Changes In Hepatic Expression Research Articles

Age-related changes in hepatic expression and activity of cytochrome P450 in male rats

Age-related changes in hepatic expression and activity of cytochrome P450 (CYP) were investigated in male rats aged 3 (weanling), 12 (young), 26 (adult), and 104 (old) weeks. Levels of microsomal protein, total CYP, and cytochrome b(5) increased fully after puberty. CYP1A1 was detected only in 3-week-old rats, and CYP1A2, CYP2B1, and CYP2E1 were maximally expressed at 3 weeks but decreased at 12 and 26 weeks. CYP2C11 and CYP3A2 increased markedly after puberty and decreased with aging. Ethoxyresorufin-O-deethylase, methoxyresorufin-O-demethylase, pentoxyresorufin-O-depenthylase, and p-nitrophenol hydroxylase activities were at their highest in 3-week-old rats, and midazolam hydroxylase activity was at a maximum in 12-week-old rats but decreased with aging. The present results show that increasing age caused significant alterations in hepatic expression/activity of CYP isoforms in an isoform-specific manner. These results suggest that age-related changes in hepatic CYP isoforms may be an important factor for deciding the efficacy and safety of xenobiotics.

Gene profiling of maternal hepatic adaptations to pregnancy

Maternal metabolic demands change dramatically during the course of gestation and must be co-ordinated with the needs of the developing placenta and fetus. The liver is critically involved in metabolism and other important functions. However, maternal hepatic adjustments to pregnancy are poorly understood. The aim of the study was to evaluate the influences of pregnancy on the maternal liver growth and gene expression profile. Holtzman Sprague-Dawley rats were mated and sacrificed at various stages of gestation and post-partum. The maternal livers were analysed in gravimetric response, DNA content by PicoGreen dsDNA quantitation reagent, hepatocyte ploidy by flow cytometry and hepatocyte proliferation by ki-67 immunostaining. Gene expression profiling of non-pregnant and gestation d18.5 maternal hepatic tissue was analysed using a DNA microarray approach and partially verified by northern blot or quantitative real-time PCR analysis. During pregnancy, the liver exhibited approximately an 80% increase in size, proportional to the increase in body weight of the pregnant animals. The pregnancy-induced hepatomegaly was a physiological event of liver growth manifested by increases in maternal hepatic DNA content and hepatocyte proliferation. Pregnancy did not affect hepatocyte polyploidization. Pregnancy-dependent changes in hepatic expression were noted for a number of genes, including those associated with cell proliferation, cytokine signalling, liver regeneration and metabolism. The metabolic demands of pregnancy cause marked adjustments in maternal liver physiology. Central to these adjustments are an expansion in hepatic capacity and changes in hepatic gene expression. Our findings provide insights into pregnancy-dependent hepatic adaptations.

Clodronate Liposomes: All Sites of Injection Are Not Equal

We appreciate the interest of Dr. Leclercq and colleagues in our article.1 In their correspondence,2 Leclercq and colleagues take issue with respect to the design and some of the outcomes of our experiments, which revealed important cross-talk between Kupffer cells and hepatocytes in the regulation of hepatic triglyceride storage. Here, we would like to take the opportunity to respond to the specific issues raised and continue to make a strong case for a relation between Kupffer cells and liver triglycerides. It should be emphasized that the chronic high-fat feeding protocol as employed in our study represents the most widely used model for diet-induced obesity and metabolic syndrome in mice. Leclercq et al. raise the point that Kupffer cells are not activated in this model, which is based on the lack of change in hepatic expression of macrophage markers Cd68 and F4/80, as well as interleukin-1beta (IL-1β). Although this may be true, it is unclear to us why Kupffer cells have to be activated to be able to study the effect of Kupffer cell deletion on hepatic lipid storage. In our study, the purpose of chronic high-fat feeding was to induce obesity-related hepatic steatosis, not to create a state of steatohepatitis. Second, Leclercq et al. assert that intraperitoneal injection of clodronate liposomes not only depletes hepatic Kupffer cells but also adipose tissue macrophages. Unfortunately, the experimental backing of this claim is weak. As revealed by the gene expression data, the effects of clodronate liposomes on expression of macrophage markers in epididymal adipose tissue are small and dwarfed by the dramatic changes observed in liver. Nevertheless, we acknowledge in the discussion that we cannot completely rule out a role of nonhepatic macrophages in the observed reduction in hepatic lipids and increase in peroxisome proliferator-activated receptor α activity but consider this explanation very unlikely. Third, it is argued that, in conflict with existing literature, the mice fed a high-fat diet do not exhibit adipose tissue inflammation. However, our expression data perfectly copy the established literature by showing marked induction of macrophage markers, including all those that were reported in the original publication by Xu et al.3 Histological data that were not included in our manuscript support enhanced macrophage infiltration in the mice fed a high-fat diet. The alleged absence of adipose tissue inflammation is based on perceived lack of induction of tumor necrosis factor-α, IL-1β, and IL-6 expression in adipose tissue of the mice. However, induction of these cytokines is rarely observed in diet-induced obesity models and does not define adipose tissue inflammation. Perhaps more importantly, it is unclear to us why induction of adipose tissue inflammation by the dietary treatment has any relevance to our findings that entirely pertain to liver. Finally, we would like to stress that a number of very recent studies reported findings similar to ours and corroborate a role for Kupffer cells in hepatic fatty acid metabolism and hepatic steatosis.4-7 Thus, we feel that a role of Kupffer cells in ameliorating steatosis has been convincingly demonstrated. Sander Kersten*, Rinke Stienstra*, Michael Müller*, * Nutrigenomics Consortium, TI Food and Nutrition, Wageningen, the Netherlands, and Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands.

Metabolic acidosis in sheep alters expression of renal and skeletal muscle amino acid enzymes and transporters1

To determine the effect of metabolic acidosis on expression of L-Gln, L-Glu, and L-Asp metabolizing enzymes and transporters, the relative content of mRNA, protein, or mRNA and protein, of 6 enzymes and 5 transporters was determined by real-time reverse transcription-PCR and immunoblot analyses in homogenates of kidney, skeletal muscle, and liver of growing lambs fed a common diet supplemented with canola meal (control; n = 5) or HCl-treated canola meal (acidosis; n = 5). Acidotic sheep had a 790% greater (P = 0.050) expression of renal Na(+)-coupled neutral AA transporter 3 mRNA and a decreased expression of renal glutamine synthetase mRNA (47% reduction, P = 0.037) and protein (57% reduction, P = 0.015) than control sheep. No change in renal cytosolic phosphoenolpyruvate carboxykinase (protein and mRNA), glutaminase (mRNA), or L-Glu dehydrogenase (protein) was found. In skeletal muscle, acidotic sheep had 101% more (P = 0.026) aspartate transaminase protein than did control sheep, whereas no change in the content of 3 Na(+)-coupled neutral AA transporters (mRNA) or 2 high-affinity L-Glu transporter proteins was found. In liver, no change in the content of any assessed enzyme or transporter was found. Collectively, these findings suggest that tissue-level responses of sheep to metabolic acidosis are different than for nonruminants. More specifically, these results indicate the potential capacity for metabolism of L-Asp and L-Glu by skeletal muscle, and L-Gln absorption by kidneys, but no change in hepatic expression of L-Gln metabolism, elaborates previous metabolic studies by revealing molecular-level responses to metabolic acidosis in sheep. The reader is cautioned that the metabolic acidosis model employed in this study differs from the increased plasma lactate-induced metabolic acidosis commonly observed in ruminants fed a highly fermentable grain diet.

Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs

The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we studied gene expression changes in peripheral lymphoid tissues as compared to hepatic expression changes, 14–18 h after lung infection in pigs. The lung infection was established with the pig specific respiratory pathogen Actinobacillus pleuropneumoniae. Quantitative real-time PCR based expression analysis were performed on samples from liver, tracheobronchial lymph node, tonsils, spleen and on blood leukocytes, supplemented with measurements of interleukin-6 and selected acute phase proteins in serum. C-reactive protein and serum amyloid A were clearly induced 14–18 h after infection. Extrahepatic expression of acute phase proteins was found to be dramatically altered as a result of the lung infection with an extrahepatic acute phase protein response occurring concomitantly with the hepatic response. This suggests that the acute phase protein response is a more disseminated systemic response than previously thought. The current study provides to our knowledge the first example of porcine extrahepatic expression and regulation of C-reactive protein, haptoglobin, fibrinogen, pig major acute phase protein, and transferrin in peripheral lymphoid tissues.

Isolation of the cDNA encoding the acid labile subunit (ALS) of the 150 kDa IGF-binding protein complex in cattle and ALS regulation during the transition from pregnancy to lactation

During the transition from pregnancy to lactation, dairy cows experience a 70% reduction in plasma IGF-I. This reduction has been attributed to decreased hepatic IGF-I production. IGF-I circulates predominantly in multi-protein complexes consisting of one molecule each of IGF-I, IGF binding protein-3 and the acid labile subunit (ALS). Recent studies in the mouse have shown that absence of ALS results in accelerated turnover and severely depressed concentration of plasma IGF-I. These observations suggest that reduced plasma ALS could be a second factor contributing to the fall of plasma IGF-I in peri-parturient cows. This possibility has not been studied due to the lack of bovine ALS reagents. To address this, we isolated the bovine ALS cDNA and used its sequence to develop a ribonuclease protection assay (RPA) and a bovine ALS antiserum. Using the RPA, ALS mRNA abundance was approximately fivefold higher in liver than in lung, small intestine, adipose tissue, kidney and heart, but was absent in muscle and brain. The antiserum detected the highest ALS levels in plasma followed by ovarian follicular fluid, lymph and colostrum. A portion of colostrum and follicular fluid ALS appears to be synthesized locally as ALS mRNA was found in mammary epithelial cells and ovarian follicular cells. Finally, we measured plasma ALS in dairy cows during the peri-parturient period (days -35 and +56 relative to parturition on day 0). Plasma ALS dropped by 50% between late pregnancy and the first day of lactation and returned to prepartum levels by day +56. To determine whether this reflected a change in hepatic expression, ALS mRNA was measured in liver biopsies collected on days -35, +3 and +56. ALS mRNA expression was significantly lower on day +3 than on day -35, but recovered completely by day +56. Finally, we examined the ability of GH to increase plasma ALS abundance at selected times before and after parturition (weeks -5, -2, +1 and +5). GH increased plasma ALS at weeks -5, -2 and +5, but not at week +1. Identical effects of GH were seen when the response considered was plasma IGF-I. We conclude that the decline in plasma ALS after parturition is a consequence of hepatic GH resistance and contributes to the associated reduction of plasma IGF-I.

Phosphatidylcholine transfer protein regulates size and hepatic uptake of high-density lipoproteins

Phosphatidylcholine transfer protein (PC-TP) is a steroidogenic acute regulatory-related transfer domain protein that is enriched in liver cytosol and binds phosphatidylcholines with high specificity. In tissue culture systems, PC-TP promotes ATP-binding cassette protein A1-mediated efflux of cholesterol and phosphatidylcholine molecules as nascent pre-beta-high-density lipoprotein (HDL) particles. Here, we explored a role for PC-TP in HDL metabolism in vivo utilizing 8-wk-old male Pctp(-/-) and wild-type littermate C57BL/6J mice that were fed for 7 days with either chow or a high-fat/high-cholesterol diet. In chow-fed mice, neither plasma cholesterol concentrations nor the concentrations and compositions of plasma phospholipids were influenced by PC-TP expression. However, in Pctp(-/-) mice, there was an accumulation of small alpha-migrating HDL particles. This occurred without changes in hepatic expression of ATP-binding cassette protein A1 or in proteins that regulate the intravascular metabolism and clearance of HDL particles. In Pctp(-/-) mice fed the high-fat/high-cholesterol diet, HDL particle sizes were normalized, whereas plasma cholesterol and phospholipid concentrations were increased compared with wild-type mice. In the absence of upregulation of hepatic ATP-binding cassette protein A1, reduced HDL uptake from plasma into livers of Pctp(-/-) mice contributed to higher plasma lipid concentrations. These data indicate that PC-TP is not essential for the enrichment of HDL with phosphatidylcholines but that it does modulate particle size and rates of hepatic clearance.

Liver-derived IGF-I regulates GH secretion at the pituitary level in mice.

We have reported that liver-specific deletion of IGF-I in mice (LI-IGF-I-/-) results in decreased circulating IGF-I and increased GH levels. In the present study, we determined how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion. The pituitary mRNA levels of GH releasing factor (GHRF) receptor and GH secretagogue (GHS) receptor were increased in LI-IGF-I-/- mice, and in line with this, their GH response to ip injections of GHRF and GHS was increased. Expression of mRNA for pituitary somatostatin receptors, hypothalamic GHRF, somatostatin, and neuropeptide Y was not altered in LI-IGF-I-/- mice, whereas hypothalamic IGF-I expression was increased. Changes in hepatic expression of major urinary protein and the PRL receptor in male LI-IGF-I-/- mice indicated an altered GH release pattern most consistent with enhanced GH trough levels. Liver weight was enhanced in LI-IGF-I-/- mice of both genders. In conclusion, loss of liver-derived IGF-I enhances GH release by increasing expression of pituitary GHRF and GHS receptors. The enhanced GH release in turn affects several liver parameters, in line with the existence of a pituitary-liver axis.