Effect of fructose and 3,5-diiodothyronine (3,5-T(2)) on lipid accumulation and insulin signalling in non-alcoholic fatty liver disease (NAFLD)-like rat primary hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is nowadays considered as one of the most serious pathological conditions affecting the liver. NAFLD is supposed to be initiated by the accumulation of lipids in the liver, which finally results in an impaired hepatic insulin signalling. Many researchers have recently focused their attention on the role played by fructose as a NAFLD-triggering agent, because of the increased diffusion of fructose-sweetened food. However, epidemiological data do not permit to evaluate the role of fructose per se, because these foods are often associated with elevated energy intake and unhealthy lifestyle. In the present work, we analysed the effects of fructose on the accumulation of lipids and insulin signalling in rat primary hepatocytes. Moreover, we investigated the effect of the thyroid hormone metabolite, devoid of thyrotoxic effects, 3,5-diiodothyronine (3,5-T2) over the same parameters. To evaluate the effect on insulin signalling we took into consideration three key proteins, such as p85 subunit of phosphatidylinositol 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), and Akt. Our results show that fructose in vitro, in the range of physiological concentrations, was not able to stimulate either lipid accumulation or to impair insulin signalling in our NAFLD-like rat primary hepatocytes. Our data thus support the idea that fructose per se may exert detrimental effects mainly triggering systemic effects, rather than directly affecting isolated hepatocytes. Moreover, we demonstrated that 3,5-T2, at physiological levels, reduces lipid content and triggers phosphorylation of Akt in an insulin receptor-independent manner, revealing new interesting properties as a biologically active molecule.

Emerging role of thyroid hormone metabolites.

Thyroid hormones (THs) are essential for the regulation of development and metabolism in key organs. THs produce biological effects both by directly affecting gene expression through the interaction with nuclear receptors (genomic effects) and by activating protein kinases and/or ion channels (short-term effects). Such activations can be either direct, in the case of ion channels, or mediated by membrane or cytoplasmic receptors. Short-term-activated signalling pathways often play a role in the regulation of genomic effects. Several TH intermediate metabolites, which were previously considered without biological activity, have now been associated with a broad range of actions, mostly attributable to short-term effects. Here, we give an overview of the physiological roles and mechanisms of action of THs, focusing on the emerging position that TH metabolites are acquiring as important regulators of physiology and metabolism.

Changes in parameters of lipoprotein metabolism during rat hepatic development.

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.

Plasma membrane changes associated with rat liver regeneration.

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.

The promoter of the rat 3-hydroxy-3-methylglutaryl coenzyme A reductase gene contains a tissue-specific estrogen-responsive region.

The isoprenoid metabolic pathway is mainly regulated at the level of conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA reductase. As estrogens are known to influence cholesterol metabolism, we have explored the potential regulation of the HMG CoA reductase gene promoter by estrogens. The promoter contains an estrogen-responsive element-like sequence at position -93 (termed Red-ERE), which differs from the ERE consensus by one mismatch in each half of the palindrome. A Red-ERE oligonucleotide specifically bound estrogen receptor in vitro and conferred receptor-dependent estrogen responsiveness to a heterologous promoter in all cell lines tested. However, expression of a reporter driven by the rat HMG CoA reductase promoter was induced by estrogen treatment after transient transfection into the breast cancer cell line MCF-7 cells but not in hepatic cell lines expressing estrogen receptor. Estrogen induction in MCF-7 cells was dependent on the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384. A functional cAMP-responsive element is located immediately upstream of the Red-ERE, but cAMP and estrogens inhibit each other in terms of transactivation of the promoter. Similarly, induction by estrogens was inhibited by micromolar concentrations of cholesterol, likely acting via changes in occupancy of the sterol-responsive element located 70 bp upstream of the Red-ERE. Thus, within its natural context, Red-ERE is able to mediate hormonal regulation of the HMG CoA reductase gene in tissues that respond to estrogens with enhanced cell proliferation, while it is not operative in liver cells. We postulate that this tissue-specific regulation of HMG CoA reductase by estrogens could partially explain the protective effect of estrogens against heart disease.

Age-related changes in lipid secretion of perfused livers from male Wistar rats donors.

Male Wistar rats show typical age-related variations in the distribution of high-density lipoprotein subfractions that include an increase in HDL1 and a decrease in HDL2 proportion. The role of liver in these variations was evaluated by studying the lipoprotein and bile secretions from perfused livers of 14 +/- 1 and 3.5 +/- 0.5 month old Wistar rats (adult and young animals, respectively). The lipid content of lipoproteins secreted from adult livers was higher in HDL2 fraction and lower in VLDL fraction. The lipid output did not show significant age-related variations in the case of HDL1 fraction. However, the lipoproteins secreted from adult livers contained a higher proportion of phospholipids, and a lower proportion of triacylglycerols in comparison with lipoproteins secreted by young livers. Therefore, the molar ratio of core to surface lipids was lower in lipoproteins secreted by adult livers. Adult livers showed a reduction in bile flow by about 37% with a significantly higher phospholipid secretion. These findings suggest that both the hepatic metabolism of glycerophospholipids and their repartition between plasma and bile compartments are affected by aging process. In conclusion, present data show that the age-related increase in plasma HDL1 proportion, previously observed in this rat strain in vivo, are not due to a higher liver secretion of these particles. Conversely, liver appears to have a major role in the age-related VLDL increase and in the variations of phospholipid lipoprotein secretion.

Changes in plasma dolichol levels, transport, and hepatic delivery during rat liver regeneration.

During the proliferative process that follows partial hepatectomy in the rat, the dolichol content increases in both plasma and liver. Its transport in the blood by lipoproteins also changes. The difference in the distribution of dolichols of various chain lengths in plasma and in the liver is further enhanced during liver regeneration. The dolichol released by perfused liver shows a homologue distribution more similar to that observable in blood than in the liver, thus confirming the importance of the liver as a regulatory site for the blood dolichol supply.

Characterization of the response of growth and differentiation to lipoproteins and agents affecting cholesterol metabolism in murine neuroblastoma cells.

Treatment with mevinolin, a competitive inhibitor of HMGCoAR, the key enzyme of isoprenoid metabolism, causes the arrest of proliferation and the differentiation of a neuroblastoma cell line (N18TG2). Mevalonate and high density lipoproteins partially restore growth. Cholesterol synthesis in the presence of mevinolin remains active, because in these cells the key enzyme HMG-CoA reductase is not completely inhibited by this drug. The fact that cell growth is reduced, while cholesterogenesis remains active, suggests that mevinolin acts by interfering with the synthesis of some unknown compound, other than cholesterol, which is necessary for proliferation.

Mevalonate-derived proteins in liver regeneration.

Sixteen hours after partial hepatectomy in the rat, the synthesis of mevalonate (MVA) is not committed to produce cholesterol and only partially utilized for dolichol formation. In order to investigate the fate of MVA in this replicative system, slices of the remaining liver were incubated with 5-3H-MVA. Labeled proteins from whole liver and purified nuclei were analyzed after extensive delipidation and separation by SDS-PAGE. Many MVA-derived proteins were identified at 16 hours, while only two labelled peptides were detectable at 24 hours. The radioactivity was localized in covalently bound lipid moieties. A highly labeled 26 kD peptide was detectable in the nucleus at 16 hours, suggesting its involvement in the cell cycle progression.

Modulation of asialoglycoprotein binding activity in livers of pregnant or estrogen-treated rats.

This report deals with the modulation of activity and expression of the hepatic asialoglycoprotein receptor, in pregnant or diethylstilbestrol-treated rats. The results show a two-fold increase in the total cell associated binding activity, both in pregnant and in estrogen-treated animals, with respect to normal values. On the contrary the surface expression was shown to be strongly enhanced only in the liver of pregnant rat. Therefore the modulation shown by this receptor system in pregnancy seems to be only partially estrogen-dependent.

Seasonal commitment of HMGCoA reductase activity to vitellogenin production.

In female frogs (Rana Esculenta) during gametogenesis the cholesterol synthesized in the liver by 3-hydroxy-3-methylglutaryl coenzyme A reductase is mostly exported into the blood and taken up by the oocytes. In order to understand the fate of the neosynthesized cholesterol, female and male frogs and estrogenized male controls were injected with the labelled precursor 14C mevalonate. In females and in estrogenized controls, mevalonate-derived radioactivity is found in a plasmatic lipoprotein that has been identified as vitellogenin by immunological detection. The increased 3-hydroxy-3-methylglutaryl coenzyme A reductase activity present in females in Fall is likely to be committed to provide cholesterol for the lipidation of this cholesterol-rich protein.

Regulation of acyl CoA: cholesterol acyl transferase (ACAT) activity by mevalonate and cholesterol in isolated rat hepatocytes during perinatal development.

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.

Short term regulation of acyl CoA: cholesterol acyl transferase (ACAT) activity in the regenerating and perinatal liver.

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.

Enhanced production of dolichol, but not dolichyl phosphate, in the earliest stages of rat liver regeneration.

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 of mevalonate 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.

Effect of HDL1 infusion on biliary secretion in perfused rat liver.

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in the in vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.

Changes in lipoprotein binding and uptake by hepatocytes during rat liver regeneration.

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 regulate the blood lipoprotein pattern and the uptake of lipoproteins by modulating the surface expression of the receptors.

Cholesterol metabolism in frog (Rana esculenta) liver: seasonal and sex-related variations.

Many aspects of lipid metabolism have been studied in amphibians, but seasonal lipid modulation in male and female frogs has not been investigated. We describe here the yearlong patterns of hepatic lipid content and enzyme activities related to cholesterol homeostasis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and acyl coenzyme A:cholesterol acyltransferase (ACAT) activity in liver of the male and female frog, Rana esculenta. Lipid storage follows distinct seasonal patterns, with an increase in June that is more pronounced in the female than in the male frog. Cholesterol content and cholesterol storage as cholesteryl ester in male liver are consistent with the activity of HMG-CoA reductase and of ACAT enzymes. HMG-CoA reductase activity of the female frog shows an extra peak in fall unrelated to cholesterol storage and probably related to the production of essential compound for oogenesis.

Functional changes of endoplasmic reticulum membranes associated with liver regeneration.

The fluidity and lipid composition of microsomal membranes have been studied at the earliest stage of liver regeneration in the rat (16 h after partial hepatectomy). The physical properties of the membranes have been measured by electron paramagnetic resonance analysis of freedom of motion of lipid and protein analogue probes. The fluidity of the hydrophobic core and of the microenvironment surrounding membrane proteins appeared to be modified, while no modifications were detectable in the fluidity at the surface or in bulk biochemical composition. The kinetic parameters of two enzymes of the endoplasmic reticulum (3-hydroxy-3-methyl glutaryl coenzyme A reductase and glucose-6-phosphatase) which are differentially localized within the membrane bilayer, were also measured. The temperature dependence of both enzymes was modified in the proliferating system, but these modifications were not consistent with the changes detectable in their specific activity. A model to explain the changes that occur in this proliferating membrane system is presented.

Circannual variations in mevalonate utilization in frog (Rana esculenta).

1. The fate of mevalonate, the product of HMGCoA reductase, was studied in male and female frogs (Rana esculenta) in order to explain the circannual variations of enzyme activity. 2. The incorporation of 2-14C MVA into unsaponifiable lipids, cholesterol and dolichol in liver, plasma and eggs was followed. 3. Labeled MVA shows a different utilization depending on season and sex. In spring and summer cholesterol synthesis is related to hepatic reserve storage in both sexes, while the peak of enzyme activity, present only in females in fall, seems committed to cholesterol export into the blood and uptake by the oocytes. 4. The presence of a MVA-derived protein identifiable with vitellogenin and labeled on the lipid moiety, suggests that HMGCoA reductase activity in fall is committed to the lipidation of this protein essential for oocyte maturation.

High density lipoproteins during rat liver regeneration.

Compositional and maturative parameters of high density lipoproteins (HDL) have been examined during the early stages of rat liver regeneration, when lecithin:cholesterol acyltransferase (LCAT) activity, responsible for the maturation of this lipoprotein class, is markedly decreased. Both HDL subclass distribution and chemical composition are not significantly different from the control, except for a slightly lower cholesterol ester content. Few disc-shaped particles are detectable by electron microscopic observation. Cholesterol ester decrease and presence of immature particles are related, but the entity of the modification is lower than suggested by the deep decrease of LCAT activity. This seems to indicate that proper HDL maturation is assured in the regenerating liver despite low LCAT activity.