Stine M. Ulven

Liver X receptors as insulin-mediating factors in fatty acid and cholesterol biosynthesis.

The nuclear receptor liver X receptor (LXR) α, an important regulator of cholesterol and bile acid metabolism, was analyzed after insulin stimulation in liver in vitro andin vivo. A time- and dose-dependent increase in LXRα steady-state mRNA level was seen after insulin stimulation of primary rat hepatocytes in culture. A maximal induction of 10-fold was obtained when hepatocytes were exposed to 400 nminsulin for 24 h. Cycloheximide, a potent inhibitor of protein synthesis, prevented induction of LXRα mRNA expression by insulin, indicating that the induction is dependent on de novo synthesis of proteins. Stabilization studies using actinomycin D indicated that insulin stimulation increased the half-life of LXRα transcripts in cultured primary hepatocytes. Complementary studies where rats and mice were injected with insulin induced LXRα mRNA levels and confirmed our in vitrostudies. Furthermore, deletion of both the LXRα and LXRβ genes (double knockout) in mice markedly suppressed insulin-mediated induction of an entire class of enzymes involved in both fatty acid and cholesterol metabolism. The discovery of insulin regulation of LXR in hepatic tissue as well as gene targeting studies in mice provide strong evidence that LXRs plays a central role not only in cholesterol homeostasis, but also in fatty acid metabolism. Furthermore, LXRs appear to be important insulin-mediating factors in regulation of lipogenesis.

Expression of the insulin-responsive glucose transporter GLUT4 in adipocytes is dependent on liver x receptor α

The insulin-responsive glucose transporter GLUT4 plays a crucial role in insulin-mediated facilitated glucose uptake into adipose tissue and muscle, and impaired expression of GLUT4 has been linked to obesity and diabetes. In this study, we demonstrate that liver X receptors (LXRs) regulate the expression of GLUT4 through direct interaction with a conserved LXR response element in the GLUT4 promoter. The expression of GLUT4 in WAT is induced by a potent LXR agonist in wild type, LXRα-/-, and LXRβ-/- mice but not in LXRα-/-β-/- mice, demonstrating that both LXRs are able to mediate ligand activated transcription of the GLUT4 gene. However, basal and insulin stimulated expression of GLUT4 in epididymal WAT is reduced only in mice carrying ablation of the LXRα isoform. The expression of GLUT4 is furthermore correlated to the induction of LXRα during mouse and human adipocyte differentiation. LXRβ is thus apparently not able to rescue basal expression of GLUT4 in the absence of LXRα. We have previously demonstrated that LXRα is down-regulated in animal models of obesity and diabetes, thus revealing a striking correlation between GLUT4 and LXRα expression in insulin-resistant conditions. This suggests that the LXRα isoform has a unique role in adipose expression of GLUT4 and suggests that alteration of adipose tissue expression of LXRα might be a novel tool to normalize the expression of a gene that is dysregulated in diabetic and insulin-resistant conditions.

Effects of an isocaloric healthy Nordic diet on insulin sensitivity, lipid profile and inflammation markers in metabolic syndrome – a randomized study (SYSDIET)

Different healthy food patterns may modify cardiometabolic risk. We investigated the effects of an isocaloric healthy Nordic diet on insulin sensitivity, lipid profile, blood pressure and inflammatory markers in people with metabolic syndrome.

PPARα activators and fasting induce the expression of adipose differentiation-related protein in liver

The adipose differentiation-related protein (ADFP)/adipophilin belongs to a family of PAT (for perilipin, ADFP, and TIP47) proteins that associate on the surface of lipid droplets (LDs). Except for LD association, a clear role for ADFP has not been found. We demonstrate that ADFP is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα) in mouse liver and rat and human hepatoma cells through a highly conserved direct repeat-1(DR-1) element. Although the ADFP mRNA is highly increased by a synthetic PPARα agonist, the ADFP protein is only substantially increased in cells containing LDs, such as hepatocytes incubated with fatty acids, and in livers of fasted mice. ADFP is induced by fasting even in the absence of a functional PPARα, in marked contrast to the PPARα target gene acyl-coenzyme A oxidase-1. Activation of LXRs, which stimulates LD formation through the activation of lipogenesis, does not affect ADFP mRNA levels. TIP47, another PAT member known to be expressed in liver, was unaffected by all treatments. This constitutively expressed PAT member seems to be less transcriptionally regulated than ADFP. These observations suggest that ADFP is primarily a fasting-induced protein in liver that coats the newly synthesized triacylglycerol-containing LDs formed during fasting.

Tissue-specific autoregulation of the LXRα gene facilitates induction of apoE in mouse adipose tissue

The functions of the liver X receptors (LXRs) are not well documented in adipose tissue. We demonstrate here that expression of the LXRα gene is highly induced in vivo and in vitro in mouse and human adipocytes in the presence of the synthetic LXR agonist T0901317. This autoregulation is caused by an identified LXR-responsive element motif in the mouse LXRα promoter, which is conserved in the human LXRα promoter. Using different LXR-deficient mice, we demonstrate that the basal expression level of LXRα is increased in LXRβ−/− mice, whereas the basal expression level of LXRβ is unchanged in LXRα−/− mice. The two LXRs can compensate for each other in mediating ligand-activated regulation of LXR target genes involved in lipid homeostasis in adipose tissue. Sterol regulatory element binding protein-1 (SREBP-1), ATP binding cassette transporter A1 (ABCA1), ABCG1, as well as apolipoprotein E (apoE) are induced in vivo by T0901317 in wild-type, LXRα−/− or LXRβ−/− mice but not in LXRα−/−β−/− mice. Although SREBP-1 and ABCG1 are induced in liver, muscle, and adipose tissue, the apoE, glucose transporter-4 (GLUT4), and LXRα genes are specifically induced only in adipose tissue. We suggest that an important aspect of LXRα autoregulation in adipose tissue may be to increase the level of LXRα over a threshold level necessary to induce the expression of certain target genes.

Intracellular Nicotinamide Phosphoribosyltransferase Protects against Hepatocyte Apoptosis and Is Down-Regulated in Nonalcoholic Fatty Liver Disease

CONTEXT Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western and non-Western countries, but its pathogenesis is not fully understood. OBJECTIVE Based on the role of nicotinamide phosphoribosyltransferase (NAMPT) in fat and glucose metabolism and cell survival, we hypothesized a role for NAMPT/visfatin in the pathogenesis of NAFLD-related disease. DESIGN AND SETTING We conducted clinical studies at a referral medical center in well-characterized NAFLD patients (n = 58) and healthy controls (n = 27). In addition we performed experimental in vitro studies in hepatocytes. MAIN OUTCOME MEASURES We examined 1) the hepatic and systemic expression of NAMPT/visfatin in patients with NAFLD and control subjects, 2) the hepatic regulation of NAMPT/visfatin, and 3) the effect of NAMPT/visfatin on hepatocyte apoptosis. RESULTS Our main findings were as follows. 1) Patients with NAFLD had decreased NAMPT/visfatin expression both systemically in serum and within the hepatic tissue, with no difference between simple steatosis and nonalcoholic steatohepatitis. 2) By studying the hepatic regulation of NAMPT/visfatin in wild-type and peroxisome proliferators-activated receptor (PPAR)alpha(-/-) mice as well as in hepatocytes, we showed that PPARalpha activation and glucose may be involved in the down-regulation of hepatic NAMPT/visfatin expression in NAFLD. 4) Within the liver, NAMPT/visfatin was located to hepatocytes, and our in vitro studies showed that NAMPT/visfatin exerts antiapoptotic effects in these cells, involving enzymatic synthesis of nicotinamide adenine dinucleotide. CONCLUSION Based on these findings, we suggest a role for decreased NAMPT/visfatin levels in hepatocyte apoptosis in NAFLD-related disease.

Oxidised fish oil does not influence established markers of oxidative stress in healthy human subjects: a randomised controlled trial.

Intake of fish oil reduces the risk of CHD and CHD deaths. Marine n-3 fatty acids (FA) are susceptible to oxidation, but to our knowledge, the health effects of intake of oxidised fish oil have not previously been investigated in human subjects. The aim of the present study was to investigate markers of oxidative stress, lipid peroxidation and inflammation, and the level of plasma n-3 FA after intake of oxidised fish oil. In a double-blinded randomised controlled study, healthy subjects (aged 18-50 years, n 54) were assigned into one of three groups receiving capsules containing either 8 g/d of fish oil (1.6 g/d EPA+DHA; n 17), 8 g/d of oxidised fish oil (1.6 g/d EPA+DHA; n 18) or 8 g/d of high-oleic sunflower oil (n 19). Fasting blood and morning spot urine samples were collected at weeks 0, 3 and 7. No significant changes between the different groups were observed with regard to urinary 8-iso-PGF2α; plasma levels of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and α-tocopherol; serum high sensitive C-reactive protein; or activity of antioxidant enzymes in erythrocytes. A significant increase in plasma level of EPA+DHA was observed in both fish oil groups, but no significant difference was observed between the fish oil groups. No changes in a variety of in vivo markers of oxidative stress, lipid peroxidation or inflammation were observed after daily intake of oxidised fish oil for 3 or 7 weeks, indicating that intake of oxidised fish oil may not have unfavourable short-term effects in healthy human subjects.

Quantitative axial profiles of retinoic acid in the embryonic mouse spinal cord: 9-cis retinoic acid only detected after all-trans-retinoic acid levels are super-elevated experimentally.

Studies using bioassays in normal mice and gene activation in transgenic reporter mice have demonstrated peaks of retinoic acid receptor (RAR) signaling in the brachial and lumbar regions of the spinal cord. Recently, Solomin et al. (Solomin et al. [ 1998 ] Nature 395:398–402) detected a retinoid X receptor (RXR) signal in the same region of the developing spinal cord at a slightly later stage than the RAR signal. This finding raises the question of which retinoid ligands underlie RAR and RXR signaling in this part of the embryo. Quantitative measurements of regional differences in retinoid profiles have not been reported previously due to limitation in the sensitivity and specificity of available retinoid detection methods. Here, by using a recently developed ultrasensitive HPLC technique (Sakhi et al. [ 1998 ] J. Chromatogr. A 828:451–460), we address this question in an attempt to identify definitively the endogenous retinoids present in different regions of the spinal cord at the stages when regional differences in RAR and RXR signaling have been reported. We find a bimodal distribution of all‐trans retinoic acid (at‐RA), the ligand for RARs, and relate this to the expression of several retinoid‐synthesizing enzymes. However, we do not detect 9‐cis‐retinoic acid (9‐cis‐RA), the putative RXR ligand, in any region of the spinal cord unless retinoid levels are massively increased experimentally by gavage feeding pregnant mice with teratogenic doses of at‐RA. This study provides for the first time quantitative profiles of endogenous retinoids along the axis of the developing spinal cord, thereby establishing a foundation for more definitive studies of retinoid function in the future. It sets definite limits on how much 9‐cis‐RA potentially is present and demonstrates that at‐RA predominates over 9‐cis‐RA by at least 30‐ to 180‐fold in different spinal cord regions.

RAR-, not RXR, ligands inhibit cell activation and prevent apoptosis in B-Lymphocytes

We have previously shown that retinoids inhibit activation of human peripheral blood B‐lymphocytes. In the present paper, we wished to explore the involvement of nuclear retinoid‐specific receptors in this process by using ligands specific for the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We found that the RAR‐specific ligand TTAB reduced anti‐IgM‐induced B‐cell activation in a dose‐dependent manner. Thus, at 100 nM of TTAB, DNA synthesis was reduced by approximately 60%. In contrast, the RXR‐selective ligand SR11217 had no effect on DNA synthesis. Similar findings were obtained when the expression of the activation antigen CD71 (appears late in G1) was examined. The role of retinoids in apoptosis of resting peripheral blood B‐lymphocytes was examined using the same receptor‐selective ligands. Again, we found that the RAR‐selective ligands were more potent effectors than were the RXR‐selective ligands. In spite of the inhibitory effects of retinoids on B‐cell proliferation, the same retinoids significantly promoted the survival of the cells. Thus, 10 nM TTAB significantly reduced spontaneous apoptosis of in vitro cultured B‐cells at day 3 from 45% to 30%, as determined by vital dye staining and DNA end‐labeling. Again, the RXR‐specific ligand SR11217 had no effect. Interestingly, we found that CD40 ligand was able to potentiate the retinoid‐mediated inhibition of apoptosis. By reverse transcriptase polymerase chain reaction (PCR), we found that peripheral blood B‐lymphocytes expressed RARα, RARγ, and RXRα, but not RARβ, RXRβ, or RXRγ. Hence, the lack of effect of the RXR‐specific ligand SR11217 on growth and apoptosis was not due to absence of RXRs. In conclusion, the ability of retinoids to inhibit growth and prevent apoptosis of normal human B‐lymphocytes indicates a dual role of retinoids in this cell compartment, and it appears that both effects of retinoids are mediated via RARs and not RXRs. J. Cell. Physiol. 175:68–77, 1998.

Arsenic in the human food chain, biotransformation and toxicology - Review focusing on seafood arsenic

Fish and seafood are main contributors of arsenic (As) in the diet. The dominating arsenical is the organoarsenical arsenobetaine (AB), found particularly in finfish. Algae, blue mussels and other filter feeders contain less AB, but more arsenosugars and relatively more inorganic arsenic (iAs), whereas fatty fish contain more arsenolipids. Other compounds present in smaller amounts in seafood include trimethylarsine oxide (TMAO), trimethylarsoniopropionate (TMAP), dimethylarsenate (DMA), methylarsenate (MA) and sulfur-containing arsenicals. The toxic and carcinogenic arsenical iAs is biotransformed in humans and excreted in urine as the carcinogens dimethylarsinate (DMA) and methylarsonate (MA), producing reactive intermediates in the process. Less is known about the biotransformation of organoarsenicals, but new insight indicates that bioconversion of arsenosugars and arsenolipids in seafood results in urinary excretion of DMA, possibly also producing reactive trivalent arsenic intermediates. Recent findings also indicate that the pre-systematic metabolism by colon microbiota play an important role for human metabolism of arsenicals. Processing of seafood may also result in transformation of arsenicals.