Carmen Marco

Comparative study of the effects of short- and long-term ethanol treatment and alcohol withdrawal on phospholipid biosynthesis in rat hepatocytes

This study describes the effects of short- and long-term ethanol treatment and withdrawal on the biosynthesis of the phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in hepatocytes isolated from rats, using isotopically labelled choline and ethanolamine as exogenous precursors. Our results demonstrate that short-term ethanol consumption increases the incorporation of exogenous polar bases into PC and PE, whereas long-term ethanol administration provokes a differential effect in both PC and PE biosynthesis via cytidine diphosphate derivatives (CDP-derivatives), decreasing PC synthesis and increasing the biosynthesis of PE. We suggest that the increased biosynthesis of PE after ethanol treatment results from changes in lipogenic substrates produced as a consequence of ethanol metabolism, whilst the specific inhibition of PC biosynthesis seems to be a consequence of alterations of enzymes involved in the CDP-choline pathway. With regard to the influence of ethanol on PE methylation to give PC, our results demonstrate that ethanol activates this pathway in short-term, as well as chronic ethanol treatment. Ethanol withdrawal returns the activity of the PC and PE pathways to control levels. The alterations in the biosynthesis of the main phospholipids, PC and PE, demonstrated in this study could be of a great physiological interest in determining the pathology of alcoholism.

Effects of different nutritional conditions on chick liver mevalonate-activating enzymes

The response of mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase of chick liver to different dietary situations has been investigated. Fasting inhibited mevalonate kinase and mevalonate-5-pyrophosphate decarboxylase activities, while mevalonate-5-phosphate kinase remained practically unaltered. Refeeding after 72 h of starvation restored mevalonate kinase activity to normal levels after 120 h of refeeding. Likewise, decarboxylase activity reached normal levels at 72 h of refeeding the standard diet and slightly supranormal levels after 120 h. In addition, the sequential response of the three enzymes to a high cholesterol diet was followed throughout a 120 h period. Feeding a 5% cholesterol diet to 13-day-old chicks previously fed with a standard diet from hatching reduced considerably the activity of mevalonate-5-pyrophosphate decarboxylase, while the kinases were less affected. The present results support the idea of a coordinate regulation of the enzymes implied in cholesterol biosynthesis and suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in this regulation.

Alterations in the homeostasis of phospholipids and cholesterol by antitumor alkylphospholipids

The alkylphospholipid analog miltefosine (hexadecylphosphocholine) is a membrane-directed antitumoral and antileishmanial drug belonging to the alkylphosphocholines, a group of synthetic antiproliferative agents that are promising candidates in anticancer therapy. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of miltefosine and other alkylphospholipids on the human hepatoma HepG2 cell line, with a special emphasis on lipid metabolism. Results obtained in our laboratory indicate that miltefosine displays cytostatic activity and causes apoptosis in HepG2 cells. Likewise, treatment with miltefosine produces an interference with the biosynthesis of phosphatidylcholine via both CDP-choline and phosphatidylethanolamine methylation. With regard to sphingolipid metabolism, miltefosine hinders the formation of sphingomyelin, which promotes intracellular accumulation of ceramide. We have demonstrated for the first time that treatment with miltefosine strongly impedes the esterification of cholesterol and that this effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to higher levels of cholesterol in the cells. Indeed, miltefosine early impairs cholesterol transport from the plasma membrane to the endoplasmic reticulum, causing a deregulation of cholesterol homeostasis. Similar to miltefosine, other clinically-relevant synthetic alkylphospholipids such as edelfosine, erucylphosphocholine and perifosine show growth inhibitory effects on HepG2 cells. All the tested alkylphospholipids also inhibit the arrival of plasma-membrane cholesterol to the endoplasmic reticulum, which induces a significant cholesterogenic response in these cells, involving an increased gene expression and higher levels of several proteins related to the pathway of biosynthesis as well as the receptor-mediated uptake of cholesterol. Thus, membrane-targeted alkylphospholipids exhibit a common mechanism of action through disruption of cholesterol homeostasis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine and sphingomyelin biosyntheses certainly alter the ratio of choline-bearing phospholipids to cholesterol, which is critical for the integrity and functionality of specific membrane microdomains such as lipid rafts. Alkylphospholipid-induced alterations in lipid homeostasis with probable disturbance of the native membrane structure could well affect signaling processes vital to cell survival and growth.

Relationship between changes in free cholesterol and pyrophosphomevalonate decarboxylase activity during myelination

The patterns of cholesterol content in chick brain and liver were studied during embryonic development and compared with the variations in the specific activities of mevalonate-activating enzymes during the same period. Total cholesterol content in both embryonic chick brain and liver increased during incubation. The relative percentage of free cholesterol was always maintained over 85% in brain, while in liver this percentage decreased to less than 10% during the later days of incubation. A strait parallelism was observed between free cholesterol and pyrophosphomevalonate decarboxylase activity in the embryonic brain. On the other hand, the hepatic decarboxylase exhibited a lower specific activity than in brain and did not show significant variations throughout the same period of incubation. Changes in brain pyrophosphomevalonate decarboxylase activity were more pronounced than those observed in both mevalonate kinase and phosphomevalonate kinase activities, in spite of that the specific activity of decarboxylase was the lowest of the three mevalonate-activating enzymes, suggesting that this reaction is one rate-limiting step for cholesterogenesis during myelination.

Disruption of cellular cholesterol transport and homeostasis as a novel mechanism of action of membrane‐targeted alkylphospholipid analogues

Background and purpose:  Alkylphospholipid (APL) analogues constitute a new class of synthetic anti‐tumour agents that act directly on cell membranes. We have previously demonstrated that hexadecylphosphocholine (HePC) alters intracellular cholesterol traffic and metabolism in HepG2 cells. We now extended our studies to analyse the effects of other clinically relevant APLs, such as edelfosine, erucylphosphocholine and perifosine on intracellular cholesterol homeostasis.

Chronic ingestion of ethanol stimulates lipogenic response in rat hepatocytes

We isolated hepatocytes from rats chronically fed with ethanol and pair-fed control rats and incubated them both in the presence and absence of 100 mM ethanol in order to analyze the uptake into their lipids of several radiolabeled exogenous substrates. The hepatocytes treated chronically with ethanol showed higher lipogenic activity both in neutral lipids and phospholipids from serine, ethanolamine, glycerol and oleate. The only exception found was in the incorporation of choline into phosphatidylcholine (PC), which was lower in the hepatocytes from ethanol-fed rats than in the controls and was concomitant with a decrease in the PC levels of the ethanol-fed hepatocytes. The results obtained after exposing the cells to 100 mM ethanol in vitro indicate that in general the hepatocytes from ethanol-fed rats exhibit a higher lipogenic activity than the control cells. The only difference in the response to ethanol in vitro was found in the biosynthesis of phosphatidylserine (PS) from serine, which rose significantly in control cells but was unaffected in alcoholic hepatocytes. We put this difference in response down to specific adaptation to ethanol feeding.

Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.

Hexadecylphosphocholine alters nonvesicular cholesterol traffic from the plasma membrane to the endoplasmic reticulum and inhibits the synthesis of sphingomyelin in HepG2 cells.

The synthetic lipid analogue, hexadecylphosphocholine is an antitumoral and antileishmanial agent that acts on cell membranes and can induce apoptosis. We have previously investigated the effect of hexadecylphosphocholine on the biosynthesis and intracellular transport of cholesterol in the human hepatoma HepG2 cell line. Here we show that the traffic of endocytosed-cholesterol from LDL to the plasma membrane and the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane were unaffected by alkylphosphocholine exposure. On the contrary, cholesterol traffic from the plasma membrane to the endoplasmic reticulum was drastically interrupted after 1 h of cell exposition to HePC and, consequently, the intracellular esterification of cholesterol was substantially decreased. Our results also demonstrate that this alkylphosphocholine exclusively affected the nonvesicular, energy-independent cholesterol traffic, without altering the vesicular transport. In addition, hydrolysis of plasma membrane sphingomyelin by exogenously added sphingomyelinase resulted in enhanced plasma-membrane cholesterol esterification, but sphingomyelinase treatment did not prevent the inhibition in cholesteryl ester formation caused by hexadecylphosphocholine. We also found that sphingomyelin synthesis was significantly inhibited in HepG2 cells after exposure to hexadecylphosphocholine. Since sphingomyelin and cholesterol are major lipid constituents of membrane raft microdomains, these results suggest that hexadecylphosphocholine could disturb membrane raft integrity and thence its functionality.

Postnatal Development of the Sterol and Nonsterol Mevalonate Metabolism in Chick Liver and Kidneys

The effect of 2% cholesterol feeding on changes throughout postnatal development of total, free, and esterified cholesterol in neonatal chick liver and kidneys was studied. The increase observed in the hepatic cholesterol content after supplementation of the diet with 2% cholesterol was mainly due to the accumulation of esterified cholesterol. Small but significant differences were also found in the esterified cholesterol content in kidneys between control and cholesterol-fed animals. In normally fed chicks, the hepatic percentage of squalene synthesized from mevalonate decreased during the first days of independent life while cholesterol percentage increased. On the contrary, the percentage of squalene recovered in kidneys immediately after hatching was minimal, increasing during postnatal development. Addition of 2% cholesterol to the diet produced a clear inhibition in the mevalonate incorporation into nonsaponifiable lipids by liver slices, especially from 4 days onwards. This inhibition was particularly clear in the percentage of cholesterol synthesized. Mevalonate incorporation by kidney slices was higher than in liver, although cholesterol supplementation had little influence on the percentage of each nonsaponifiable lipid formed. In normally fed chicks, kidneys metabolized mevalonate by the shunt pathway not leading to sterols at a rate more than 50 times that of liver. Cholesterol feeding produced a clear enhancement of the hepatic shunt pathway while in kidneys it had practically no effect.

Ethanol and lipid metabolism Differential effects on liver and brain microsomes

We have determined the effect of prolonged ethanol treatment on several enzyme activities related to lipid metabolism in chick‐brain and liver microsomes. Ethanol increased microsome cholesterol levels in both organs. The treatment caused a marked increase in the hepatic HMG‐CoA reductase and ACAT activities while in the brain a clear decrease was found in these enzyme activities. At the same time the activity of reacylation of phospholipids, was clearly modified in both brain and liver. Thus, while in the liver the turnover of acyl moieties of phosphatidylethanolamine, sphingomyelin and phosphatidylinositol was enhanced by ethanol consumption, in the brain only the reacylation of phosphatidylserine increased to any significant extent. These results indicate that ethanol exerts a differential action in brain and liver, namely cholesterol synthesis and esterification decreased in brain and increased in chick liver. Ethanol also induces faster phospholipid metabolism in both brain and liver microsomes.