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Nonalcoholic fatty liver disease (NAFLD) is an emerging disease with a broad spectrum of liver conditions. The complex molecular pathogenesis of NAFLD is still unclear. In this study, we conducted an analysis of microRNA (miRNA) expression profiles in liver of rats made NAFLD by different diets. To this aim, Sprague-Dawley rats were fed ad libitum for 3 months with different diets: standard diet (SD), diet enriched in fats and low in carbohydrates (HFD), SD with high fructose (SD-HF) and diet with high levels of fats and fructose (HFD-HF). Our results demonstrated that the treatment with different dietetic regimens caused a significant increase of the body weight and the alteration of some metabolic parameters compared with control animals, as well as various liver injuries. The miRNAs analysis showed the significant downregulation of three miRNAs (miR-122, miR-451 and miR-27) and the upregulation of miR-200a, miR-200b and miR-429 in HFD, SD-HF and HFD-HF rats. Besides, miR-21 expression was significantly decreased only in fructose-enriched diets. These miRNAs target molecules involved in the control of lipid and carbohydrate metabolism, signal transduction, cytokine and chemokine-mediated signaling pathway and apoptosis. Western blot analysis of PKCδ, LITAF, ALDOLASE-A, p38MAPK, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 showed a diet-induced deregulation of all these proteins. Interestingly, the expression pattern of LITAF, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 might be well explained by the trend of their specific mRNAs, by potentially regulatory miRNAs, or both. In conclusion, we highlight for the first time the potential involvement of novel determinants (miRNAs and proteins) in the molecular pathogenesis of diet-induced NAFLD.

We investigate the effect of EGF on IP3 production, PLCγ phosphorylation, calcium transients in rat hepatocytes isolated in quiescent liver (G0 phase of cell cycle) and at 4 h (G1 phase of cell cycle) and 24 h (M phase of cell cycle) after partial hepatectomy. Our results show that EGF does not utilize IP3 and calcium as its signal transduction molecules when the hepatocytes are in vivo stimulated to entry in the cell cycle. In particular the growth factor does not phosphorylate PLCγ and induces a decrease in IP3 content. These data suggest that EGF utilizes different signal transduction to send information from receptor to nucleus during PH with respect to the quiescent liver.

High-fat and high-carbohydrate diets may predispose to simple steatosis, alone or associated with necroinflammation and fibrosis (steatohepatitis). However, there are few reports about the real effect of these nutrients on hepatocyte redox homeostasis and consequent molecular derangement. Here, we investigated whether different diets would induce oxidative damage in primary rat hepatocytes and thereby affect the activity of phosphatase and tensin homolog (PTEN). We used Sprague–Dawley rats fed, for 14 weeks, a standard diet (SD), a high-fat/low-carbohydrate diet (HFD-LC), a normal-fat/high-fructose diet (NFD-HF), or a high-fat/high-fructose diet (HFD-HF). Metabolic and histological parameters were analyzed in blood and liver samples, while oxidative stress markers and related posttranscriptional modification of PTEN were analyzed in isolated hepatocytes. Our results indicate that different dietetic hypercaloric regimens caused liver damage and a significant increase of body and liver weight, as well as elevated plasma levels of alanine aminotransferase, triglycerides and insulin. Hepatocytes from NFD-HF and HFD-HF rats displayed a decrement of cell viability and proliferation rate. Hepatocytes from animals treated with hypercaloric regimens also exhibited oxidative stress greater than SD hepatocytes. Finally, NFD-HF and HFD-HF hepatocytes showed an increased PTEN phosphorylation and decreased PTEN activity, which seem strongly correlated to an increased glutathionylation of the protein. In conclusion, we demonstrate that fructose-enriched diets cause a tissue and hepatocyte damage that might exacerbate those observed in the presence of high-fat alone and might render, via redox homeostasis imbalance, the hepatocytes more prone to posttranslational modifications and activity alteration of PTEN.

[1-14C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP3) and calcium, is concerned, substantial differences were found: EGF increased IP3 production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP3 and calcium transients, suggesting that this transduction pathway is not activated during fetal life.

The relationship between cell proliferation and inositol lipid turnover has been studied by comparing the steady state of inositol derivative metabolism in quiescent and regenerating rat hepatocytes isolated at 4 h (G1 phase of first cell cycle) and 24 h (onset of M phase) after partial hepatectomy. The effect of two hormones able to regulate hepatic regeneration, insulin and vasopressin, has been considered, and the results can be summarized as follows: (i) at 4 h after partial hepatectomy, the precursor incorporation into inositol polyphosphates and the particulate phospholipase C activity increase with respect to quiescent hepatocytes, whereas the content of I1,4,5P3 does not change, suggesting an increased turnover of this molecule in this step of cell cycle priming; (ii) 24 h after partial hepatectomy, the radioactivity linked to IP3 and IP4, as well as soluble and particulate phospholipase C activity, and IP3 content increase, suggesting the presence, at the onset of M phase, of second messenger accumulation; (iii) only 24 h after partial hepatectomy, the inositol derivative metabolism is affected by vasopressin; and (iv) insulin exerts a modulatory role on inositol polyphosphate production without involving membrane-bound PLC activity or phosphoinositide hydrolysis. These data suggest that inositol-derived signal molecules are associated with hepatic regneration; moreover, the metabolic pathway of such compounds seems to be regulated so that only specific inositol phosphates are present in each step of the cell cycle. © 1992 Wiley-Liss, Inc.

Cadmium, a toxic heavy metal, expresses its toxicity by affecting several cellular functions, such as enzyme activities, DNA repair systems, redox state of the cell and signal transduction. Although the liver is a known target organ, the mechanisms involved in cadmium toxicity are not yet clarified, especially during prenatal development. Here we consider the effects of cadmium on viability, proliferation, adhesion and defence mechanisms in primary adult and fetal rat hepatocytes. Fetal hepatocytes are less sensitive to cadmium toxicity, they appear to be unaffected or even stimulated by treatments that strongly inhibit DNA synthesis in adult cells. The behaviour of proteins involved in cell cycle regulation also differs from adult cells, according to the proliferative state. In addition, following Cd exposure, E-cadherin/beta-catenin complex disassembles in both cell types, with fetal cells being influenced at higher doses. The beta-catenin is not found in the nucleus, ruling out a direct role on DNA synthesis stimulation. Finally, metallothionein is more easily inducible in fetal hepatocytes, while Cd intracellular concentrations and HSP protein levels are not differentially affected. In conclusion, multiple cellular targets are affected by Cd in primary hepatocytes and the adverse effects of the metal are always better counteracted by fetal cells.

The glycosylation pattern in isolated rat hepatocytes during pre- and post-natal development and senescence has been studied by following: the [14C]glucosamine and [3H]galactose incorporation into cellular glycoproteins and glycolipids and the activity of two microsomal enzymes, N-acetyl-glucosaminyl-1-P transferase and galactosyl transferase. The data show a lowered precursor incorporation into lipids and proteins in the fetus, newborn and old rats versus the adult. Only the galactosyl transferase activity is enhanced on the 19th and 22nd day of fetal life. The glucosamine and N-acetyl-glucosamine content in both soluble and protein bound fractions was increased, while the galactose content in lipids and proteins decreased in the fetal stage. The different sugar composition of the proteins, and the decreased glucosamine and galactose incorporation into the proteins, observed in the fetus, newborn and old rat, suggest a post-translational modification which may cause alterations in functions usually mediated by glycoproteins.

The purpose of this study was to define the distribution of the asialoglycoprotein receptor (ASGP-R) main peptide, rat hepatic lectin (RHL)-1, within the rat liver lobule and to investigate its possible modulation in physiological states characterised by marked changes of receptorial expression. In particular, we chose livers from rats partially hepatectomised or at the end of pregnancy, as models, respectively, of decreased or increased expression of the ASGP-R, and used the in situ hybridisation and immunocytochemistry techniques to analyse in parallel the lobular distributions of RHL-1 mRNA and protein. In normal rat liver, although the RHL-1 mRNA was homogeneously distributed, the RHL-1 peptide was predominantly localised on the surface of pericentral hepatocytes with a gradient of expression towards the periportal zone. This gradient of expression of RHL-1 peptide was reduced in regenerating livers, in which the positive stain was restricted to a few layers of cells around the central vein. In contrast, livers at the end of pregnancy showed an overall increase of the peptide with a concomitant flattening of the gradient across the liver plate. In all the conditions, we never observed important changes in the pattern of expression of the specific mRNA. These findings indicate that the distribution of ASGP-R is heterogeneous across the liver lobule, with a pattern of expression prevalently modulated at the posttranscriptional level.

In rats, various growth factors and hormones, as well as partial hepatectomy (PH) are able to trigger the proliferative response of hepatocytes. Although recent evidence highlights the important role of thyroid hormones and thyroid status in regulating the growth of liver cells in vitro and in vivo models, the mechanism involved in the pro-proliferative effects of thyroid hormones is still unclear. Here we have investigated how in rats made hypo- and hyperthyroid after prolonged treatment respectively with propylthiouracil (PTU) and triiodothyronine (T3), the thyroid status affects liver regeneration after PH by regulating cell cycle and apoptosis proteins. Our results show that both in control and partially hepatectomized animals hyperthyroidism increases the cyclin D1, E and A levels and the activity of cyclin-cdk complexes, and decreases the levels of cdk inhibitors such as p16 and p27. On the contrary hypothyroidism induces a down-regulation of the activity of cyclin cdk complexes decreasing cyclin levels. Thyroid hormones control also p53 and p73, two proteins involved in apoptosis and growth arrest which are induced by PH. In particular, hypothyroidism increases and T3 treatment decreases p73 levels. The analysis of the phosphorylated forms of p42/44 and p38 MAPK revealed that they are induced during hepatic regeneration in euthyroid and hyperthyroid rats whereas they are negatively regulated in hypothyroid rats. In conclusion our data demonstrate that thyroid status can affects liver regeneration, altering the expression and the activity of the proteins involved in the control of cell cycle and growth arrest.

Liver proliferation occurs in the presence of mitogenic stimuli such as partial hepatectomy or growth factors. In this work we investigate how partial hepatectomy and Epidermal Growth Factor (EGF) affect hepatocyte proliferation by modulating cell cycle regulators. EGF administered to non-operated rats increased PCNA (proliferating cell nuclear antigen) expression, whereas when EGF was administered to partially hepatectomized rats it was able to anticipate the increase in PCNA expression to 18h after PH and to prolong it to 34h. Cell cycle progression was examined by monitoring specific markers of late G1- and S-phases. Western blot analysis showed that both treatment with EGF alone and treatment with EGF after PH induce the expression of cyclins D1 and A and of p21(cip1), but inhibites the expression of cyclin E and p27(kip1). EGF administration after PH also significantly affected the activity of the cyclin D1-cdk4 and cyclin E-cdk2 complexes, mainly by changing their time progression: it accelerated the increase in activity to 18h and caused a subsequent drop in activity after 34h; it delayed the activity of the cyclin A-cdk2 complex to 34h. In conclusion we observed that EGF modulates the activity of cdk complexes and induces a different linkage with inhibitory proteins that demonstrates their dual role, depending on their association with different cyclin-cdk complexes.

Glycerophosphoinositols are metabolites formed by a phosholipase A2 and a lysolipase specifically acting on membrane phosphoinositol lipids. High levels of these compounds characterize epithelial cells and fibroblasts transformed by ras and other cellular oncogenes. Here we have analyzed the glycerophosphoinositol levels in cells that are considered models of cell differentiation. Using rat hepatocytes at different stages of liver development we have shown that the glycerophosphoinositol basal levels of fetal cells were up to fourfold higher than in adult hepatocytes. No changes in glycerophosphoinositol were observed in regenerating rat liver, a model of differentiated cells proliferating in a synchronous manner, where only glycerophosphoinositol 4-phosphate increased by 80%. Similarly to fetal hepatocytes, a modest but significant increase (30%) in the levels of glycerophosphoinositols was observed in undifferentiated NG-108-15 cells as compared to the same cells induced to differentiate by cAMP. In a different neuronal cell line, PC12 cells, increased glycerophosphoinositol levels characterized the differentiated cells. Based on these observations we suggest that high glycerophosphoinositol levels characterize cellular phenomena associated with the activation of ras/mitogen-activated protein kinase pathways.

The modulation by epidermal growth factor (EGF) of the Na+/H+ antiport in fetal and adult rat hepatocytes was studied in nominally HCO3- free solution. EGF (10 nM) activated the antiport in adult rat hepatocytes by 0.22 +/- 0.03 (mean +/- SD;n=10) pH units over basal value, measured with the fluorescent pH-sensitive intracellular probe, 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein (BCECF). The effect of EGF was inhibited by amiloride analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA), by ouabain, inhibitor of the Na+ pump, and by erbstatin analogue, an inhibitor of the tyrosine kinase activity of the EGF receptor. The effect of EGF on Na+/H+ antiport in adult rat hepatocytes appeared to be mediated by both protein kinase C (PKC) and G protein system. No effect of EGF and phorbol 12-myristate 13-acetate, an activator of PKC, on the Na+/H+ antiport was observed in fetal hepatocytes of 20 and 22 days. A different sensitivity of the antiport to high concentrations of amiloride and EIPA suggests that altered amount of the Na+/H+ antiport units or different isoforms could be expressed in fetal compared with adult cells.

Stimulation of resident peritoneal macrophages with S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-C ysSerLys4 or S(-)[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-++ +CysAlaLys4, two synthetic bacterial lipopeptides, promoted the expression of the inducible form of nitric oxide synthase, exhibiting a temporal pattern of nitric oxide release that was delayed with respect to the induction elicited by bacterial lipopolysaccharide. Treatment of macrophages with genistein blocked the nitric oxide synthesis triggered by the lipopeptides or lipopolysaccharide. Simultaneous incubation with lipopolysaccharide and lipopeptide resulted in an antagonistic effect on nitric oxide synthase mRNA levels and on nitrite plus nitrate release to the medium. Triggering with bacterial lipopeptides induced macrophage programmed cell death. In macrophages activated with lipopeptide, apoptosis was observed even in the absence of nitric oxide synthesis, therefore indicating the existence of alternative pathways in the control of programmed cell death in these cells.

Amino acid uptake mediated by system A was studied in cultured fetal and adult hepatocytes, subjected to growth stimulation by EGF and insulin, or to growth inhibition by high cell density. The mitogenic stimulation induced a strong transport increase only in fetal cells, while the cell density-dependent growth inhibition, probably mediated by molecules present on adult hepatocyte membranes, provoked the decrease of amino acid uptake only in the adult cells. The results indicate that the different modulation of amino acid transport by cell growth is dependent on the age and the differentiation stage of hepatocytes.

The content and the percent distribution of dolichol and dolichyl phosphate homologues were measured by high-performance liquid chromatography in perinatal rat livers. Short dolichol chains and no dolichyl phosphate are detectable in the liver at foetal stages; dolichol content progressively increases during liver development. A good correlation is observable between the changes of the dolichol, dolichyl phosphate and the activity of dolichyl-phosphate phosphatase.

The effect of epinephrine on the amino acid transport mediated by system A was investigated by determining the uptake of 2-amino [1-14C]isobutyric acid (AIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the hormone increased AIB uptake, enhancing the Vmax, while Km was unchanged. This effect was evident in cells from adult, 18- to 20-day-old fetus, and neonate rat. Actinomycin D or cycloheximide abolished the hormone dependent increase. Experiments carried out with alpha- and beta-antagonists showed that the effect of epinephrine was beta-mediated in fetal life and alpha-mediated in adult life. Membrane binding experiments showed a higher value for epinephrine and beta-agonist dihydroalprenolol in the fetus versus the adult. The calcium depletion obtained after cell incubation with EGTA or calcium ionophore A23187 reduced the hormonal stimulation in the adult, and was ineffective in the prenatal period. An involvement of cAMP was present in the epinephrine modulation of AIB transport, both in adult and in fetal life.

The lipid composition and fluidity of plasma membranes have been studied at different stages of liver regeneration (4, 15 and 24 h after surgery). The phospholipid and fatty acid composition is not modified, whereas the cholesterol/phospholipid ratio is lower with respect to control membranes. The modification of the physical properties of the membranes has been studied directly by EPR analysis and indirectly by temperature dependence and cooperativity of some membrane-bound enzymes (Mg2+-ATPase, (Na+ + K+)-ATPase and 5'nucleotidase). Surgical operation or anaesthesia alone causes an early increase in fluidity; such an effect appears to be markedly reduced at a later stage. There seems to be a marked effect of regeneration on plasma membrane fluidity 15 h after partial hepatectomy when several parameters--surface fluidity, cholesterol/phospholipid ratio, and 5'-nucleotidase activity in the presence of concanavalin A -- are modified and indicate an increase in membrane fluidity. It is suggested that this modification of membrane properties could be related to the proliferative process.

Cholesterogenesis pathway during pre- and postnatal development was studied in isolated rat hepatocytes. No modified activity of cytosol acetoacetyl coenzyme A (CoA), thiolase, or 3-hydroxy-3-methylglutaryl CoA (HMGCoA) synthase was detectable at the different stages examined. Minimal levels of 1(14)C-acetate incorporation into cholesterol and HMGCoA reductase activity were present at 16 days of fetal development in newborn and suckling rats, whereas both parameters increased rapidly before birth. The pattern of NaF nonsuppressible reductase activity showed a different activation state of the enzyme, suggesting the appearance of a modulation state, probably related to the development of some short-term regulatory mechanisms.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.

Acyl CoA: cholesterol acyl transferase (ACAT) activity presents marked oscillations and differential sensitivity to the “in vitro” stimulation of the kinase-phosphatase modulatory system in the perinatal rat liver. The regulation of this enzyme activity by some modulators generally active in adulthood, such as cholesterol, lipoproteins and mevalonate, has been studied in hepatocytes isolated at different developmental stages. A lack of effect of mevalonate and a positive effort of lipoprotein cholesterol have been observed at the fetal and neonatal stages. A differential prevalence is suggested of one of the two modulatory mechanisms (phosphorylation-dephosphorylation system, or substrate effect) at each developmental stage.

1. The liver of female frogs shows a significantly higher dolichol content in May (134 micrograms/g) than in November (90 micrograms/g). The male frog liver (134 micrograms/g May; 119 micrograms/g November) does not show any significant change. 2. The rat liver does not show any significant change in dolichol content from May to November (30 micrograms/g). 3. The dolichyl-phosphate level was the same in male and female frog livers and in the rat liver. 4. Data is given of the distribution of dolichols according to the number of their isoprene residues.

The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na+−K+)-ATPase and Mg2+-ATPase activities but not 5′-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.

Acyl coenzyme A:cholesterol acyltransferase (ACAT), the enzyme catalyzing the hepatic cholesterol esterification could be involved in the modified availability of cholesterol detectable in proliferating systems. While no significant variations are detectable in the regenerating liver, the modified ACAT activity during liver development and its differential sensitivity to the in vitro stimulation of modulatory systems suggest an involvement of the enzyme in this proliferating process.

The regenerating liver presents a changed ability to use mevalonate 16 hr after partial hepatectomy. The dolichol content and its synthesis from mevalonate is increased, while no variation of dolichyl-P and ubiquinone parameters are detectable. The greater amount ofmevalonate utilized to form dolichol, but not dolichyl-P, in this proliferating system, raises some questions about the physiological significance of these isoprenoid compounds and about their biosynthetic sequence.

Colchicine effect has been tested on rat liver plasma membrane-bound enzymes after in vitro or in vivo treatment. It appears that the in vitro treatment does not affect 5'-nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase, whereas adenylate cyclase is sensitive to both in vitro and in vivo treatment, the latter condition being also effective for 5'-nucleotidase.

The regenerating rat liver provides a unique in vivo synchronized system for study of the interrelationships between mevalonate and sterol metabolism during the cell cycle. The regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, cholesterol synthesis and acyl coenzyme A: cholesterol acyltransferase during the first cell cycle was investigated. At 8 h postoperative and prior to onset of DNA synthesis or S phase, cholesterol synthesis was depressed in the regenerating liver relative to that in sham-operated controls. This suppression was observed whether assayed in vitro with liver homogenates utilizing radiolabeled acetate, mevalonate or water or in vivo with tritium water. In contrast, at this time point, 3-hydroxy-3-methylglutaryl-CoA reductase activity was increased in microsomes prepared both in the presence and absence of NaF. By 24 h, well into S phase and approaching mitosis, reductase activity and cholesterol synthesis both approached levels observed in the sham-operated control animals. There were no detectable changes in acyl-CoA: cholesterol acyltransferase activity at any time point. Thus, at the 8 h time point, the regulation of the three processes appeared uncoupled. The increased levels of in vitro expressed 3-hydroxy-3-methylglutaryl-CoA reductase activity compared with the decrease in the rate of both cholesterol and squalene biosynthesis suggested diversion of mevalonate into products other than squalene or sterols. We propose that this may reflect the needs of the cell for a nonsterol metabolite of mevalonate necessary for entry of cells into S phase.

The binding and uptake of cholesterol enriched lipoproteins by isolated hepatocytes was decreased at 16 hours after partial hepatectomy, with a tendency to return to control values as the regeneration proceeds. The number of lipoprotein binding sites of total cellular membranes remained similar to control at 16 and 24 hours. The plasma lipoprotein pattern, determined by electrophoretic analysis, showed a lower per cent of very low density lipoproteins (VLDL) and a higher per cent of low density lipoproteins (LDL) at 16 and 24 hours post-partial hepatectomy. At these times, plasma lecithin: cholesterol acyltransferase (LCAT) activity was decreased. It is intriguing to suggest that the regenerating liver could regulated the blood lipoprotein pattern and the uptake of lipoproteins by modulating the surface expression of the receptors.

Maintenance of whole body cholesterol homeostasis is determined in part by the liver. Thus, changes in expression of hepatic parameters important in the regulation of cholesterol metabolism may play key roles in determining how homeostasis is maintained. The expression of hepatic lipoprotein uptake systems was studied during development using as a ligand very-low density lipoproteins rich in apolipoprotein E that had been obtained from hypercholesterolemic adult rats. These lipoproteins can serve as ligands for cell surface receptors recognizing apolipoproteins B and/or E. Uptake was lowest in freshly isolated fetal rat hepatocytes, increased substantially in hepatocytes from neonates and was intermediate in those from adults. Binding of these lipoproteins to liver membranes prepared from fetal, neonatal, suckling, weaned and adult rats was lowest in fetal preparations, while those from suckling, weaned and adult livers behaved similarly. Numbers of binding sites in neonatal liver membranes were similar to those in adult, but showed a different affinity. On the basis of this data, the ability of hepatocytes to recognize and remove apolipoprotein B/E-containing lipoproteins from the plasma appears to be a function of the differential expression or regulation of lipoprotein-uptake systems during development.

The 2-aminoisobutyric acid transport in hepatocytes isolated from 3- and 24- month-old rats studied and some age-related differences were observed. The basal uptake appeared to be almost constant in cells from old animals during the incubation time, while, in the cells from adults, it showed a progressive increase, interpreted as being due to a depression mechanism. Epinephrine and glucagon increased the transport in hepatocytes from animals of both ages, even if with a slightly different pattern; the hormones increased the V max , while the K m was unchanged at each age tested. However, the glucagon-induced increase in V max was lower in the older animals. The mechanism of hormonal action appeared to be similar in adult and old rats. In fact the uptake stimulation by glucagon and epinephrine showed a dependence on protein synthesis. The epinephrine effect was mediated by α-adrenergic receptors. No effect was exerted by extracellular amino acids on hepatocytes rom 24-month-old animals, suggesting a loss of adaptative regulation mechanism with aging. This behaviour was reflected in the kinetic parameters; in the fact the V max was not modified by extracellular amino acids at 24 months of age, while it appeared to be strongly decreased in the adult.

The effect of glucagon and insulin on the incorporation of 1-14C-acetate into cholesterol and fatty acids and on the enzymes involved in the first steps of cholesterol synthesis (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase, and acetoacetyl-coenzyme A thiolase) was investigated. Isolated rat hepatocytes at different stages of fetal and postnatal development were employed. Data obtained show the appearance of hormonal control on the 18th day of fetal life, indicating the same pattern, as regards acetate incorporation and HMGCoA reductase prepared and assayed in the presence of NaF. On the contrary, HMGCoA reductase, prepared without NaF, HMGCoA synthase, and acetoacetyl CoA thiolase, does not appear to respond to hormonal stimulation. In the perinatal period, the hormonal effect is no longer detectable, probably because of a hormone resistance of this metabolic pathway.