Nonalcoholic fatty liver disease is associated with an altered hepatocyte microRNA profile in LDL receptor knockout mice

MicroRNAs modulate processes associated with cell cycle control and differentiation. Here we explored the potential of microRNAs in the modulation of hepatic lipid metabolism and the development of nonalcoholic fatty liver disease.MicroRNA profiles of hepatocytes from low-density lipoprotein (LDL) receptor knockout mice fed a chow diet or a hypertriglyceridemia/fatty liver-inducing Western-type diet (WTD) were determined using quantitative real-time polymerase chain reaction. Ninety-seven of 103 microRNAs measured were expressed by hepatocytes and low variability between hepatocyte pools was observed. Feeding WTD coincided with a marked fivefold decrease in the relative expression level of miR-216 (P<.05) and miR-302a (P<.01). Interestingly, an increased hepatic miR-216 expression was detected in response to fasting. MicroRNA/biological function linkage analysis suggested that the change in hepatocyte microRNA profiles in response to high dietary lipid levels is associated with changes in cell cycle control and proliferation. In accordance with a diminished miR-302a expression on the WTD, hepatocyte mRNA expression levels of miR-302a target genes ABCA1 and in particular ELOVL6 were increased in response to WTD (twofold to ninefold). This suggests a role for miR-302a in hepatic cholesterol, fatty acid and glucose metabolism.In conclusion, we have shown that fatty liver development in LDL receptor knockout mice is associated with a significant change in the hepatocyte microRNA profile, i.e., a fivefold decrease in miR-216 and miR-302a expression. Based upon our comparative gene and microRNA expression studies it is anticipated that miR-302a may prove to be a valuable therapeutic target in the regulation of hepatic fatty acid utilization and insulin resistance.     

Adenovirus-mediated hepatic overexpression of scavenger receptor class B type I accelerates chylomicron metabolism in C57BL/6J mice

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of HDL cholesteryl esters is well established. In SR-BI-deficient mice, we recently observed a delayed postprandial triglyceride (TG) response, suggesting an additional role for SR-BI in facilitating chylomicron (CM) metabolism. Here, we assessed the effect of adenovirus-mediated hepatic overexpression of SR-BI (Ad.SR-BI) in C57BL/6J mice on serum lipids and CM metabolism. Infection of 5 x 10(8) plaque-forming units per mouse of Ad.SR-BI significantly decreases serum cholesterol (>90%), phospholipids (>90%), and TG levels (50%), accompanied by a 41.4% reduction (P < 0.01) in apolipoprotein B-100 levels. The postprandial TG response is 2-fold lower in mice treated with Ad.SR-BI compared with control mice (area under the curve = 31.4 +/- 2.4 versus 17.7 +/- 3.2; P < 0.05). Hepatic mRNA expression levels of genes known to be involved in serum cholesterol and TG clearance are unchanged and thus could not account for the decreased plasma TG levels and the change in postprandial response. We conclude that overexpression of SR-BI accelerates CM metabolism, possibly by mediating the initial capture of CM remnants by the liver, whereby the subsequent internalization can be exerted by additional receptor systems such as the LDL receptor (LDLr) and LDLr-related protein 1.     

Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90 degrees C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80 degrees C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80 degrees C. Interestingly, addition of the dye to the denatured VHH-R2 at 80 degrees C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibody-antigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications.     

Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

Dynamic shear stress in parallel-plate flow chambers

An in vitro model using a parallel-plate fluid flow chamber is supposed to simulate in vivo fluid shear stresses on various cell types exposed to dynamic fluid flow in their physiological environment. The metabolic response of cells in vitro is associated with the wall shear stress. However, parallel-plate flow chambers have not been characterized for dynamic fluid flow experiments. We use a dimensionless ratio h / lambda(v), in determining the exact magnitude of the dynamic wall shear stress, with its oscillating components scaled by a shear factor T. It is shown that, in order to expose cells to predictable levels of dynamic fluid shear stress, two conditions have to be met: (1) h / lambda(v) < 2, where h is the distance between the plates and lambda(v) is the viscous penetration depth; and (2) f(0) < f(c) / m, where the critical frequency f(c) is the upper threshold for this flow regime, m is the highest harmonic mode of the flow, and f(0) is the fundamental frequency of fluid flow.     

Specific gene expression of ATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal,endothelial, and Kupffer cells

Hepatic cholesterol(ester) uptake from serum coupled to intracellular processing and biliary excretion are important features in the removal of excess cholesterol from the body. ATP-binding cassette (ABC) transporters play an important role in hepatic cholesterol transport. The liver consists of different cell types, and ABC transporters may exert different physiological functions dependent on the individual cell type. Therefore, in the current study, using real time PCR we compared the mRNA expression of ABC transporters and genes involved in the regulation of cholesterol metabolism in liver parenchymal, endothelial, and Kupffer cells. It appears that liver parenchymal cells contain high expression levels compared with endothelial and Kupffer cells of scavenger receptor class BI ( approximately 3-fold), peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma (8-20-fold), cholesterol 7alpha-hydroxylase A1 (>100-fold), and ABCG5/G8 ( approximately 5-fold). Liver endothelial cells show a high expression of cholesterol 27-hydroxylase, liver X receptor (LXR)beta, PPARdelta, and ABCG1, suggesting a novel specific role for these genes in endothelial cells. In Kupffer cells, the expression level of LXRalpha, ABCA1, and in particular ABCG1 is high, leading to an ABCG1 mRNA expression level that is 70-fold higher than in parenchymal cells. It can be calculated that 51% of the total liver ABCG1 expression resides in Kupffer cells and 24% in endothelial cells, suggesting an intrahepatic-specific role for ABCG1 in Kupffer and endothelial cells. Because of a specific stimulation of ABCG1 in parenchymal cells by a high cholesterol diet, the contribution of parenchymal cells to the total liver increased from 25 to 60%. Our data indicate that for studies of the role of ABC transporters and their regulation in liver, their cellular localization should be taken into account, allowing proper interpretation of metabolic changes, which are directly related to their (intra)cellular expression level.     

Ecotoxicological threshold levels of a mixture of herbicides (atrazine, diuron and metolachlor) in freshwater microcosms

Twelve indoor, plankton-dominated, freshwater microcosms (600 l) were used to study the effect of a mixture of herbicides on structural and functional aspects of these ecosystems. The EC50, 72 h values of the most susceptible standard test alga Selenastrum capricornutum (EC50, atrazine=54 μg l−1, EC50, diuron=15 μg l−1, EC50, metolachlor=56 μg l−1) were used as a starting point for the dosage applied in the microcosms (dosages: 0, 0.01, 0.03, 0.1, 0.3, 1× EC50). The microcosms were exposed to chronic levels for 28 days and subsequently monitored for 4 more weeks. The following effects were observed: (1) direct effects became apparent from an initial drop in photosynthesis efficiency, pH and oxygen concentration and a decrease in the abundance of several phytoplankton taxa at the 0.3 × EC50 treatment level and higher. (2) Fourteen days post application an increase in the abundance of several phytoplankton taxa (Chlamydomonas sp. and Stephanodiscus/Cyclotella) was observed; oxygen concentrations recovered while alkalinity, conductivity and total inorganic nitrogen were elevated. (3) Effects on fauna were minor. Daphnia galeata showed a decreasing trend and the cyclopoid copepods an increasing trend at the end of the experiment. Multivariate statistical analyses demonstrated no effects of any treatment level on the zooplankton community. Effects were reported for the phytoplankton community at dose levels of 0.3 × EC50 and higher. On species level the most sensitive taxon was Chlorophyceae coccales. For this taxon a NOEC at the dose level of 0.01 × EC50 was calculated. This effect however was relatively small in magnitude and merely based on an increase in numbers in the control and lowest treated microcosms rather than a decrease in numbers in all other treatments. The standards based on algal toxicity data, as adopted by the Uniform Principles, consist of a safety factors of 0.1 to be multiplied with the EC50. The NOEC of coccales was lower than 0.1 × EC50. All other observed variables in this aquatic ecosystem were sufficiently protected against the mixture of herbicides by the safety factor as proposed in the Uniform Principles. This revised version was published online in August 2006 with corrections to the Cover Date.     

Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation

In addition to glutathione (GSH) conjugating activity, glutathione S-transferases (GSTs) catalyze "reverse" reactions, such as the hydrolysis of GSH thiol esters. Reverse reactions are of interest as potential tumor-directed pro-drug activation strategies and as mechanisms for tissue redistribution of carboxylate-containing drugs. However, the mechanism and specificity of GST-mediated GSH thiol ester hydrolysis are uncharacterized. Here, the GSH thiol esters of ethacrynic acid (E-SG) and several nonsteroidal antiinflammatory agents have been tested as substrates with human GSTs. The catalytic hydrolysis of these thiol esters appears to be a general property of GSTs. The hydrolysis of the thiol ester of E-SG was studied further with GSTA1-1 and GSTP1-1, as a model pro-drug with several possible fates for the hydrolysis products: competitive inhibition, covalent enzyme adduction, and sequential metabolism. In contrast to hydrolysis rates, significant isoform-dependent differences in the subsequent fate of the products ethacrynic acid and GSH were observed. At low [E-SG], only the GSTP1-1 efficiently catalyzed sequential metabolism, via a dissociative mechanism.     

You can't always get what you want: size assortative mating by mutual mate choice as a resolution of sexual conflict

Background  

Assortative mating patterns for mate quality traits like body size are often observed in nature. However, the underlying mechanisms that cause assortative mating patterns are less well known. Sexual selection is one important explanation for assortment, suggesting that i) one (usually the female) or both sexes could show preferences for mates of similar size or ii) mutual mate choice could resolve sexual conflict over quality traits into assortment. We tested these hypotheses experimentally in the socially monogamous cichlid fish Pelvicachromis taeniatus, in which mate choice is mutual.     

Hepatic Adaptation Compensates Inactivation of Intestinal Arginine Biosynthesis in Suckling Mice

Suckling mammals, including mice, differ from adults in the abundant expression of enzymes that synthesize arginine from citrulline in their enterocytes. To investigate the importance of the small-intestinal arginine synthesis for whole-body arginine production in suckling mice, we floxed exon 13 of the argininosuccinate synthetase (Ass) gene, which codes for a key enzyme in arginine biosynthesis, and specifically and completely ablated Ass in enterocytes by crossing Ass ^fl and Villin-Cre mice. Unexpectedly, Ass ^fl/fl /VilCre ^tg/- mice showed no developmental impairments. Amino-acid fluxes across the intestine, liver, and kidneys were calculated after determining the blood flow in the portal vein, and hepatic and renal arteries (86%, 14%, and 33%, respectively, of the transhepatic blood flow in 14-day-old mice). Relative to control mice, citrulline production in the splanchnic region of Ass ^fl/fl /VilCre ^tg/- mice doubled, while arginine production was abolished. Furthermore, the net production of arginine and most other amino acids in the liver of suckling control mice declined to naught or even changed to consumption in Ass ^fl/fl /VilCre ^tg/- mice, and had, thus, become remarkably similar to that of post-weaning wild-type mice, which no longer express arginine-biosynthesizing enzymes in their small intestine. The adaptive changes in liver function were accompanied by an increased expression of genes involved in arginine metabolism (Asl, Got1, Gpt2, Glud1, Arg1, and Arg2) and transport (Slc25a13, Slc25a15, and Slc3a2), whereas no such changes were found in the intestine. Our findings suggest that the genetic premature deletion of arginine synthesis in enterocytes causes a premature induction of the post-weaning pattern of amino-acid metabolism in the liver.