA composition of the muscle, and affected growth performance (based on lipid source and inclusion level) and hepatic gene expression of several lipid metabolism-relevant genes. Multiple regression analysis revealed that dietary lipid source affected growth performance, even when inclusion level (as a replacement of FO) was not a factor; suggesting that either inclusion or removal of certain FA in particular lipid sources, even at minimal levels, influenced growth performance. A breakpoint analysis of final weight indicated that dietary levels of <1% each of 20:5n−3 and 22:6n−3 resulted in lower final weight. A multidimensional scaling plot showed that the muscle tissue FA composition of salmon fed diets with CO were distinguishable from salmon fed CA and PF, mainly on account of 18:3n−3, while salmon fed FO were distinct from the terrestrial-based treatments. Salmon fed CO diets maintained 22:6n−3 levels in muscle tissue, and increased in n−3 and n−6 intermediary biosynthesis products in the muscle suggesting endogenous production of 22:6n−3 via dietary 18:3n−3. In addition, significant correlations were observed among dietary and/or muscle tissue FA with elovl2, fadsd5, and srebp1 transcript expression, further supporting FA biosynthesis. Significant relationships were also observed among lipid and cholesterol metabolism-related genes with dietary and tissue sterol and 20:4n−6 content, and they were most often related to cholesterol catabolism (or reduction in synthesis), such as hmgcr, dhcr7, and pparβ1. Correlations among gene expression with dietary and tissue lipid classes and FA content provided evidence to support the link between dietary FA input and phenotypic response.

Fish oil improves lipid profile in juvenile rats with intrauterine growth retardation by altering the transcriptional expression of lipid-related hepatic genes

Objective: To determine whether maternal intrauterine undernutrition and post-weaning fish oil intake influence lipid profile in juvenile offspring, and explore the possible mechanisms at transcriptional levels.Methods: After weaning, 32 control offspring and 24 intrauterine growth retardation (IUGR) offspring were randomly allocated to standard chow or fish oil diet. At 10 weeks, fasting plasma glucose, triglycerides, total cholesterol and expressions of related hepatic genes were examined.Results: IUGR offspring without catch-up growth tended to develop hyperglycemia, dyslipidemia and hepatic steatosis. Down-regulation of CPT-1 and LDLR at transcriptional levels were found in IUGR offspring. Early short-term fish oil intervention reversed these unfavorable changes in juvenile rats with IUGR. The mechanisms might be mediated by decreased expression of ACC-1, increased expression of CPT-1, LDLR and ABCG5.Conclusion: These data suggest that IUGR offspring already present lipid abnormality in juvenile stage, and early short-term fish oil consumption is beneficial to prevent these unfavorable changes.

Effect of Cottonseed Meal Fermented with Yeast on the Lipid-related Gene Expression in Broiler Chickens

Fermented cottonseed meal (FCSM) is widely used in poultry diets in China. This study was conducted to investigate the effect of FCSM on lipid-related gene expression in broilers. Initially, 180 broiler chickens (21-days-old, equal number of males and females) were randomly divided into three groups, with six pens per group and 10 birds per pen. The chickens in the control group were fed a diet containing unfermented cottonseed meal, and those in the treatment groups were fed with diets including either CSM fermented by Candida tropicalis (Ct group) or CSM fermented by Candida tropicalis plus Saccharomyces cerevisae (Ct-Sc group) until 64 days old. The results revealed that, compared with the control group (p 0.05). Likewise, the expressions of peroxisome proliferator-activated receptor gamma (PPAR-g) and LPL in the abdominal fat were not altered by the FCSM-supplemented diets (p>0.05). The results in this study indicate that CSM fermented by Candida tropicalis and Saccharomyces cerevisiaeeffectively regulated the genes involved in fatty acid b-oxidation and triglyceride hydrolysis in male broiler chickens. Furthermore, the effects of the FCSM-supplemented diets were significantly different between bird sexes and between yeast strains used in the fermentation process.

Perinatal deiodinase 2 expression in hepatocytes defines epigenetic susceptibility to liver steatosis and obesity

Thyroid hormone binds to nuclear receptors and regulates gene transcription. Here we report that in mice, at around the first day of life, there is a transient surge in hepatocyte type 2 deiodinase (D2) that activates the prohormone thyroxine to the active hormone triiodothyronine, modifying the expression of ∼165 genes involved in broad aspects of hepatocyte function, including lipid metabolism. Hepatocyte-specific D2 inactivation (ALB-D2KO) is followed by a delay in neonatal expression of key lipid-related genes and a persistent reduction in peroxisome proliferator-activated receptor-γ expression. Notably, the absence of a neonatal D2 peak significantly modifies the baseline and long-term hepatic transcriptional response to a high-fat diet (HFD). Overall, changes in the expression of approximately 400 genes represent the HFD response in control animals toward the synthesis of fatty acids and triglycerides, whereas in ALB-D2KO animals, the response is limited to a very different set of only approximately 200 genes associated with reverse cholesterol transport and lipase activity. A whole genome methylation profile coupled to multiple analytical platforms indicate that 10-20% of these differences can be related to the presence of differentially methylated local regions mapped to sites of active/suppressed chromatin, thus qualifying as epigenetic modifications occurring as a result of neonatal D2 inactivation. The resulting phenotype of the adult ALB-D2KO mouse is dramatic, with greatly reduced susceptibility to diet-induced steatosis, hypertriglyceridemia, and obesity.

Breed-specific lipid-related gene expression in the subcutaneous fat of Large White and Erhualian pigs at weaning

Abstract. The Erhualian (EHL) pig possesses significantly lower growth rates and higher adipose deposition compared with the Large White (LW) pig. To further understand the mechanism of breed lipid deposition difference at the early postnatal age, we employed an animal model of EHL and LW pigs at weaning age to compare the lipid metabolism differences in subcutaneous fat. The result showed that serum triglyceride in EHL was significantly higher (P &lt; 0.05) than that of LW. Peroxisome proliferator-activated receptor-γ protein level in EHL was significantly higher (P &lt; 0.01) though CCTTA enhancer-binding protein level demonstrated no change compared with LW pigs. Hormone sensitive lipase, adipose tissue triglyceride lipase mRNA expression and the lipase activity were significantly lower (P &lt; 0.05) in EHL. Uncoupling protein-2 protein content was significantly lower (P &lt; 0.01) in EHL than that in LW pigs. We first cloned the nucleotide sequence of Zinc-α2-glycoprotein (ZAG) with 1090 bp and found that both ZAG mRNA expression and protein level in EHL pigs was significantly lower (P &lt; 0.01) than that of LW pigs. β3 adrenergic receptor mRNA expression in EHL pigs was significantly higher (P &lt; 0.01) than that of LW pigs, though tumour necrosis factor α gene expression demonstrated no significant difference. Therefore, the significant breed lipid metabolism difference in subcutaneous fat exists at an early postnatal age between EHL and LW pigs, and this difference may originate from two causes including the increased lipid synthesis and reduced lipid mobilization in EHL pigs compared with LW pigs.

Irisin has no effect on lipolysis in 3T3-L1 adipocytes or fatty acid metabolism in HepG2 hepatocytes

Irisin, a newly identified myokine responsible for browning of white or beige adipocytes, has been reported to be present at reduced levels in diabetic patients and associated with obesity, serum triglyceride (TG) levels, and intrahepatic TG levels. We wondered whether irisin could directly affect fatty acid and TG metabolism in adipocytes and hepatocytes. We examined the effects of various concentrations of irisin on lipolysis (according to Oil Red O staining, free fatty acid release, and glycerol release), protein expression of HSL and ATGL, and mRNA expression of other lipid-related genes (UCP-1, PPARγ, FABP-4, HSL, ATGL, PPARα, and CPT-1) in mature 3T3-L1 adipocytes, as well as mRNA levels of genes involved in the synthesis (SREBP-1C and FAS) and β-oxidation (PPARα and CPT-1) of fatty acids in HepG2 hepatocytes under physiological or hyperglycemic conditions. Our results revealed that although irisin significantly increased the mRNA levels of UCP-1 and PPARα, it failed to show detectable effects on lipolysis, HSL or ATGL protein levels, or the mRNA expression of other lipid-related genes in mature 3T3-L1 adipocytes. In HepG2 hepatocytes, high glucose induced the upregulation of SREBP-1C and FAS and the downregulation of PPARα; however, no significant effect of irisin on gene expression was observed under either physiological or hyperglycemic conditions. We therefore conclude that irisin has no significant direct effect on lipolysis in 3T3-L1 adipocytes or on fatty acid metabolism in HepG2 hepatocytes.

Hepatitis C virus infection alters lipid metabolism depending on IL28B polymorphism and viral genotype and modulates gene expression in vivo and in vitro

Hepatitis C virus (HCV) interacts with lipid receptors to enter the cell, circulates as lipoviroparticle and is secreted as VLDL. We aimed to investigate the role of the rs12979860 polymorphism in the IL28B gene in 143 with chronic hepatitis C genotype 1, 144 infected with genotype 3, 90 genotype 4 and 413 noninfected individuals on lipid profile and to test the impact of HCV infection in an in vitro model on VLDL biosynthesis-related gene expression rs12979860 polymorphism was analysed using real-time PCR coupled to Fluorescence Resonance Energy Transfer (FRET). Huh7.5 (rs12979860 CT) and Huh7 (genotype CC) cells were infected with JFH-1 particles and serum from patients infected with genotypes 1 and 3. Gene expression of apolipoprotein B (apoB), microsomal triglyceride transfer protein (MTP), acetyl CoA carboxylase (ACC), diacylglycerol acyltransferase 2 (DGAT2), diacylglycerol acyltransferase 1 (DGAT1) and low-density lipoprotein receptor (LDLr) genes were determined by semiquantitative RT-PCR in vivo and in vitro. Genotype CC rs12979860 polymorphism was associated with significantly higher serum LDL and total cholesterol levels in patients with hepatitis C genotype 1 but not in patients with hepatitis C genotype 3, genotype 4 and control (noninfected) population. Genotype CC was more often seen in genotype 3 and healthy people in comparison with genotype 1; P=0.001. In vitro results showed that HCV infection promotes lipid metabolism gene expression induction depending on viral genotype, but to a lesser extent in cells with CT genotype. These results demonstrate that IL28B genotype influences lipid metabolism in patients with hepatitis C but not in noninfected and it seems to be viral genotype-mediated. HCV infection modifies lipid-related genes expression (DGAT1 and DGAT2) in cultured cells based on viral genotype and IL28 polymorphism.

Ginseng Extracts Restore High-Glucose Induced Vascular Dysfunctions by Altering Triglyceride Metabolism and Downregulation of Atherosclerosis-Related Genes

The king of herbs, Panax ginseng, has been used widely as a therapeutic agent vis-à-vis its active pharmacological and physiological effects. Based on Chinese pharmacopeia Ben Cao Gang Mu and various pieces of literature, Panax ginseng was believed to exert active vascular protective effects through its antiobesity and anti-inflammation properties. We investigated the vascular protective effects of ginseng by administrating ginseng extracts to rats after the induction of diabetes. We found that Panax ginseng can restore diabetes-induced impaired vasorelaxation and can reduce serum triglyceride but not cholesterol level in the diabetic rats. The ginseng extracts also suppressed the expression of atherosclerosis-related genes and altered the expression of lipid-related genes. The results provide evidence that Panax ginseng improves vascular dysfunction induced by diabetes and the protective effects may possibly be due to the downregulation of atherosclerosis-related genes and altered lipid metabolism, which help to restore normal endothelium functions.

Ursodeoxycholic acid lowers bile lithogenicity by regulating SCP2 expression in rabbit cholesterol gallstone models.

Aims: We designed this study to get insight into the disorder of lipid metabolism during cholesterol gallstone formation and evaluate the effect of ursodeoxycholic acid on the improvement of bile lithogenicity and on expression of lipid related genes. Methods: Rabbit cholesterol gallstone models were induced by high cholesterol diet. Bile, blood and liver tissues were obtained from rabbits after 0, 1, 2, 3, 4 and 5 weeks. Bile and blood lipids were measured enzymatically. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1) and sterol carrier protein 2 (SCP2) mRNA expressions were detected by using quantitative real-time RT-PCR. Cholesterol saturation index (CSI) was calculated by using Carey table to represent the bile lithogenicity. Results: Rates of gallstone formation of the 4 and 5 week treatment groups were 100 %, but that of the ursodeoxycholic acid treatment group was only 33.3 %. Expression of HMGCR and SCP2 mRNA in the 4 week group was upregulated and that of CYP7A1 mRNA decreased as compared with the 0 week group. Ursodeoxycholic acid could significantly extend nucleation time of bile and lower CSI. Ursodeoxycholic acid could reduce the expression of SCP2, but couldn't influence expression of HMGCR and CYP7A1. Conclusions: Abnormal expression of HMGCR, CYP7A1 and SCP2 might lead to high lithogenicity of bile. Ursodeoxycholic acid could improve bile lipids and lower bile lithogenicity, thereby reducing the incidence of gallstones. So it might be a good preventive drug for cholesterol gallstones.

Alteration of lipids and the transcription of lipid-related genes in myelodysplastic syndromes via a TP53-related pathway

Myelodysplastic syndromes (MDS) are clonal stem cell diseases of the bone marrow characterized by abnormalities in maturation of hematopoietic cells of all lineages. MDS patients frequently have lower lipids and high rates of apoptosis and p53 (TP53) expression. An association between the reduced lipids in MDS and the expression of lipid-related genes was sought. We further evaluated whether 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGcoAR) and low-density lipoprotein receptor (LDL-R) are regulated by TP53 invivo and invitro. Gene expression was measured using real-time reverse transcription polymerase chain reaction on RNA extracted from bone marrow and peripheral blood from eight newly diagnosed MDS patients and eight controls and from mice livers. Serum lipid profile was measured using colorimetric enzymatic procedures. Total- and LDL cholesterol were lower in MDS patients in comparison to controls (p = 0.04 and p = 0.01, respectively). HMGcoAR messenger RNA increased in peripheral blood and bone marrow of MDS patients compared to controls (p = 0.04 and p = 0.01, respectively). LDL-R messenger RNA was higher only in the peripheral blood of MDS patients (p = 0.05). Comparable results were obtained invivo. The transcription of these genes correlates with TP53 activation as documented by p21 messenger RNA elevation, a surrogate for TP53 activation and by using TP53 temperature-sensitive cells treated with adriamycin. To conclude, an association between reduced lipids in MDS and expression of HMGcoAR and LDL-R genes was documented. The transcription of these genes can be regulated by TP53.

The effect of combining linseed oil and sesamin on the fatty acid composition in white muscle and on expression of lipid-related genes in white muscle and liver of rainbow trout (Oncorhynchus mykiss)

Sesamin (S) is a known lipid modulator and has been shown to increase the conversion of α-linolenic acid (ALA) to docosahexaenoic acid in rainbow trout (Oncorhynchus mykiss) fed vegetable oil mixtures including linseed oil. In this study, we evaluated the effects of S supplementation in linseed oil-based diets, content of α- and γ-tocopherols, fatty acid (FA) composition, as well as the gene expression of lipid-related genes. Fish with an average weight of 36.5 g were fed different combinations of commercial linseed oil (LO), purified linseed oil triacylglycerols (TAG) with polar fraction removed and a mixed linseed-sunflower oil (6:4 v/v) (MO). S was added at 0.58 g 100−1g feed and fed to the fish for a period of 58 days. Expression of PPARα was downregulated in white muscle of fish fed S containing diets (P < 0.05). The expression of PPARβ1A was not affected by S supplementation except where TAG oil was used. The expression of PPARβ1A declined significantly in TAG + S fed group (P < 0.05), which indicates that some minor compounds in linseed oil might suppress the effect of S on the expression of PPARβ1A. The expression of PPARγ(long) declined in LO + S and MO + S fed group (P < 0.05). The β-oxidation-related genes CPT1 and ACO were upregulated by vegetable oils compared to fish oil. S decreased percentage of ALA in white muscle of fish fed LO + S (P < 0.05). The increased desaturation index and the decreased ALA levels suggest that S may increase the biosynthesis of highly unsaturated FA in rainbow trout.

Acute effects of orexigenic antipsychotic drugs on lipid and carbohydrate metabolism in rat.

ObjectiveThis study aims to investigate whether orexigenic antipsychotic drugs may induce dyslipidemia and glucose disturbances in female rats through direct perturbation of metabolically active peripheral tissues, independent of prior weight gain.MethodsIn the current study, we examined whether a single intraperitoneal injection of clozapine or olanzapine induced metabolic disturbances in adult female outbred Sprague–Dawley rats. Serum glucose and lipid parameters were measured during time-course experiments up to 48 h. Real-time quantitative PCR was used to measure specific transcriptional alterations in lipid and carbohydrate metabolism in adipose tissue depots or in the liver.ResultsOur results demonstrated that acute administration of clozapine or olanzapine induced a rapid, robust elevation of free fatty acids and glucose in serum, followed by hepatic accumulation of lipids evident after 12–24 h. These metabolic disturbances were associated with biphasic patterns of gluconeogenic and lipid-related gene expression in the liver and in white adipose tissue depots.ConclusionOur results support that clozapine and olanzapine are associated with primary effects on carbohydrate and lipid metabolism associated with transcriptional changes in metabolically active peripheral tissues prior to the development of drug-induced weight gain.Electronic supplementary materialThe online version of this article (doi:10.1007/s00213-011-2397-y) contains supplementary material, which is available to authorized users.

Effects of perilla extract on productive performance, serum values and hepatic expression of lipid-related genes in Shaoxing ducks

1. The aim of this study was to identify the effect of perilla extract, a source of polyunsaturated fatty acids, on lipid metabolism and expression of lipid-related genes in livers of Shaoxing ducks. 2. Two hundred and forty 28-week-old laying ducks received a commercial diet with perilla extract added at 0 (control) or 200 mg/kg of feed. 3. Ducks fed on a diet with perilla extract had increased laying rates compared with control ducks. 4. Serum concentrations of triglycerides were reduced by perilla extract, while high-density lipoprotein cholesterol and total serum cholesterol increased. 5. The expression of genes involved in hepatic lipogenesis, sterol regulatory element-binding protein-1, acetyl CoA carboxylase, stearoyl CoA desaturase, fatty acid synthase, apolipoprotein B, and apolipoprotein very low density lipoprotein, were decreased in the perilla group. 6. The mRNA expression of peroxisome proliferators-activated receptor alpha and acyl-coenzyme A oxidase was enhanced following treatment with perilla extract, and a similar tendency was observed in the expression of liver fatty acid-binding protein. 7. The results show that a diet with 200 mg/kg perilla extract regulated fat metabolism of Shaoxing ducks by improving egg laying, altering serum lipid profiles, stimulating lipid catabolic gene expression and inibiting lipogenic gene expression in the liver.

Effects of dietary lipids and Clostridium butyricum on serum lipids and lipid-related gene expression in broiler chickens

The effect of dietary lipids and Clostridium butyricum supplementation on circulating lipids and the transcription of selected genes involved in lipid metabolism were investigated in broiler chickens. One hundred and sixty-day-old broilers (Arbor Acres) were divided into four treatment groups in a 2 × 2 factorial arrangement and fed four diets with two lipid sources (soybean oil or fish oil) and two levels of C. butyricum (0 or 5 g/kg) for a period of 6 weeks. Serum concentrations of total cholesterol and low-density lipoprotein cholesterol were lower (P < 0.05) in broilers fed diets containing fish oil than in those fed diets containing soybean oil. Fish oil significantly reduced (P < 0.05) hepatic fatty acid synthase and lipoprotein lipase (LPL) activities in abdominal fat. In contrast, fish oil significantly increased (P < 0.05) breast muscle LPL activity and hepatic peroxisome proliferator-activated receptor-α and LPL gene expression. The addition of C. butyricum significantly increased (P < 0.05) LPL activity in abdominal fat and liver-type fatty acid-binding protein gene expression in jejunal mucosa. The results of this study indicated that the reduced abdominal fat in broilers fed fish oil as observed may be due to augmented hepatic fatty acid catabolism and lower hepatic fat synthesis. The increased intramuscular fat content in breast muscle of broilers fed C. butyricum as described may be the result of enhanced fatty acid uptake.

Culture, immortalization, and characterization of human meibomian gland epithelial cells.

Meibomian gland epithelial cells are essential in maintaining the health and integrity of the ocular surface. However, very little is known about their physiological regulation. In this study, the cellular control mechanisms were explored, first to establish a defined culture system for the maintenance of primary epithelial cells from human meibomian glands and, second, to immortalize these cells, thereby developing a preclinical model that could be used to identify factors that regulate cell activity. Human meibomian glands were removed from lid segments after surgery, enzymatically digested, and dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, the cells were exposed to retroviral human telomerase reverse transcriptase (hTERT) and/or SV40 large T antigen cDNA vectors, and antibiotic-resistant cells were selected, expanded, and subcultured. Analyses for possible biomarkers, cell proliferation and differentiation, lipid-related enzyme gene expression, and the cellular response to androgen were performed with biochemical, histologic, and molecular biological techniques. It was possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free medium. These cells proliferated, survived through at least the fifth passage, and contained neutral lipids. Infection with hTERT immortalized these cells, which accumulated neutral lipids during differentiation, expressed multiple genes for lipogenic enzymes, responded to androgen, and continued to proliferate. The results show that human meibomian gland epithelial cells may be isolated, cultured, and immortalized.

Imatinib Mesylate Affects the Expression of Lipid Metabolism Genes in K562, a Chronic Myeloid Leukemia Cell Line

Background: Imatinib mesylate (IM) is a tyrosine kinase inhibitor designed to inhibit the BCR-ABL fusion protein, the hallmark of chronic myeloid leukemia (CML). Interestingly, IM treatment possesses a rapid lipid-lowering effect on patients with hyperlipidemia, particularly those receiving 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR) inhibitors. While it is known that IM may competitively inhibit statin metabolism, the lipid-lowering effect of IM is not fully understood. Therefore, we have studied the effects of IM on a family of genes critically involved in lipid metabolism, including low density lipoprotein receptor (LDL-R), HMGCoAR, cholesteryl ester transfer protein (CETP), apolipoprotein B (apoB) and apolipoprotein B editing complex (apobec1), in K562, a CML cell line model.Methods:Cell cultures and treatments: K562 cells were grown in DMEM supplemented with 10% FCS. Cells were treated with IM or DMSO (1mM) and were harvested after 24–96 hours. Untreated cells served as a control as did the DMSO-treated cells.RNA extraction and cDNA synthesis: RNA was extracted using Trizol reagent method and reverse transcribed using random primers and M-MLV.Real-time PCR assays: Real-time PCR assays were conducted using assays on demand for the different genes (Applied Biosystems, Foster City, CA, USA) and performed on Applied Biosystems 7900HT Prism® real-time PCR instrument with a 100ng equivalent of cDNA, in a total volume of 20 ml 1X Taqman master mix (Applied Biosystems). Results were quantified using the delta-delta cycle threshold method, normalized to actin beta and expressed as “fold-change from control”.Statistics: Results were analyzed using unpaired students' t-test.Results: LDL-R, HMGcoAR, CETP, apobec1 and apoB genes expression were evaluated in untreated K562 cells (control) and following treatment with DMSO (negative control) or IM for 24 to 96 hours. IM treatment of K562 cells significantly affected the expression of lipid-related genes. Namely, a time-dependent increase in LDL-R (3–6 fold), HMGcoAR (2–5 fold) and CETP (4–50 fold) expressions were documented following IM treatment. No increase in expression of these genes was observed in untreated and DMSO treated controls. Basal apobec1 and apoB expression detected in untreated K562 cells was abolished following IM but not DMSO treatment.Discussion: Putative mechanisms for the decrease in serum cholesterol and triglycerides observed in some patients treated with IM include reduced intestinal absorption and inhibition of platelet derived growth factor receptor (PDGFR), resulting in decreased lipoprotein lipase (LPL) synthesis. Our novel observation proposes another explanation, namely a direct effect of IM on central lipid-related genes.Conclusions: The direct effect of IM on lipid-related genes, together with the fact that in cell culture IM inhibits proliferation and induces apoptosis, suggest a possible role of lipids in apoptosis/cell cycle pathways. This possibility is currently the subject of further investigation.

Galanin Preproprotein Is Associated With Elevated Plasma Triglycerides

There is increasing physiological evidence in rodents connecting the neuropeptide galanin to triglyceride (TG) levels. We hypothesized that variation in the galanin preproprotein (GAL) gene may contribute to hypertriglyceridemia (HTG) in humans. We investigated GAL as a TG candidate gene by genotyping 4 tagSNPs in Dutch, Finnish, and Mexican familial combined hyperlipidemia (FCHL) families as well as in white combined hyperlipidemia cases/controls (n=2471). The common allele of rs2187331, residing in the promoter region of GAL, was significantly associated with HTG (probability value=0.00038). In an unascertained population sample of 4463 Finnish males, the rare allele of rs2187331 was associated with higher TGs (probability value=0.0028 to 0.00016). We also observed an allele specific difference with rs2187331 in reporter gene expression and nuclear factor binding in vitro. Furthermore, we detected differential expression of many key lipid genes in adipose tissue based on rs2187331 genotypes. The SNP rs2187331 is associated with HTG in FCHL and white combined hyperlipidemia cases/controls and influences TG levels in the population. Further studies are warranted to elucidate the allelic difference observed between FCHL and the general population. Functional evidence shows that rs2187331 has an allele specific cis-regulatory function and influences the expression of lipid related genes in adipose.

Posttranscriptional Regulation of Mammalian Circadian Clock Output

Circadian clocks are present in many different cell types/tissues and control many aspects of physiology. This broad control is exerted, at least in part, by the circadian regulation of many genes, resulting in rhythmic expression patterns of 5-10% of the mRNAs in a given tissue. Although transcriptional regulation is certainly involved in this process, it is becoming clear that posttranscriptional mechanisms also have important roles in producing the appropriate rhythmic expression profiles. In this chapter, we review the available data about posttranscriptional regulation of circadian gene expression and highlight the potential role of Nocturnin (Noc) in such processes. NOC is a deadenylase-a ribonuclease that specifically removes poly(A) tails from mRNAs-that is expressed widely in the mouse with high-amplitude rhythmicity. Deadenylation affects the stability and translational properties of mRNAs. Mice lacking the Noc gene have metabolic defects including a resistance to diet-induced obesity, decreased fat storage, changes in lipid-related gene expression profiles in the liver, and altered glucose and insulin sensitivities. These findings suggest that NOC has a pivotal role downstream from the circadian clockwork in the post-transcriptional regulation genes involved in the circadian control of metabolism.