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M Llobera

Publications and source records attributed to M Llobera.

At least 19 recordsLinked to original sources
PubMed1997

Growth and differentiation factors inhibit the migratory phenotype of cultured neonatal rat hepatocytes induced by HGF/SF.

Previous studies have demonstrated that neonatal rat hepatocytes cultured in a serum-free medium adopt a fibroblast-like morphology and form a well-developed vimentin network. By immunoblotting, phase-contrast microscopy, and immunolocalization studies we report here that hepatocyte growth factor (HGF) induces a pronounced migratory/scatter phenotype in neonatal hepatocytes in primary culture. These fibroblast-like cells are highly elongated and adopt a pronounced spindle shape in the presence of this growth factor. Most of them coexpress vimentin and cytokeratin intermediate filaments. Both EGF and TGF-beta1 also induced vimentin expression in cytokeratin-positive cells but this effect was not correlated with a change of epithelial phenotype. Only in DMSO-supplemented cultures was vimentin expression of hepatocytes inhibited and no coexpression was observed in the presence of this factor. Remarkably, the effect induced by HGF was totally inhibited when DMSO, TGF-beta1, or EGF was added to HGF-supplemented cultures. Epithelial sheets of well-defined hepatocytes were observed with these combinations although the complete epithelial morphology was achieved by combining DMSO with a growth factor. Taken together these results suggest that growth and differentiation factors modulate the migratory effect of HGF/SF in hepatocytes.

Animals↗
PubMed1997

Epithelial-mesenchymal transition of cultured rat neonatal hepatocytes is differentially regulated in response to epidermal growth factor and dimethyl sulfoxide.

Neonatal rat hepatocytes cultured in the absence of added growth factors dedifferentiate by epithelial-mesenchymal transition (EMT). This involves the loss of their typical differentiation markers, the acquisition of a migrating morphology, and a change in the expression of the intermediate filament (IF) proteins. We attempted to determine whether the EMT of cultured neonatal rat hepatocytes could be modulated by factors that maintain and promote the differentiation state of adult and fetal hepatocytes such as epidermal growth factor (EGF) and dimethyl sulfoxide (DMSO). By (3H)-thymidine incorporation, Western blotting analysis, flow cytometry, and double-immunofluorescence studies, we found that both factors have marked but opposite effects on the EMT and on proliferation of neonatal liver cells. In DMSO treatment, albumin levels were higher than in the nontreated cells at all days studied. Moreover, DMSO reduced cytokeratin levels and inhibited cell proliferation, acquisition of the fibroblast-like morphology, and vimentin expression typical of the EMT. The increase in vimentin-positive cells in serum-free medium was not observed in DMSO cultures. EGF also increased albumin levels at all days studied. In contrast, EGF treatment induced hepatocyte proliferation and enhanced vimentin and cytokeratin expression. However, the increase in vimentin levels did not correlate with a significant increase in the number of vimentin-positive cells. Moreover, vimentin-positive cells in EGF treatment were also cytokeratin-positive and albumin-positive, and they maintained epithelioid morphology in spite of the vimentin network. These results indicate that EMT of cultured rat neonatal hepatocytes is differentially regulated in response to EGF and DMSO.

Albumins↗
PubMed1996

Hepatic regeneration induces changes in lipoprotein lipase activity in several tissues and its re-expression in the liver.

We examined the expression of lipoprotein lipase (LPL) gene and LPL activity following a two-thirds hepatectomy and during liver regeneration. In most of the tissues studied, LPL activity increased a few hours after partial hepatectomy, but soon returned to normal levels. The greatest increase was found in the adrenal glands, plasma and liver. This increase in LPL activity in the liver could be partially due to an increase in the influx of the enzyme from extrahepatic tissues. There is, however, also a re-expression of LPL mRNA in the liver after partial hepatectomy (during the first hours). It is well known that LPL is expressed in the liver of neonatal animals, but progressively decreases during post-natal development, to reach adult levels around the time of weaning. Our results show by the first time that the remaining liver re-expresses LPL gene during the regeneration process and that the hepatocytes de-differentiate and acquire some of the neonatal characteristics. The increase in LPL mRNA will contribute to the rise in LPL activity after hepatectomy. This presence of LPL could enable the liver to take up fatty acids from the circulating triacylglycerols, which are needed as energetic and plastic substrates during the process of hepatic regeneration.

Adipose Tissue↗
PubMed1996

Decrease in the expression of hepatic lipase activity following partial hepatectomy.

Variations in hepatic lipase (HL) activity were studied for the first time in liver, plasma and adrenal glands of partially hepatectomized (70%), sham-operated and intact rats. Activity profiles performed during 7 days in liver, plasma and adrenal glands of sham-operated rats were similar to those obtained in intact animals. However, HL activity in intact animals appeared to be slightly higher during the first 24 hours. Following surgery, hepatectomized animals showed a reduction of about 300 U in liver HL activity which persisted for 7 days. Plasma HL activity of hepatectomized rats was undetectable at 6 hours post-surgery but in increased afterwards. A high correlation between liver and adrenal gland HL activity was found in hepatectomized but not in sham-operated animals. HL mRNA levels in hepatectomized rats showed a 40% decrease during the first 24 hours after surgery, but they returned to the normal range later. On the other hand, HL mRNA values increased in sham-operated rats but no increase in HL activity was detected in these animals. To conclude, our results show that HL activity decreases dramatically during hepatic regeneration due to a concomitant decrement in the expression of the gene that encodes the enzyme and to other undetermined factors.

Adrenal Glands↗
PubMed1996

Vimentin filaments follow the preexisting cytokeratin network during epithelial-mesenchymal transition of cultured neonatal rat hepatocytes.

Changes in cell cytoskeleton are known to play an important role in differentiation and embryogenesis and also in carcinogenesis. Previous studies indicated that neonatal hepatocytes undergo an epithelial-mesenchymal transition when cultured in a serum-free medium for several days. Here we show by Western blotting of neonatal rat liver cells cultured for 3 days that vimentin and cytokeratin were expressed by these cells. Epidermal growth factor treatment induced high coexpression of vimentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribution of cytokeratin and vimentin intermediate filament networks. Vimentin-expressing hepatocytes were mainly located on the periphery of epithelial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell-cell contact. Short vimentin filaments were mainly located at the cytoplasmic sites behind the extending lamella. Horizontal and vertical dual imaging of double immunofluorescence with anti-vimentin and anti-cytokeratin antibodies indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthesized vimentin distributed following the preexisting cytokeratin network and, when present, both filament scaffolds codistributed inside cultured hepatocytes. Immunoelectron microscopy performed in whole-mount-extracted cultured cells revealed that both filaments are closely interrelated but independent. However, a high degree of immunogold colocalization was found in the knots of the filament network. Further experiments with colcemide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intact microtubule network. These results are consistent with a mechanism of vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmic sites, in close relation to the cytokeratin cytoskeleton and to changes in cell-cell contact and cell shape.

Albumins↗
PubMed1996

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.

Animals↗
PubMed1995

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.

Aging↗
PubMed1995

Epithelial-mesenchymal transition in cultured neonatal hepatocytes.

When hepatocyte-enriched fractions from neonatal rat livers were cultured for different times in the absence of added growth factors, a population of highly proliferating and migrating fibroblastlike cells appeared. Double immunofluorescence with antibodies to cytokeratin and to vimentin showed a progressive reduction in the number of cytokeratin-positive cells parallel to an increase in the vimentin-positive cells. Some cells with transitional epithelial or migrating morphology coexpressed both intermediate filament proteins. Immunofluorescence with antibodies against hepatocyte differentiation markers showed that shortly after seeding most of the cells were positive to anti-albumin antibodies, but after 1 week in culture, only 10% were positive. Cells presenting albumin and cytokeratin appeared morphologically epithelial. Fibroblastlike cells were not positive for albumin, but some cells with transitional epithelial morphology presented some labels for albumin and for vimentin. Immunofluorescence with antibodies to glutathione-S-transferase subunit Pi and vimentin showed that many fibroblastlike cells were positive for both markers, some of them binucleate. Cultures performed in the presence of dexamethasone, absence of arginine, or on collagen type I matrix had no effect on the behavior of neonatal hepatocytes. The appearance of fibroblastlike cells was ontogenically regulated because the highest increase in the percentage of vimentin-positive cells was observed in cell cultures from livers of 7- and 15-day-old animals. These data provide evidence that neonatal hepatocytes in culture have the potential to dedifferentiate by epithelial-mesenchymal transition and contribute to an understanding of hepatic growth development.

Aging↗
PubMed1994

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 only 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.

Adrenal Glands↗
PubMed1994

Coexisting subdural and intercostal haemangiomata.

Haemangioma in the chest wall is a very rare entity, seldom described in the literature. We report the case of a 23 year old woman presenting with independent, cervical subdural and intercostal haemangiomata. She suffered several episodes of recurrent, self-limiting subdural haemorrhage before diagnosis was made by magnetic nuclear resonance, aortography and selective intercostal angiography. Surgical excision was performed in both locations with excellent results.

Adult↗
PubMed1993

Epidermal growth factor interferes with the effect of adrenaline on glucose production and on hepatic lipase secretion in rat hepatocytes.

We studied the interaction of epidermal growth factor (EGF) and adrenaline in the control of several metabolic functions in isolated hepatocytes from fed rats. EGF did not modulate glucose release, urea production or hepatic lipase secretion, but interfered with the stimulatory effect of adrenaline on both glucose and urea production and also with the inhibitory effect of this hormone on hepatic lipase secretion. EGF also interfered with the effect of both angiotensin II and vasopressin on glucose release and on hepatic lipase secretion. While the effect of EGF interfering with the action of adrenaline on glucose release was potentiated in the absence of extracellular calcium, the effect on the inhibition of hepatic lipase secretion was abolished. These results suggest that EGF interfered with catecholamine actions in the liver at a site distal from the generation of the calcium signal.

Animals↗
PubMed1993

Lipoprotein lipase in developing rat tissues: differences between Wistar and Sprague-Dawley rats.

Lipoprotein lipase in animal tissues is known to be affected by fasting, but contradictory results have been published concerning this effect in particular tissues. For example, we reported that lipoprotein lipase activity expressed in the liver of neonatal rats was either increased or not affected by fasting. To evaluate the influence of the rat strain used as experimental animal model, we studied differences between Wistar and Sprague-Dawley rats in the development and in the effect of fasting on lipoprotein lipase and hepatic lipase activities in tissues of neonatal rats. Beside some minor differences in the development of lipoprotein lipase in some tissues like brown adipose tissue and lungs, we found quite remarkable differences between both strains in the development of lipoprotein lipase and hepatic lipase activities in the liver. In 1-day-old neonates, differences between both strains were also observed in the effect of fasting on lipoprotein lipase activity both in liver and lungs. In the liver of Wistar pups lipoprotein lipase activity was increased by fasting by 350%, but only by 50% in the liver of Sprague-Dawley pups. In contrast, in the lungs of Wistar pups lipoprotein lipase activity was increased by fasting by 280%, but by 580% in lungs of Sprague-Dawley rats. Therefore, our results indicate that quantitative differences exist between Wistar and Sprague-Dawley rats in the regulation of lipoprotein lipase.

Adipose Tissue, Brown↗
PubMed1992

Neonatal extinction of liver lipoprotein lipase expression.

In contrast to the complete absence of lipoprotein lipase (LPL) mRNA in adult rat liver, fetal and neonatal rat liver contain substantial amounts of LPL mRNA, which is translated in active LPL protein as can be deduced from the presence of LPL activity in this organ. At this neonatal stage, both the relative abundance of LPL mRNA and LPL activity increased with starvation. During the suckling period, LPL mRNA and LPL activity gradually decreased until both parameters were undetectable. While the administration of L-thyroxine or hydrocortisone enhanced the disappearance of LPL mRNA, induced hypothyroidism delayed its disappearance. In adult animals induced hypothyroidism could not reactivate LPL mRNA production in the liver. The data presented suggest that liver LPL production responds to changes in the nutritional state and becomes extinguished during development, in a fashion reminiscent to the extinction of alpha-fetoprotein. This extinction of LPL gene expression is influenced by hormonal factors.

Animals↗
PubMed1992

Lipoprotein lipase and hepatic lipase activities are differentially regulated in isolated hepatocytes from neonatal rats.

Lipoprotein lipase and hepatic lipase are members of the lipase gene family sharing a high degree of homology in their amino acid sequences and genomic organization. We have recently shown that isolated hepatocytes from neonatal rats express both enzyme activities. We show here that both enzymes are, however, differentially regulated. Our main findings are: (i) fasting induced an increase of the lipoprotein lipase activity but a decrease of the hepatic lipase activity in whole liver, being in both cases the vascular (heparin-releasable) compartment responsible for these variations. (ii) In isolated hepatocytes, secretion of lipoprotein lipase activity was increased by adrenaline, dexamethasone and glucagon but was not affected by epidermal growth factor, insulin or triiodothyronine. On the contrary, secretion of hepatic lipase activity was decreased by adrenaline but was not affected by other hormones. (iii) The effect of adrenaline on lipoprotein lipase activity appeared to involve beta-adrenergic receptors, but stimulation of both beta- and alpha 1-receptors seemed to be required for the effect of this hormone on hepatic lipase activity. And (iv), increased secretion of lipoprotein lipase activity was only observed after 3 h of incubation with adrenaline and was blocked by cycloheximide. On the contrary, decreased secretion of hepatic lipase activity was already significant after 90 min of incubation and was not blocked by cycloheximide. We suggest that not only synthesis of both enzymes, but also the posttranslational processing, are under separate control in the neonatal rat liver.

Animals↗
PubMed1991

Lipoprotein lipase enables triacylglycerol hydrolysis by perfused newborn rat liver.

Fasted 1-day-old rat liver has high heparin-releasable (endothelial) lipoprotein lipase (LPL) activity, and its hepatocytes synthesize LPL protein. To test the physiological role of this LPL, we perfused the isolated organ with a 0.8 mM triacylglycerol (TAG) (Intralipid + glycerol tri[3H]oleate) 6.3% serum medium. Samples of the recirculated perfusate were taken at different times to determine 3H in TAG, free fatty acid (FFA), and water-soluble (WS) fractions. In the medium [3H]TAG disappeared and [3H]FFA and [3H]WS fractions appeared linearly with time. This TAG hydrolysis was 1) absent when medium was recirculated without liver, 2) not affected by chloroquine addition, 3) inhibited by anti-LPL immunoglobulins, 4) absent when serum was omitted from the medium, and 5) restituted when apolipoprotein CII was added to the medium without serum. Therefore, lysosomal lipase is not involved in this TAG hydrolysis, the features of which are characteristic of LPL, not of the so-called "hepatic endothelial lipase." Thus LPL activity enables the neonatal rat liver to hydrolyze and take up circulating TAG, i.e., has the same function as extrahepatic LPL.

Animals↗
PubMed1991

Involvement of catecholamines in the effect of fasting on hepatic endothelial lipase activity in the rat.

The effect of fasting on hepatic endothelial lipase activity in the liver of adult rats was investigated. We found that, both in male and female rats, fasting produced a progressive decrease of the hepatic endothelial lipase activity. Upon refeeding, the activity returned to control values in 48 h. In isolated livers from fed male rats, a sharp peak of hepatic endothelial lipase activity appeared in the perfusate upon heparin addition. It accounted for 75% of the total activity (heparin-released + residual) of the tissue. Fasting (24 h) decreased the heparin-releasable activity, and this effect was responsible for most of the decrease found in whole tissue. We suggest that the effect might be due to a decreased synthesis and/or secretion of the enzyme by hepatocytes, since isolated hepatocytes from fasted rats, incubated at 37 C, released 65% less activity to the incubation medium than hepatocytes from fed rats. Adrenaline, but not insulin, glucagon, dexamethasone, epidermal growth factor, or T3, decreased the amount of hepatic endothelial lipase activity released by hepatocytes isolated from fed rats. The effect of adrenaline appears to be mediated by alpha 1-receptors since phenylephrine but not isoprenaline reproduced, and prazosin but not propranolol blocked, the effect of the catecholamine. In the presence of cycloheximide, adrenaline also decreased the amount of activity released. We suggest that, in our incubation conditions (up to 3 h), the hormone affects the posttranslational processing of the enzyme. In vivo administration of prazosin blocked the effect of both noradrenaline and fasting on hepatic endothelial lipase activity in whole liver. Those results suggest that catecholamines are involved in the decreased hepatic endothelial lipase activity found in the liver of fasted rats, and points out the role of these hormones in the acute modulation of an enzyme involved in reverse cholesterol transport.

Animals↗