Miquel Llobera

Effects of growth and differentiation factors on the epithelial-mesenchymal transition in cultured neonatal rat hepatocytes

BACKGROUND/AIMS Loss of specific differentiation markers, adoption of a migrating morphology and progressive replacement of the cytokeratin network by vimentin intermediate filaments characterize the epithelial-mesenchymal transition of cultured neonatal rat hepatocytes. In a previous study (Hepatology 1997; 25: 598-606), we reported that this process can be differentially regulated by EGF and DMSO, two agents that affect hepatocyte growth and differentiation. The aim of the present study was to determine if growth activation or differential gene expression could explain the differences in EMT observed between these two factors. METHODS We compared the effects of EGF, HGF, TGF-beta1 and DMSO on growth, proto-oncogene expression, epithelial-mesenchymal transition markers and expression of liver transcription factors in cultured neonatal rat hepatocytes using thymidine incorporation, Northern blotting and Western blotting analysis. RESULTS When TGF-beta1 or DMSO was added to the cultures supplemented with EGF and HGF, the mitogenic activity induced by these factors was inhibited. DMSO down-regulated c-myc and c-fos expression. mRNA levels of some liver-specific genes such as albumin, or liver-enriched transcription factors such as C/EBPdelta, HNF-4 and HNF-1beta were slightly different in cultures supplemented with DMSO or TGF-beta1. However, no differences were found when DMSO or TGF-beta1 was added to the cultures supplemented with EGF. Western blotting analysis showed that TGF-beta1 decreased cytokeratin and increased vimentin levels, while DMSO decreased both cytokeratin and vimentin. When DMSO or TGF-beta1 was added in combination with EGF or HGF, both factors maintained the increase in albumin and cytokeratin induced by the growth factors although DMSO, but not TGF-beta1, inhibited vimentin expression. CONCLUSIONS Activation of vimentin expression produced in cultures supplemented with the mitogenic factors (EGF and HGF) is independent of the activation of cell growth, because DMSO but not TGF-beta1 can abolish vimentin synthesis, although both inhibited growth. Moreover, the vimentin expression in these cultures seems to be independent of the mRNA levels of transcription factors associated with the differentiated liver phenotype.

Circadian rhythms of lipoprotein lipase and hepatic lipase activities in intermediate metabolism of adult rat

Although intermediate metabolism is known to follow circadian rhythms, little information is available on the variations in lipoprotein lipase (LPL) and hepatic lipase (HL) activities during the 24-h period, and there is also a lack of adequate statistical analysis. Here, adult male rats were fed ad libitum and kept at 21 degrees C under 12:12-h light-dark cycles. They were killed in batches every 3 h over a 24-h period. Lipase activities were determined in plasma and fresh homogenates of epididymal white adipose tissue (EWAT), interscapular brown adipose tissue (IBAT), heart, skeletal muscle, and liver. Plasma insulin, corticosterone, glucose, triacylglycerol (TAG), cholesterol, glycerol, beta-hydroxybutyrate, and liver and muscle glycogen were determined. Cosinor analysis was used to evaluate the presence (significance of fit of cosine curve to data and variance explained by rhythm) and characteristics of possible circadian rhythms [acrophase (phi), mesor, and amplitude]. Statistically significant circadian rhythms were detected for 1) all metabolites studied, except TAG, cholesterol, and liver HL activity; 2) LPL and HL activity in plasma (both phi in light phase); and 3) LPL activity in all tissues studied (phi: heart in light phase; skeletal muscle, IBAT, and EWAT in dark phase). Liver also showed a circadian rhythm of LPL activity, with phi near that in plasma. These findings demonstrate for the first time that, in physiological conditions, LPL activities in plasma and various tissues, including liver, and HL activity in plasma follow circadian rhythms. Their metabolic significance is discussed.Although intermediate metabolism is known to follow circadian rhythms, little information is available on the variations in lipoprotein lipase (LPL) and hepatic lipase (HL) activities during the 24-h period, and there is also a lack of adequate statistical analysis. Here, adult male rats were fed ad libitum and kept at 21°C under 12:12-h light-dark cycles. They were killed in batches every 3 h over a 24-h period. Lipase activities were determined in plasma and fresh homogenates of epididymal white adipose tissue (EWAT), interscapular brown adipose tissue (IBAT), heart, skeletal muscle, and liver. Plasma insulin, corticosterone, glucose, triacylglycerol (TAG), cholesterol, glycerol, β-hydroxybutyrate, and liver and muscle glycogen were determined. Cosinor analysis was used to evaluate the presence (significance of fit of cosine curve to data and variance explained by rhythm) and characteristics of possible circadian rhythms [acrophase (φ), mesor, and amplitude]. Statistically significant circadian rhythms were detected for 1) all metabolites studied, except TAG, cholesterol, and liver HL activity; 2) LPL and HL activity in plasma (both φ in light phase); and 3) LPL activity in all tissues studied (φ: heart in light phase; skeletal muscle, IBAT, and EWAT in dark phase). Liver also showed a circadian rhythm of LPL activity, with φ near that in plasma. These findings demonstrate for the first time that, in physiological conditions, LPL activities in plasma and various tissues, including liver, and HL activity in plasma follow circadian rhythms. Their metabolic significance is discussed.

Lipoprotein lipase and hepatic lipase in Wistar and Sprague-Dawley rat tissues. Differences in the effects of gender and fasting

To evaluate the effects of strain, gender and fasting in the regulation of lipoprotein lipase (LPL) and hepatic lipase (HL) activities were measured in tissues of male and female Wistar and Sprague-Dawley rats after feeding or a 24-h starvation period. It is noteworthy that an effect of gender on LPL activity was observed in Wistar, but not in Sprague-Dawley rats, not in the basal (fed) activity in several tissues, such as white and brown adipose tissues, heart, and brain, but also in response to fasting which affected LPL activity in brown adipose tissue, heat and lung of female but not of male Wistar rats. By contrast, HL activity in liver, plasma and adrenals of Sprague-Dawley rats was higher in females than in males. No effect of gender on HL activity was observed in Wistar rats. Our results indicate that differences exist between Wistar and Sprague-Dawley rats in the regulation of both LPL and HL. Some of the contradictory results found in the literature may be explained by the differences between rat strains and gender, as well as differences in the nutritional status of the animals.

Localization of lipoprotein lipase to discrete areas of the guinea pig brain

Lipoprotein lipase is a key enzyme in lipoprotein metabolism present primarily in extrahepatic tissues with high turnover of fatty acids. Using immunocytochemistry we have explored where lipoprotein lipase is localized in guinea pig brain. The enzyme was found to be associated with neuronal cells and vascular endothelial surfaces. The distribution was strikingly uneven with intense reaction in some areas, and virtually no reaction in adjacent areas. The highest reactivity was in neocortex, in hippocampus, in Purkinje cells of the cerebellum and in some motor nuclei of the brainstem. The results suggest marked differences between individual brain areas in utilization of plasma lipoproteins.

Effects of prenatal ethanol exposure on physical growth, sensory reflex maturation and brain development in the rat.

Effects of prenatal ethanol exposure on physical growths, sensory reflex maturation and brain development in the rat

Ethanol administration in the drinking fluid to pregnant rats as a model for the fetal alcohol syndrome.

Addition of ethanol (ET) to the drinking fluid of pregnant rats has been questioned as an experimental model for the fetal alcohol syndrome (FAS). This model, however, closely simulates human alcohol intake, and in this study we used a modified version of previous protocols to overcome their major defects. A group of female rats was given 10% ET in drinking fluid for one week, 15% for the second week, 20% for the third, and 25% for the fourth, at the end of which they were mated with non-treated males and given 25% ET throughout gestation. Three groups of non-ET treated sex and age-matched rats were studied in parallel: (1) normal controls receiving solid diet ad lib, (2) paired fed rats, and (3) rats fed ad lib the solid diet mixed with 50% fiber. In the ET group, food intake decreased as ET consumption augmented, the ET calories comprising over 30% of the total energy intake during pregnancy. Total energy intake was similar for ET group and normal controls, and was higher than in paired fed animals or those on 50% fiber diet. Body weight gain in ET rats was similar to those on 50% fiber diet, lower than in normal controls and higher than in paired fed animals. At the 21st day of gestation, rats on ET had plasma ethanol levels of 147 +/- 18 mg/dl and higher plasma osmolality than in the other groups studied. In ET rats, fetal body weight was lower than in either normal controls or rats on 50% fiber diet, and fetal body length was shorter than in any other group.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein Lipase Activity in Developing Rat Brain Areas

Lipoprotein lipase (LPL) is a key extracellular enzyme that enables tissue to import fatty acids from triacylglyceride-rich lipoproteins. LPL is present in most tissues of the body, but in the brain its functional significance remains unclear. Lipids constitute the main components of myelin and undergo significant changes during maturation. However, nothing is known of the postnatal evolution of LPL activity in the brain areas during postnatal development. Here we found that LPL activity is relatively high in the newborn brain and peaks between the 5th and the 10th days after birth, reaching activities 5 times higher than in the adult brain. In all the areas studied (olfactory bulbs, cortex, thalamus, cerebellum, hippocampus, striatum, brain-stem and spinal cord) LPL also increases sharply during postnatal development. Hippocampus shows the highest LPL activity levels, which are between 5 and 11 times higher than in the other regions. The significance of these high LPL activity levels is discussed.

HORMONAL REGULATION OF LIPOPROTEIN LIPASE ACTIVITY FROM 5-DAY-OLD RAT HEPATOCYTES

Lipoprotein lipase (LPL) activity is known to be synthesized, active and functional in the 1-day-old rat liver: it peaks just at birth triggered by parturition. During suckling LPL mRNA, LPL synthesis and LPL activity are still high at 5 days and then fade reaching adult values at weaning. How LPL expression is gradually extinguished is not known. Therefore we studied the effect of different doses of several hormones on LPL activity released by incubated hepatocytes from 5-day-old rats. In the presence of heparin the release of LPL activity in the medium was linear until 3 h and was always significantly increased vs. without heparin. At 3 h in the presence of heparin the main hormonal effects were: dose-dependent increase (30-60%) with dexamethasone; dose-dependent increase (20-60%) with glucagon; dose-independent decrease (50-60%) with ethinylestradiol, testosterone, progesterone and prolactin; no effect with insulin; 20-40% increase with adrenaline < 1 mM but 40-50% decrease with noradrenaline < 10 microM. Increase of LPL release by glucagon and adrenaline agrees with the increased LPL expression we previously found in an undifferentiated hepatoma cell line when the adenylate cyclase/protein kinase A pathway was activated. The effect of glucagon is concordant with our previous observations that fasting increases liver LPL activity in neonatal rats. The high estradiol levels known to be present in male and female 9-19-day-old rats might contribute to liver LPL extinction during suckling.

Morphological recovery of hippocampal pyramidal neurons in the adult rat exposed in utero to ethanol

Reduced numbers of dendritic spines on the secondary apical dendritic branches and basilar dendrites of CA1 and CA3 pyramidal neurons were observed in ethanol-treated rats during embryonic life aged 15 days when compared with age-matched controls. However, differences were no longer present at the age of 90 days. These results suggest that recovery of some morphological parameters of pyramidal hippocampal neurons may occur in rats exposed in utero to ethanol.

Hepatic endothelial lipase activity in neonatal rat liver

Hepatic endothelial lipase (HEL) activity is as high in the neonatal (1-day old) rat liver as in adults. Most of the HEL activity is located at the capillaries since 75% of the total activity is released by heparin or collagenase perfusion. The residual activity (non-releasable) is located in hepatocytes and not in hemopoietic cells, which are the major cell type in neonatal liver. Per mg of protein, the HEL activity is 50% higher in neonatal than in adult hepatocytes. We suggest that neonatal hepatocytes have an increased capacity to synthesize and secrete HEL activity, so maintaining a high activity in the whole organ. it might contribute to the hepatic uptake of cholesterol from circulating lipoproteins, in a period in which endogenous cholesterol synthesis is known to be inhibited in the liver.