Interleukin-6 and tumour necrosis factor-α differentially regulate lincRNA transcripts in cells of the innate immune system in vivo in human subjects with rheumatoid arthritis.

lincRNAs recently have been discovered as evolutionary conserved transcripts of non-coding DNA sequences and have been implicated in the regulation of cellular differentiation. In humans, molecular studies have suggested a functional role for lincRNAs in cancer development. The aim of the present study was to examine whether these novel molecules are specifically regulated by different cytokines in cells of the innate immune system in humans in vivo and whether lincRNAs thereby might be involved in the pathophysiology of rheumatoid arthritis (RA). Therefore, CD14(+) monocytes were isolated from RA patients before and after anti-IL-6R (tocilizumab) or anti-TNF-α (adalimumab) therapy and lincRNA transcription was analysed by a microarray based experiment. As expected, we found lincRNAs to be present in CD14(+) monocytes of RA patients. However, of the total number of 7.419 lincRNAs examined in this study only a very small number was significantly regulated by either IL-6 or TNF-α (85 lincRNAs, corresponding to 1.1%). The numbers of lincRNAs regulated was higher due to TNF-α compared to IL-6. Interestingly, none of the identified lincRNAs was influenced by both, IL-6 and TNF-α, suggesting the regulation of lincRNA transcription to be highly specific for distinct cytokines. Taken together, our results suggest (1) that lincRNAs are novel intracellular molecular effectors of specific cytokines in cells of the innate immune system in humans in vivo and (2) that lincRNAs might be involved in the molecular pathophysiology of RA.

Identifying evolutionarily conserved genes in the dietary restriction response using bioinformatics and subsequent testing in Caenorhabditis elegans.

Dietary restriction (DR) increases life span, health span and resistance to stress in a wide range of organisms. Work from a large number of laboratories has revealed evolutionarily conserved mechanisms that mediate the DR response. Here, we analyzed the genome-wide gene expression profiles of Caenorhabditis elegans under DR versus ad libitum conditions. Using the Ortho2ExpressMatrix tool, we searched for C. elegans orthologs of mouse genes that have been shown to be differentially expressed under DR conditions in nearly 600 experiments. Based on our bioinformatic approaches, we obtained 189 DR-responsive genes, and 45 of these are highly conserved from worm to man. Subsequent testing of sixteen genes that are up-regulated under DR identified eight genes that abolish the DR-induced resistance to heat stress in C. elegans. Further analyses revealed that fkb-4, dod-22 and ikb-1 genes also abolish increased life span in response to DR. The identified genes that are necessary for the DR response are sensitive to certain stress signals such as metabolic perturbances (dod-22, fkb-4 and nhr-85), DNA damage (ikb-1), heat shock (hsp-12.6) and cancer-like overgrowth (prk-2 and tsp-15). We propose that most of the DR-responsive genes identified are components of the recently discovered cellular surveillance-activated detoxification and defenses pathway, which is, among others, important for the survival of organisms in times of food deprivation.

Expression of fatty acid binding proteins 3 and 5 genes in rat pancreatic islets and INS-1E cells: regulation by fatty acids and glucose.

Fatty acids binding proteins (FABPs) are involved in uptake, binding, transport and metabolism of fatty acids (FAs). Although FAs are known to stimulate insulin secretion from pancreatic islets when transiently elevated, while contributing to islet loss of function, lipotoxicity and apoptosis when chronically elevated, almost nothing is known regarding the FABPs in this tissue. The present study aimed at exploring the expression pattern and regulation of FABPs in rat islets and the insulin-secreting INS-1E cells. Rat islets and INS-1E cells expressed the heart/muscle type (FABP3) and the epidermal type (FABP5) genes. Different FAs significantly enhanced the expression of both FABPs. High glucose concentration induced a similar elevation of both FABP mRNA levels, and similarly to its effect on insulin 1 mRNA. Addition of oleic or palmitic acids to glucose did not render a further effect. FABP3 gene expression increased in response to PPARα agonist, while FABP5 increased in response to PPARα and PPARγ agonists, and decreased in response to a PPARß agonist. Beta-oxidation of FAs is required for the gene expression of both FABP genes in INS-1E cells. Inhibition of CPT-1 by etomoxir inhibited the oleic acid-induced FABP 3 and 5 gene expression, while activation of AMPK by metformin amplified the oleic-induced expression of both FABPs. FABP3 and 5 gene transcription required de novo protein synthesis, since inhibition by cycloheximide significantly decreased both FABP mRNAs. These data show a complex interrelationship between glucose and FAs in the control of FABP gene expression and that FABP3 and 5 may play a role in insulin secretion.

Oleate regulates genes controlled by signaling pathways of mitogen-activated protein kinase, insulin, and hypoxia.

Oleate (C18:1) is, besides palmitate (C16:0), the most abundant fatty acid in the human diet, and its involvement in the development of insulin resistance is broadly discussed. Because its influence on gene expression is poorly defined in mammalian cells, we performed whole genome expression profiling and quantitative real-time polymerase chain reaction in the human hepatocyte cell line HepG2 to identify oleate-regulated genes. In this respect, HepG2 cells were exposed for 24 hours to a physiologic concentration of oleate coupled to bovine serum albumin (BSA) (200 micromol/L) or BSA alone. Subsequent microarray analysis revealed 14 genes that were significantly (single-sided permutational t test, P < .05) regulated after oleate treatment. To decipher the functional and regulatory connections of these genes, a text mining approach combined with transcription factor binding site analysis was performed using Genomatix BiblioSphere (Munich, Germany) and MatInspector (Munich, Germany). The oleate-inducible genes encoding early growth response 1, c-fos, S-phase kinase-associated protein 2, and splicing factor 2 are mapped into a network, which is controlled by signaling pathways of mitogen-activated protein kinase, insulin, or hypoxia. Comparative in silico promoter analysis revealed putative regulation of oleate-sensitive genes through v-ets erythroblastosis virus E26 oncogene homolog 1 and retinoid X receptor family. In sum, a physiologic oleate concentration modulates genes expression in a very sensitive way as 14 genes were regulated.

Identification of palmitate-regulated genes in HepG2 cells by applying microarray analysis.

Palmitate is the most abundant saturated fatty acid in the human diet and the major one synthesized de novo. To identify palmitate-regulated genes we performed whole genome mRNA expression profiling by using human hepatoma HepG2 cells. We identified eleven genes which are significantly (single-sided permutational t-test, p<0.05) regulated by low concentration of palmitate (50 microM). We observed a decreased expression of five metallothioneins, and an increased expression of liver expressed plasminogen activator inhibitor-1 protein and insulin-like growth factor II, which play a prominent role in the development of the metabolic syndrome. Comparative promoter analysis in-silico revealed common transcriptional regulation of differentially expressed genes through erythroid kruppel-like factor and members of the zinc binding protein factor family. In conclusion, low physiological palmitate concentrations changed expression of very responsive genes.

L-FABP T94A is associated with fasting triglycerides and LDL-cholesterol in women.

To determine the possible role of the common FABP1 T94A polymorphism in modulating susceptibility to traits of the metabolic syndrome, we analysed a random sample of 826 subjects from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. Multivariate adjusted linear trend regression analysis of metabolic, anthropometric and blood pressure variables in FABP1 T94A genotypes were performed in both genders. In women, a significant trend of higher plasma triglyceride (P=0.01) and LDL-cholesterol (P=0.02) concentrations were seen for A-allele carriers after adjustment for age, menopausal status, hormone intake and Apo E genotype. Because elevated triglyceride and cholesterol levels are important risk factors of cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM), we additionally analysed the association of the T94A variant and disease risks in two studies enrolling 220 incident CVD and 192 incident T2DM patients of the EPIC-Potsdam cohort. After adjusting for age, sex, BMI and other covariates, we found no association between FABP1 T94A and CVD or T2DM. In conclusion, our study provides evidence for an association of the FABP1 T94A polymorphism and fasting triglycerides and LDL-cholesterol levels in females. These results support previous findings in fenofibrate-treated individuals and thereby provide some additional indication of the functional relevance of the FABP1 T94A SNP in hepatic fatty acid and lipid metabolism in humans.

Cytosolic COOH terminus of the peptide transporter PEPT2 is involved in apical membrane localization of the protein.

The peptide transporter PEPT2 is a polytopic transmembrane protein that mediates the cellular uptake of di- and tripeptides and a variety of peptidomimetics. It is widely expressed in mammalian tissues, including kidney, lung, mammary gland, choroid plexus, and glia cells. In renal tubular cells, PEPT2 is exclusively found at the apical membrane. The molecular mechanisms underlying this polarized expression and targeting to the brush-border membrane are not known. We have explored the role of the 36 COOH-terminal amino acid residues in PEPT2 trafficking and apical expression. EGFP-tagged PEPT2 wild-type transporter and various truncated and mutant proteins were expressed in the polarized proximal tubule cell lines SKPT and OK, and the cellular distribution of the fusion proteins was assessed using confocal microscopy. Whereas deletion of the last seven amino acids (delC7) did not alter PEPT2 surface expression, deletion of the next residue (delC8) or up to 30 terminal amino acids resulted in impaired apical expression and distinct accumulation of mutant proteins in endosomal and lysosomal vesicles. Truncation of more amino acids (delC36) containing tyrosine-based motifs led to a rather diffuse intracellular distribution pattern. Mutations introduced at isoleucine-720 (I720A) and leucine-722 (I722A) also caused an impaired surface appearance. Internalization assays revealed a higher endocytotic rate of the PEPT2 mutants I720A, L722A, and delC36. Our data suggest that a three-amino acid stretch (INL) and tyrosine-based motifs within the COOH tail of PEPT2 are involved in PEPT2's apical membrane localization and membrane steady-state level.

Hypothyroidism induces expression of the peptide transporter PEPT2.

The kidney is a target organ for thyroid hormone action and a variety of renal transport processes are altered in response to impaired thyroid functions. To investigate the effect of thyroid hormone on the expression of the renal proximal tubular high-affinity-type H(+)-peptide cotransporter (PEPT2) in rats, hypothyroidism was induced in animals by administration of methimazole (0.05%) via drinking water. After 7 weeks of treatment, hypothyroidism was confirmed by determining serum free T(3) and free T(4) concentrations. Northern blotting was used to examine the expression of PEPT2 mRNA in kidney tissues from hypothyroid rats compared to control rats. Hypothyroidism resulted in an increased level of total renal PEPT2 mRNA (121.1+/-3.3% vs. control 100+/-2.8%; p=0.008). The mRNA results were confirmed by immuno-blotting, which demonstrated significantly increased protein levels (162% vs. control 100%; p<0.01). Immunohistochemistry also revealed increased PEPT2 protein levels in the proximal tubules of treated compared to non-treated rats. In summary, PEPT2 is the first proximal tubule transporter protein that shows increased expression in states of hypothyreosis. As PEPT2 reabsorbs filtered di- and tripeptides and peptide-like drugs, the present findings may have important implications in nutritional amino acid homeostasis and for drug dynamics in states of altered thyroid function.