Development in lipid drugs.

Lipopeptide lipid moieties induce dendritic cell (DC) internalization and epitopes are recognized by MHC, the major histocompatibility complex. HIV-1 (human immunodeficiency virus type 1) lipopeptide vaccine candidate elicits immune responses, and sustains HIV control after highly active antiretroviral therapy (HAART). Mp- and Dp-MART (anti-melanoma lipopeptides) induce strong CTL (cytolytic T lymphocyte) response. New BGTC, BGDA, TGKC lipoplexes mediate gene delivery, e.g., into mouse pancreatic tumor nodules. Triterpene glycyrrhizic acid (GL) inhibits SARS-CoV (severe acute respiratory syndrome associated coronavirus) replication. Compared to CDV (cidofovir), CDV ether lipid esters have enhanced activity against vaccinia (VV) and cowpox (CV) viruses in vitro. Oral treatment of VV and CV infected mice with CDV ether lipid esters, as effective as i.p. CDV, may be useful against orthopoxvirus infections in humans.

Lipids and retroviruses.

The role that lipids may play in enveloped viruses is reviewed. Small lipid molecules can influence retrovirus binding to cell receptors, plasma membrane fusion, and transcription. Palmitoylation following myristoylation of viral glycoproteins is required at the transmembrane level for signal transduction as well as for virion budding and maturation. Cholesterol, ether lipids, phospholipids, platelet-activating factor, phosphatidic acids, diacylglycerols, and several analogs and derivatives influence human immunodeficiency virus (HIV) activity; when conjugated with inhibitors of the viral reverse transcriptase (RT) or aspartyl protease these compounds increase drug effectiveness. On the other hand, L-carnitine, in association with the mitochondrial cardiolipins, inhibits myopathy due to continued prescription of drugs [AZT (zidovudine), ddl (didanoside), or ddC (zalcitabine)], and the redox couple of alpha-lipoic-dihydrolipoic acid prevents production of the reactive oxygen species that trigger apoptosis of infected cells, with sphingomyelin breakdown to ceramides. Retroviral infection induces a shift from phospholipid to neutral fat synthesis in host cells, and a long antiviral, i.e., antiprotease, treatment may lead to lipodystrophy. Multitherapy involving lipids and their analogs in association with anti-RT and antiproteases might enhance the inhibition of growth and proliferation of retroviruses.

Fructose-induced enhanced mitogenicity of diploid human cells: possible relationship with cell differentiation.

Fructose strongly stimulates the growth of normal diploid human skin fibroblasts (SFs) and induces marked changes in their morphology and lipid accumulation. This mitogenic effect occurs despite very low fructose consumption and depends on the presence of glutamine. The cell kinetics of cultured fructose-fed human skin fibroblasts were different from those fed on glucose: in the presence of fructose a high proliferative index persisted at Day 14 of culture and the duration of the total cell cycle and of the G1 + 1/2 M and S phases was slightly shorter. The mitogenic effect of fructose on SF was largest in the presence of human serum: it was small or undetectable when fibroblasts were cultured in media supplemented with dialyzed human serum, fetal bovine serum, or serum substitutes. This suggests that serum growth factor(s) mediate the mitogenic effect of fructose. Only normal diploid human cells seem to be sensitive to this mitogenic effect of fructose: the long-term growth of normal human liver cells on fructose was slightly better or similar to that on glucose. In contrast, fructose could only support limited growth of hamster fibroblastic Nil cells and of a transformed human fibroblastic line, which grew better with glucose.

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.

Effects of polyunsaturated fatty acids on human and rat cells. In vitro versus in vivo experiments.

Human infant skin fibroblasts and liver cells were subcultured with 250 microM PUFA (polyunsaturated fatty acid), and primary cultures of glial brain cells from new-born rats with 100 microM; oleic acid was added to controls. Minimum essential medium (MEM) supplemented with bovine serum was used as a reference. During the short-term experiment (18-24 h), control liver cells showed a regular increase in protein level, while protein increment was more rapid in linoleic and especially in arachidonic acid-treated cells, but only for the first 3 hours. During the long-term experiment (7 d), control skin fibroblasts showed a faster growth rate (increase in number of cells) than reference or fibroblasts cultured with the added PUFAs. Lipid droplets were seen in the PUFA-treated liver cells and skin fibroblasts, and ultrastructural modifications were observed in fibroblasts, but without growth rate alteration. During the long-term experiment (2 w), control glial brain cells showed faster protein increment (measuring growth rate) than PUFA-treated cells, particularly than arachidonic acid-treated cells. HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) activity, determined after 6 h (liver cells) or 1 and 2 w (brain cells) of culture, was low in controls and reference, whilst higher in PUFA-treated-cells, and was especially high in arachidonic acid-treated brain cells. The present study indicates than the high HMGR activity may correspond to cultures of cells rapidly stopped in their protein increment, and to cultures of cells showing a slow rate of proliferation. This contrasts with results obtained from in vivo experiments; it also emphasizes the high mevalonate (MVA) level as a possible sign of nutritional medium imbalance.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol and prostaglandin synthesis by cultured human skin fibroblasts in the Alagille syndrome involving paucity of interlobular bile ducts.

Children with Alagille syndrome show high serum cholesterol (15-20 mmol/L). To establish correlation of this unusual level of cholesterol with the regulation of cholesterol metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity and synthesis of cholesterol, fatty acids and acidic steroids from [14C]acetate were determined in cultured skin fibroblasts from 2-3 year old children. Prostaglandin E2 (PGE2) synthesis and nucleic acid synthesis were determined in cells when they were growing in medium containing normal, Alagille or fetal bovine serum. These values were similar to values of controls. HMGR activity was found to be similar in cells of control and children with the syndrome, whether the cells were incubated in lipoprotein-deficient or normal medium. Incorporation of acetate into cholesterol was inhibited to a greater extent by lipoprotein-containing medium in control than in children with the syndrome. Fatty acid synthesis was similar in all conditions. 1-7% of the recovered lipid radioactivity in cells and medium separated as acidic steroids. Serum from a donor patient, when included in the medium, did not affect PGE2 or nucleic acid synthesis compared with normal human or fetal bovine serum. The data suggest that cells of children with Alagille syndrome may have a membrane defect of transfer of cholesterol (LDL receptor defect) leading to excessive cholesterol synthesis. Also, synthesis of acidic steroids (bile acid-like material) and their secretion into the medium occurs in normal fibroblasts and those from children with the syndrome.

Permissive role of n-6 polyunsaturated fatty acids on carbohydrate oxidation in human infant skin fibroblasts: one possible mechanism of their intervention on coronary heart disease and diabetes.

UNLABELLED: Many publications indicate the beneficial effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) in the control of coronary heart disease and diabetes, although the mechanism is not clear. Some of our previous results suggest that, in contrast to other lipids, n-6 PUFAs could have a permissive effect on carbohydrate oxidation. To check this hypothesis, we determined pyruvate dehydrogenase (PDH, decarboxylase: EC 1.2.4.1) activity in infant skin fibroblasts (ISF) incubated 6 hours in the presence of 0.25 mM linoleic (LI) or arachidonic (AR) acid, compared to oleic acid (OL) and control ISF incubated without addition of fatty acids. The four groups of cells were preincubated 36 hours either in the presence of fetal bovine serum (FBS), or in the presence of lipoprotein-deprived serum (LPDS). RESULTS: (1) When the ISF were maintained in the medium containing FBS, the two PUFAs had little inhibitory effect on PDH activity, in contrast with the effect of OL. (2) When the ISF were kept in the lipoprotein-deficient medium, PDH activity was low in controls and in the OL cells, but the addition of LI or AR increased the activity. This suggests the role of n-6 PUFAs in enhancing carbohydrate oxidation, under certain conditions.

Extent of 14C incorporation into cholesterol of infant skin fibroblasts incubated with carboxyl-labeled oleic, -linoleic or -arachidonic acid.

In order to determine the incorporation of C1-14C derived from mono- and poly-unsaturated fatty acids into cholesterol of human cells cultured in exponential phase, infant skin fibroblasts (SF) were used at the 5th passage. On Day 6, the SF were preincubated 36 h in a medium containing 5 per cent lipoprotein-deficient serum, and thereafter [1-14C] oleic, -linoleic or -arachidonic acid-without (OL1, LI1 and AR1 group SF), or with the addition of 0.25 mM cold fatty acids (OL2), LI2 and AR2 group SF). Cholesterol specific radioactivity (SRA) peaked 1 h after, and leveled off afterwards in the OL1, LI1 and AR1 groups. Cholesterol-SRA was relatively low in the other groups, but increased progressively, giving a biphasic response: C1-14C derived from from linoleic and arachidonic acids was actively incorporated into cholesterol during the first hours, as compared to C1-14C derived from oleic acid, but stabilized between 6 and 12 h for the LI2 and AR2 group SF incubation. This result appears to be due to the stimulation of pyruvate decarboxylation, observed elsewhere, and consequently to the dilution of the radioactive units in a large pool of non-labeled acetyl-CoA units derived from glucose, when these SF were incubated with 0.25 mM polyunsaturated fatty acids.

Effects of methyl mercury and triethyllead on Na+K+ATPase and pyruvate dehydrogenase activities in glioma C6 cells.

The effect of methyl mercury (MeHg) and triethyllead (Et3Pb) on the membrane bound SH-enzymes Na+K+ATPase and pyruvate dehydrogenase (PDH) was studied in relation to the effect on the galactosyl ceramide sulfotransferase (CST) and to morphological changes in glioma C6 cells. Two-day-old cultures were incubated for 1 or 20 hrs with 5-30 microgram MeHgC1 and 2-8 microgram Et3PbC1/mg cell protein. The results show that both compounds induced morphological changes and a reduction of CST activity at growth inhibitory concentrations. A less marked reduction of Na+K+ATPase was induced with increasing exposure time only in MeHgC1 treated cultures, and PDH activity was not affected by either of the compounds under the experimental conditions. Thus, an interference with Na+K+ATPase and PDH activities do not appear to be a primary effect of MeHg and Et3Pb intoxication.

Pyruvate dehydrogenase activity in the liver, brain and adipose-tissue of lipid-deprived developing rats. Effect of minute amounts of polyunsaturated fatty acids.

UNLABELLED: The present experiment was carried out using the following diets: FF, fat-free, and LP in same diet with 0.7% sunflower oil - given to the progeny of females kept on the FF diet since the mating. after 10 mM Mg2+ activation of the PDH phosphatase, and rate of [1-14C[ pyruvate decarboxylation into acetyl-CoA ester units was determined in the liver, brain and adipose-tissue of the pair-fed developing rats. RESULTS: In the male progeny, pyruvate dehydrogenase (PDH) activity was higher (61%) in the LP group livers than in the FF group livers, at the end of the 13 week experiment. Such a difference was not observed in the two group brains up to the 91 days postweaning, but was even larger (94%) between adipose-tissues of the LP and FF groups. In the female progeny kept 12 weeks on the diets, PDH activity in the LP group tissues was also higher than in the FF group tissues: 63% in the liver, 43% in adipose-tissues, and less than 10% in the brain. Therefore, a minute amount of lipids high in linoleic acid appeared to increase PDH activity, and especially in the liver and adipose-tissues of animals kept on a strictly fat-free diet. This stimulation of the PDH activity seems closely related to the phospholipid rehabilitation in the tissues (decrease in the trienoic, tetraenoic acid ratio values).

Cholesterol synthesis in the liver, kidneys and brain after injection of uniformly 14C-labeled oleic or linoleic acid to developing rats.

6-day-old suckling rats, born to females kept on a fat-free diet, were used to determine cholesterol and fatty acid specific radioactivity (SRA) in the liver, kidneys and brain, after injection of 3 microCi uniformly 14C-labeled linoleic acid (ULI) or oleic acid (UOL). 1 h after injection, cholesterol SRA was highest in the liver and kidneys, and then decreased when UOL was injected. Cholesterol SRA peaked 3 h after injection of ULI in liver and kidneys. The delay in appearance of the ULI (over UOL) peak of cholesterol SRA may be due to differences in the rates of oxidation of these two labeled fatty acids into acetyl-CoA ester, according to the structural role of ULI. In the period between 3 and 56 h, cholesterol was more radioactive in the three tissues after injection of ULI than after injection of UOL. The radioactivity of saturated fatty acids was low in the ULI and UOL groups of these very young animals. Therefore, cholesterol synthesis seemed to happen at a faster rate than the other lipid syntheses in liver and kidneys, where the rate of linoleic acid elongation into arachidonic acid was also slower than cholesterol synthesis. Different results were obtained in the brain, where arachidonic acid SRA increased rapidly after ULI injection.

[Cholesterol ester storage disease in children. Comparative biochemistry of hepatocyte and fibroblast cultures]. / Maladie de surcharge à esters du cholestérol chez l'enfant. Etude biochimique comparative de cultures d'hépatocytes et de fibroblastes.

The results of biochemical studies in three children with cholesterol ester storage disease are reported. This rare disease (13 published cases) and the related Wolman's disease are characterised by a deficiency of acid lipase. Affected children mostly present with isolated hepatomegaly. Hepatic cells (one patient) and fibroblasts (two patients) were cultured and cholesterol accumulation measured. Hepatic cells contained more cholesterol than fibroblasts but the enzyme deficiency, assessed by the abnormal degree of esterification was the same in both cell types.

Children sea-blue histiocytosis (2 cases) compared with phospholipidosis induced by 4-4' DET (4-4' p (diethylamino-2-ethoxy phenyl) 3-4 hexane) in rat.

Two cases of children with liver and spleen enlargement are reported. Sea-blue histiocytes and Pick cells were found in both cases in liver, spleen, bone marrow and blood. Further more, lysobisphodphatidic acids were identified in phospholipid analysis of liver biopsies and cultived liver cells. Absence of neurologic involvement at 14 and 18 years fo age suggest a Crocker type C of Niemann-Pick disease, i.e. a not yet well defined entity. Resemblance of these morphological and biochemical abnormalities with certain cases of drug poisoning (especially the well-known intoxication by 4-4' DET) is discussed on the basis of results from experimental studies with this drug in the rat.

[Fetal and neo-natal development of brown adipose tissue in guinea pigs and rats. Feto-maternal or milk transfer of essential fatty acids : lipogenesis and morphology (author's transl)]. / Développement foetal et néo-natal du tissu adipeux brun du cobaye et du rat. Transfert foeto-placentaire ou lacté des acides gras essentiels : lipogenèse et morphologie

Brown adipose tissue (BAT) lipogenesis (fatty acid, glycerol and CO2 synthesis) and its morphology determined by optical microscopy, were studied in guinea pigs and rats during intra-uterine life and during the suckling period. Following the receptor induction and after the commencement of the hormone sensitive adenylate-cyclase/lipase system (i.e. on the 60th day in guinea pigs, on the 20th day in rats), the fetal BAT releases fatty acids (NEFA) and is capable of allowing the non-shivering thermogenesis. When the maternal diet and, consequently, the fetal or neonatal BAT are supplied with considerable linoleic acid, NEFA contain a large proportion of essential fatty acids. In vitro, the greater the linoleic acid concentration in these NEFA, the less inhibited is the lipogenesis from (2-14C) pyruvate. Thus, in periods just preceding or succeeding birth, fatty acid and glycerol synthesis are higher when the feto-maternal and/or the milk supply are enriched in linoleic acid than when they contain a large proportion of endogenous fatty acids. Morphological studies indicate that the adipose cell evolution could be nonidentical in BAT more or less enriched in essential fatty acids. Linoleic enriched BAT (of animals born to females kept on a sunflower oil diet) seemed to be in a healthy physiological state at birth, perhaps due to rapid lipid renewal and synthesis in their membranes. The control BAT (of animals born to females kept on a lard diet) appeared loaded with fats and in a worse conservation state at the same age.

[Adenylate cyclase/Lipase. Hormone receptor induction]. / Adénylate-cyclase/Lipase. Induction des récepteurs hormonaux

An adenylate cyclase activity (AC) was found in guinea pig brown adipose tissue (BAT), since the tissue's apparition. This enzymatic activity increased during the development and showed high values at the end of gestation. An increase of AC units per cell was observed, in addition to the cell multiplication. A norepinephrine stimulation of AC activity was observed at the end of gestation : this regulating action disappeared in the first days of extrauterine life. Neither glucagon nor ACTH had any regulating role upon AC activity during fetal and newborn life. The basal lipolytic activity which was observed in BAT of fetuses (61rst day) and neonate dramatically around the 15th day. A potent lipolysis activation by norepinephrine was observed, but only after birth. The correlation observed between these enzymatic activities in presence of norepinephrine seems to indicate that the AC/lipase system was involved in the neonatal thermogenesis of guinea pigs.

[A possible essential metabolite of linoleic acid: lipoic acid, the universal coenzyme of alpha-keto acid oxidation]. / Un dérivé vraisemblablement essentiel de l'acide linoléique: l'acide lipoïque, coenzyme universel de l'oxydation des acides alpha-cétoniques

The metabolism of [14C-U] linoleci acid (LI) and [14C-U] oleic acid were compared by injecting these fatty acids into growing rats and then homogenizing livers into a three phase system (chloroform/methanol/water). The radioactivities of these phases were equilibrated by extracting them X times with the same solvents. The lipid lower phase was discarded and the analysis was carried out on the evaporated hydroalcoholic upper phase. The residue was extracted again with methanol and hydrolyzed (HCl 6N). The acidic solution was evaporated, treated with HCl/methanol, extracted with chloroform and analysed by thin layer chromatography. One of the most radioactive intermediates detected after injecting LI was purified again and identified as lipoic acid, on the basis of: a. retention time in gas-liquid chromatography; b. Rf in thin layer chromatography; c. molecular weight as determined by mass spectrometry. Thus, the most important fate of essential fatty acids (except for their part in the prostaglandin synthesis and membrane formation) seems to be that of a precursor for this covalently bound alpha keto-acid dehydrogenation coenzyme--the link between lipid and carbohydrate metabolisms.