Differential intracellular signalling induced by TGF-beta in rat adult hepatocytes and hepatoma cells: implications in liver carcinogenesis.

The transforming growth factor-beta (TGF-beta) regulates hepatocyte growth, inhibiting proliferation and inducing apoptosis. Indeed, escaping from the TGF-beta suppressor actions might be a prerequisite for liver tumour progression. In this work we show that TGF-beta plays a dual role in regulating apoptosis in FaO rat hepatoma cells, since, in addition to its pro-apoptotic effect, TGF-beta also activates survival signals, such as AKT, the epidermal growth factor receptor (EGFR) being required for its activation. TGF-beta induces the expression of the EGFR ligands transforming growth factor-alpha (TGF-alpha) and heparin-binding EGF-like growth factor (HB-EGF) and induces intracellular re-localization of the EGFR. Cells that overcome the apoptotic effects of TGF-beta undergo morphological changes reminiscent of an epithelial-mesenchymal transition (EMT) process. In contrast, TGF-beta does not activate AKT in adult hepatocytes, which correlates with lack of EGFR transactivation and no response to EGFR inhibitors. Although TGF-beta induces TGF-alpha and HB-EGF in adult hepatocytes, these cells show very low expression of TACE/ADAM 17 (TNF-alpha converting enzyme), which is required for EGFR ligand proteolysis and activation. Furthermore, adult hepatocytes do not undergo EMT processes in response to TGF-beta, which might be due, at least in part, to the fact that F-actin re-organization induced by TGF-beta in FaO cells require the EGFR pathway. Finally, results indicate that EGFR transactivation does not block TGF-beta-induced cell cycle arrest in FaO cells, but must be interfering with the pro-apoptotic signalling. In conclusion, TGF-beta is a suppressor factor for adult quiescent hepatocytes, but not for hepatoma cells, where it plays a dual role, both suppressing and promoting carcinogenesis.

Utilidad del spect en la embolia grasa cerebral / Cerebral fat embolism and spect usefulness

El síndrome de la embolia grasa es una entidad frecuente en las Unidades de Cuidados Intensivos (UCI). En el caso del paciente politraumatizado con afectación neurológica, lo habitual es que la lesión neurológica sea por el propio traumatismo craneal, pero en algunos casos es secundario al síndrome de la embolia grasa, incluso pueden asociarse. Ante todo paciente politraumatizado que presente un deterioro neurológico conviene establecer un diagnóstico precoz.En ocasiones las pruebas neurorradiológicas habituales no muestran alteraciones en este síndrome de la embolia grasa cerebral. Recientemente hemos tenido ocasión de asistir a dos pacientes polifracturados que presentaron una sintomatología compatible con embolismo graso cerebral, siendo la tomografía computarizada (TC) normal en ambos casos. Para corroborar la sospecha diagnóstica se realizó una gammagrafía cerebral tomográfica (SPECT = Photon Single Emission Computed Tomography) que fue claramente demostrativa de lesión cerebral, sugiriendo que es una exploración a tener en cuenta en el diagnóstico y control evolutivo del embolismo graso cerebral. (AU)

Enzyme distributions in subcellular fractions of BHK cells infected with Semliki forest virus: evidence for a major fraction of sphingomyelin synthase in the trans-golgi network.

BHK cells either untreated or infected with Semliki Forest virus have been fractionated on sucrose density gradients. Virus infection caused an increase in density of a membrane fraction enriched in sphingomyelin (SM), cholesterol, SM synthase and sialyltransferase activity. This increase in density was related to incorporation of viral proteins into this fraction, which is likely to contain trans-Golgi network (TGN) membranes. In contrast, glucosylceramide synthase and galactosyltransferase activities (markers for cis/medial and trans-Golgi respectively) underwent no density shift and alkaline phosphodiesterase, a plasma membrane marker, was only slightly density-shifted in infected cells. When cells were incubated with NBD-ceramide to enable them to synthesise NBD-SM and then washed with albumin to remove surface label, fluorescence in untreated cells was concentrated in a single juxtanuclear spot but in infected cells this region of bright fluorescence was larger and extended around the nucleus. After fractionation of these cells, NBD-SM (but only a small proportion of the NBD-ceramide) was found to be shifted into the higher density fraction in infected cells. This work provides further evidence that SM synthase is not mainly localised in the early Golgi cisternae as previously thought, but is associated more with a cholesterol-rich compartment which could be the TGN.

The subcellular sites of sphingomyelin synthesis in BHK cells.

The subcellular distributions of the enzymes which synthesise sphingomyelin (SM) and glucosylceramide (GluCer) from ceramide have been assessed in BHK cells. On a sucrose density gradient GluCer synthase (a marker of the cis/medial Golgi apparatus) and the trans-Golgi marker galactosyltransferase showed an similar monotonic distribution. In contrast, SM synthase showed two peaks of activity, a minor one which migrated with the Golgi markers and a major one which had a density close to that of plasma membrane markers (sphingomyelin, cholesterol, PtdSer, ganglioside GM3 and alkaline phosphodiesterase). When cell homogenates were treated with digitonin, the sedimentation characteristics of the Golgi markers was largely unaffected whereas the plasma membrane markers and the main peak of SM synthase activity were shifted to higher density. In contrast, when cells were treated with brefeldin A (BFA) the Golgi markers were shifted to higher density but not the plasma membrane markers or the main peak of SM synthase. These results suggest that the bulk of SM synthase activity in BHK cells is not associated with the Golgi cisternae but with a cell compartment which is relatively rich in cholesterol (e.g., plasma membrane, endosomes or trans-Golgi network.) Further experiments in which cells were treated with sphingomyelinase provided evidence that SM synthase activity was in an internal compartment and not at the plasma membrane.

Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells.

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.

Differential effect of dietary fat saturation and cholesterol on hepatic apolipoprotein gene expression in rats.

The effects of dietary cholesterol and fat saturation on hepatic apolipoprotein A-I, A-II, A-IV, B, C-I, C-III, E and LDL receptor mRNA levels were studied in male rats. Animals were maintained for 2 months on a high fat diet (40% w/w) containing 0.1% cholesterol. Two groups of control animals received either chow diet or chow plus 0.1% cholesterol, while experimental groups received as their fat supplement coconut, corn or olive oil. Olive oil fed animals had higher levels of hepatic apo A-I than the control cholesterol group (1.6 +/- 0.3 vs. 0.8 +/- 0.2). Apo E mRNA levels were 50% and 72% higher in animals consuming the saturated (coconut) and unsaturated (corn and olive) fat diet than the control cholesterol group. Apo B and apo C-I mRNA levels were not affected by the experimental conditions. Apo A-IV mRNA increased between 66% and 127% in groups in which cholesterol was present. LDL receptor mRNA increased 2 times in the corn fed group compared with the control groups. These results indicate that the expression of genes coding for products involved in lipoprotein metabolism have a differential susceptibility to dietary fat saturation and cholesterol.

Microsomal sphingomyelin accumulation in thioacetamide-injured regenerating rat liver: involvement of sphingomyelin synthase activity.

The purpose of this work was to determine whether alterations in the lipid composition of rat liver microsomal membranes existed during thioacetamide-induced injury prior to the development of hepatic cancer and biochemical mechanisms involved. Rats were injected intraperitoneally with (50 mg/kg body wt per day) thioacetamide or diluent for 8 days. Liver homogenates and microsomal membranes from liver homogenates were obtained. Incorporation of [32P]orthophosphate into whole liver lipids and hepatic microsomal lipids was evaluated 75 min after isotope administration. These determinations were made after two separate periods of treatment (3 and 8 days). Activity of sphingomyelin synthase was assayed in rat liver homogenates as well as in the purified microsomal fractions. Results demonstrated a maintenance of liver and hepatic microsomal contents of phosphatidylcholine during thioacetamide-induced injury even when the biosynthesis of this glycerophospholipid in both liver and their microsomal fractions appeared decreased. Also observed was a considerable increase of microsomal sphingomyelin, as well as an increased hepatic biosynthesis of sphingomyelin caused by thioacetamide treatment. The microsomal sphingomyelin/phosphatidylcholine radioactivity ratio significantly increased. Sphingomyelin synthase activity in liver homogenate appeared stimulated. In conclusion, our data are consistent with a thioacetamide-induced increase in microsomal sphingomyelin by a stimulation of sphingomyelin synthase. Based on this and previous studies, accumulation of sphingomyelin in the microsomal purified fraction is associated with the number of thioacetamide doses and is an early event clearly detected prior to tumoral characteristics of hepatocytes.

Thioacetamide alters the regulation of cytidylyltransferase activity.

Using the weak hepatocarcinogen thioacetamide, we found a 73% decrease in microsomal CTP: Phosphocholine cytidylyltransferase (CT) activity after two months of treatment. We investigated the effect of different effectors on this enzyme activity. Results show that incubation of liver homogenates of treated animals in the presence of either oleate or spermine restored control values of microsomal activity. Incubation of thioacetamide homogenates in the presence of cAMP showed no changes in CT activity, while incubation of control and thioacetamide homogenates in the presence of Ca2+ induced an activation in microsomal activity, although of less intensity in thioacetamide homogenates than in control preparations. Results suggest that thioacetamide alters the regulation of cytidylyltransferase activity.

Alterations in lipid characteristics of lysosomes are involved in liver necrosis induced by thioacetamide.

To determine whether lysosomal lipid composition is altered in hepatic necrosis, we studied this parameter in thioacetamide-induced necrosis and in its regenerating stage as well as in the recovery of thioacetamide-induced injury. Results showed that in liver necrosis there is an increase in the protein and phospholipid lysosomal contents. This effect may be related to an increased number of lysosomes. These organelles also suffered a decrease in cholesterol content. When liver necrosis was recovered either pharmacologically or spontaneously all three parameters recovered their normal values. These results suggest that lysosomes and their lipid composition play a role in progression of hepatic necrosis.

Hepatic expression of apolipoprotein A-I gene in rats is upregulated by monounsaturated fatty acid diet.

The effect of the degree of dietary fat saturation on the hepatic expression of apolipoprotein A-I mRNA was studied in male rats. Animals were maintained for two months on a high fat diet (40% w/w) containing 0.1% cholesterol. Two groups of control animals received either chow diet or chow plus 0.1% cholesterol, while experimental groups received their fat supplement as coconut, corn or olive oil respectively. Dietary cholesterol did not affect apolipoprotein A-I mRNA levels as compared to control animals. Corn oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA than those receiving cholesterol, or coconut oil plus cholesterol. Olive oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA when compared to all other dietary groups. Our data indicate that monounsaturated fatty acids supplied as olive oil play a major role in regulating the hepatic expression of apolipoprotein A-I in male rats.

Effect of acute thioacetamide administration on rat brain phospholipid metabolism.

Brain phospholipid composition and the [32P]orthophosphate incorporation into brain phospholipids of control and rats treated for 3 days with thioacetamide were studied. Brain phospholipid content, phosphatidylcholine, phosphatidylethanolamine, lysolecithin and phosphatidic acid did not show any significant change by the effect of thioacetamide. In contrast, thioacetamide induced a significant decrease in the levels of phosphatidylserine, sphingomyelin, phosphatidylinositol and diphosphatidylglycerol. After 75 minutes of intraperitoneal label injection, specific radioactivity of all the above phospholipids with the exception of phosphatidylethanolamine and phosphatidylcholine significantly increased. After 13 hours of isotope administration the specific radioactivity of almost all studied phospholipid classes was elevated, except for phosphatidic acid, the specific radioactivity of which did not change and for diphosphatidylglycerol which showed a decrease in specific radioactivity. These results suggest that under thioacetamide treatment brain phospholipids undergo metabolic transformations that may contribute to the hepatic encephalopathy induced by thioacetamide.

Association between rat serum cholinesterase and some phospholipid components of lipoproteins in thioacetamide-induced hepatic injury.

Rat serum lipoprotein phospholipids and serum cholinesterase activity in control and thioacetamide-treated rats (50 mg/kg/day for 30 days) were studied. Analyses were done after 1, 3, 8 and 30 intraperitoneal doses of thioacetamide or 0.15 mol/l NaCl. Cholinesterase activity significantly increased with thioacetamide treatment. Only two phospholipids: LDL-phosphatidylcholine and HDL-lysophosphatidylcholine appeared associated with cholinesterase activity. LDL-phosphatidylcholine increased through the action of hepatotoxic thioacetamide while HDL-lysophosphatidylcholine significantly decreased. Because of the high statistically significant association between changes in these lipoprotein phospholipids and in cholinesterase in this model of hepatic injury, we conclude that cholinesterase could be involved in the regulation of these phospholipid levels.

Changes in serum cholinesterase (EC 3.1.1.8) activity in rats consuming a high-fat diet.

Adult male rats were fed on a control diet containing (g/kg) carbohydrate 600, lipid 35 and protein 190, or on a high-fat diet containing carbohydrate 360, lipid 420 and protein 120. After 30 d, the high-fat diet provoked a decrease in serum cholinesterase (EC 3.1.1.8) activity which was reversed by feeding rats on the control diet. The observed decrease after 90 d on the high-fat diet was not seen if a simultaneous daily intraperitoneal injection of a lipotrophic agent containing (mg/kg) S-adenosyl-L-methionine 3, coenzyme A 0.1, UDP-glucose 30 and CDP-choline 1.5 was given to rats on the high-fat diet. The findings are discussed in relation to the apparent susceptibility of serum cholinesterase to dietary components and its possible role in lipid metabolism.

Lyso-phosphatidylcholine is implicated in thioacetamide-induced liver necrosis.

Thioacetamide is a weak hepatocarcinogen. To determine whether alterations in lysophosphatidylcholine are implicated in thioacetamide-induced hepatic necrosis, rats were injected i.p. with this agent (50 mg/Kg body weight per day) or diluent for 1, 3, 8 and 30 days. Serum catalytic activities of aminotransferases were determined. Incorporation of (32P)-orthophosphate into hepatic lysophosphatidylcholine was also evaluated in animals killed 75 minutes or 13 hours after isotope administration. Results demonstrate that: A significant increase in hepatic lysolecithin concentration occurs when a maximum level of serum aminotransferases is present. An increase of (32P)-orthophosphate radioactive incorporation in lysolecithin was observed at the two assayed labelling periods, which suggest an activation of phospholipase A. The radioactivity present in lysolecithin after 13 h isotope injection showed a close correlation with serum level of aminotransferases. From these results it can be deduced that lysolecithin is implicated in TAA-induced necrosis and may be generated by increase in either phospholipase A activity and/or synthesis.