Pierluigi Biagi

Polyunsaturated fatty acids: From diet to binding to ppars and other nuclear receptors.

Dietary polyunsaturated fatty acids (PUFAs) function not only by altering membrane lipid composition, cellular metabolism, signal transduction, but possess also effects on gene expression by regulating the activity/abundance of different nuclear transcription factors: peroxisome proliferator activated receptors, retinoid X receptors, liver X receptors, hepatic nuclear factors-4a, and sterol regulatory binding proteins 1 and 2. PUFAs regulate the expression of genes in various tissues, including the liver, heart, adipose tissue, and brain, playing a major role in carbohydrate, fatty acid, triglyceride, and cholesterol metabolism. Before binding to transcription factors, PUFAs must be absorbed in the intestine and delivered to cells, and then they must enter the cell and the nucleus. PUFA concentration within the cell depends on many different factors, and regulate their possibility to act as transcription modulators. The aim of this review is to summarize recent knowledge about PUFAs destiny from dietto nuclear factors binding, examining the different variables which can modulate their interaction with nuclear factors themselves and therefore their effect on gene expression.

Age-related changes in linoleate and α-linolenate desaturation by rat liver microsomes

The first and rate limiting step in the conversion of linoleic and alpha-linolenic acid is catalyzed by the delta - 6 - desaturase (D6D) enzyme. Rat liver microsomal D6D activity decreases on linolenic acid at a rate proportional to the animal age; on alpha-linolenic acid the decrease in D6D activity begins only later than on linoleic acid. The fatty acid composition of liver microsomes determined by gas chromatographic analysis confirms the impairment of the enzymatic activity directly measured. Our data indicate a correlation between aging and D6D activity impairment. The loss of D6D activity may be a key factor in aging through altering the eicosanoid balance.

Dual influence of aging and vitamin B6 deficiency on delta-6-desaturation of essential fatty acids in rat liver microsomes.

Delta-6-desaturase (D6D) activity is influenced by many nutritional and non-nutritional factors, among which one of the most important is aging. D6D activity could be susceptible to the dual influence of aging itself and of nutritional deficiencies, due to the reduced intake and/or absorption of essential nutrients. Particularly, vitamin B6 deficiency might be a crucial factor for D6D activity in aged people. Using 20 month old Sprague-Dawley rats fed a diet with a subnormal level of vitamin B6, we evaluated D6D activity for linoleic acid (LA) and alpha-linolenic acid (ALA) in liver microsomes, and the fatty acid composition of microsomal total lipids. We observed a diminished D6D activity for LA and also for ALA in vitamin B6-deficient animals, being approximately 63% and 81% respectively of the corresponding activity in control rats. As a consequence, significant modifications in the relative molar content of microsomal fatty acids were observed. The content of arachidonic and docosahexaenoic acid, the main products of the conversion of LA and ALA respectively, decreased, LA content increased and a decrease in the unsaturation index was observed in liver microsomes of B6-deficient rats. The foregoing results suggest that the impairment of D6D activity by vitamin B6 deficiency might be an important factor in decreasing the synthesis of n-6 and n-3 PUFAs. This may be particularly important in aging, where D6D activity is already impaired.

The impairment of essential fatty acid metabolism as a key factor in doxorubicin-induced damage in cultured rat cardiomyocytes

The clinical use of the antitumoral doxorubicin (DOX) is limited by its cardiotoxicity, which is mediated through different mechanisms. The membrane lipid peroxidation induced by DOX may cause disruption of the unsaturated fatty acyl chains; in the endoplasmic reticulum, containing the system catalyzing the desaturation/elongation of fatty acids, DOX could interfere with the metabolism of linoleic and alpha-linolenic acids. Using primary cultures of neonatal rat cardiomyocytes we demonstrated that the exposure to different concentrations of DOX (10(-5) and 10(-7) M) for 24 h caused an increase in the production of conjugated dienes, an impairment in the desaturation/elongation of essential fatty acids, and a reduction in the cellular content of highly unsaturated fatty acids. Conversely, 1 h exposure to 10(-5) M DOX was sufficient to induce alterations in the desaturation/elongation of linoleic and alpha-linolenic acids, but did not cause either formation of conjugated dienes or modification of the fatty acyl pattern. Therefore, DOX has a dual negative effect, depending on its concentration and on the time of exposure, one directed against the membrane highly unsaturated fatty acids, the other against the system which is required for the synthesis of these fatty acids themselves. These two effects synergically act in causing heart cell damage.

N − 3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure

In cardiac cells the effects of n − 3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n − 3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n − 3 PUFAs is related to a direct modulation of genes in cardiac cells.

Effect of age and extent of dietary restriction on hepatic microsomal lipid peroxidation potential in mice

Lipid peroxidation potential in hepatic microsomes from young and old mice following two different caloric restriction regimens was measured by a colorimetric thiobarbituric acid method under conditions where Fe2+ autoxidation and free oxygen radical production were undetectable. Peroxidation was highest in the young (3.5-month-old) slightly restricted group (caloric intake 75% of ad libitum mice) but very low in young severely restricted (caloric intake 50% of ad libitum mice) and in both old (27-month-old) slightly and severely restricted groups. Very old (45-month-old) severely restricted animals had intermediate lipid peroxidation potentials. Fatty acid composition of liver homogenates was also determined. Significant differences between groups were found for only three fatty acids. Linoleic acid (18:2(n-6)) decreased in aged slightly restricted animals while it remained stable in severely restricted animals during aging. Dihomo-gamma-linolenic acid (20:3(n-6)) was higher in very old restricted animals than in old slightly restricted animals. Docosahexaenoic acid (22:6(n-3)) decreased in old slightly restricted animals. These results indicated that the effect of diets on hepatic fatty acid composition and the potential for microsomal lipid peroxidation in mice was dependent on the degree of caloric restriction and age.

Delta-6-desaturase activity of human liver microsomes from patients with different types of liver injury

The delta-6-desaturase (D6D) activity was evaluated in microsomes from liver fragments of cholecystectomized subjects without any liver pathology and from explanted liver of patients affected by cirrhosis of different etiologies. We observed a significant decrease in D6D activity, evaluated by a radiochemical technique using 1-[14C]-linoleic acid as substrate, in cirrhotic patients with no correlation with the etiology of the cirrhosis. The D6D activity within the pathological group was quite similar. No alteration in the 20:4/18:2 ratio obtained by gas chromatographic analysis of fatty acid methyl esters of microsomal membranes was found. Liver disease seems to be the main cause of the decreased enzyme activity independent of its etiology.

EPA or DHA Supplementation Increases Triacylglycerol, but not Phospholipid, Levels in Isolated Rat Cardiomyocytes

It is well recognized that a high dietary intake of long-chain polyunsaturated fatty acids (LC-PUFA) has profound benefits on health and prevention of chronic diseases. In particular, in recent years there has been a dramatic surge of interest in the health effects of n-3 LC-PUFA derived from fish, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Notwithstanding, the metabolic fate and the effects of these fatty acids once inside the cell has seldom been comprehensively investigated. Using cultured neonatal rat cardiomyocytes as model system we have investigated for the first time, by means of high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy in combination with gas chromatography (GC), the modification occurring in the cell lipid environment after EPA and DHA supplementation. The most important difference between control and n-3 LC-PUFA-supplemented cardiomyocytes highlighted by HR-MAS NMR spectroscopy is the increase of signals from mobile lipids, identified as triacylglycerols (TAG). The observed increase of mobile TAG is a metabolic response to n-3 LC-PUFA supplementation, which leads to an increased lipid storage. The sequestration of mobile lipids in lipid bodies provides a deposit of stored energy that can be accessed in a regulated fashion according to metabolic need. Interestingly, while n-3 LC-PUFA supplementation to neonatal rat cardiomyocytes causes a huge variation in the cell lipid environment, it does not induce detectable modifications in water-soluble metabolites, suggesting negligible interference with normal metabolic processes.

Dietary manipulation of Δ-6-desaturase modifies phospholipid arachidonic acid levels and the urinary excretion of calcium and oxalate in the rat: Insight in calcium lithogenesis

An anomalous n-6 polyunsaturated fatty acid composition in plasma and erythrocyte membrane phospholipids, namely increased levels of arachidonic acid (AA), has been reported in calcium nephrolithiasis and has been proposed to play an important role in its pathogenesis. To confirm this, in rats we modified phospholipid AA levels by dietary manipulation of the delta-6-desaturase, the rate-limiting enzyme of the fatty acid biosynthetic pathway, and evaluated the effect on cellular and renal functions predisposing to lithogenesis. Increased AA levels led to conditions at risk for nephrolithiasis: higher oxalate flux and lower sodium cotransport in erythrocytes and a rise in urinary prostaglandin E2, calcium, sodium, and oxalate levels; reduced AA levels reversed these changes. In vitro, in human erythrocytes the incorporation of exogenous AA into membranes increased band 3 protein phosphorylation directly activating the Ser/Thr protein kinase CK1 and induced a parallel raise in band 3-mediated oxalate transport. These findings demonstrate the pivotal role of phospholipid AA in modulating erythrocyte and renal transport of calcium and oxalate.

Effect of a Hyperlipidic Diet on Lipid Composition, Fluidity, and (Na+-K+)ATPase Activity of Rat Erythrocyte Membranes

Feeding rats a hyperlipidic diet in which animals were offered daily a variety of high-energy food resulted in a significant increase of serum free fatty acids and a decrease of phospholipids with respect to controls. On the contrary, there were no significant differences in erythrocyte membrane total lipid composition between the two groups. Erythrocyte membranes showed a significant decrease in saturated fatty acid content and a significant increase in (n-6) polyunsaturated fatty acid content; (n-3) polyunsaturated fatty acids significantly decreased. Membrane fluidity, investigated by fluorescence polarization of diphenylhexatriene, significantly increased in the erythrocyte membranes of the experimental group. These results seem compatible with the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+)ATPase activity occurred in erythrocyte membranes of the experimental group rats with respect to the controls.