The effect of essential fatty acid deficiency on eicosanoid production in the inflamed rat colonic mucosa

Eicosanoids are potent mediators of inflammation and are synthesized in increased quantity in active ulcerative colitis. To elucidate the role of prostaglandin E2, thromboxane A2, prostaglandin I2, and leukotriene B2 in acute chemical colitis induced by 4% acetic acid, we utilized an animal model which has a deficiency of arachidonic acid, the precursor of eicosanoids due to an essential fatty acid deficient diet. Forty-eight hours after colitis was induced, mucosal synthesis of the cyclooxygenase products, prostaglandin E2, thromboxane A2, and prostaglandin I2, was significantly decreased in essential fatty acid deficient rats compared to normal controls. However, the 5-lipoxygenase product, leukotriene B4, was not different between groups. The decrease in cyclooxygenase products did not correlate with any change in the severity of colonic inflammation as assessed by gross morphology, histology, or myleoperoxidase activity. Thus inhibition of formation of the cyclooxygenase products of arachidonate metabolism does not appear to improve the degree of inflammation under the experimental conditions employed in this study.     

The effect of essential fatty acid deficiency upon fatty acid uptake by the brain.

Young adult rats, either control or essential fatty acid deficient, were administered either [3-H] oleic acid or [3-H] arachidonic acid by stomach tube. In addition, a group of control rats was given [3-H] palmitic acid. The rats were killed at various times therafter, and the radioactivity of the lipids of brain and plasma was examined. In confirmation of previous work, the blood lipid label was found to rise rapidly and then fall, wheras the activity of brain lipids increased slowly and did not show a decline through the 24-h period studied. Analysis of the brain uptake data according to first-order kinetics confirmed the impressions gained from visual inspection of the data. The initial rate of uptake of arachidonic acid was about 4.5 times that of oleic acid in control animals and in deficient animals. Essential fatty acid deficiency, however, did not induce an altered rate of uptake for either oleic acid or arachidonic acid. The rate of uptake of palmitic acid by control rats was not significantly different from that of oleic acid. Even though the initial rates of incorporation of oleic and arachidonic acids were not changed during essential fatty acid deficiency, the final levels of radioactivity obtained in brain lipids were higher in deficient rats with both fatty acids. The plateau value obtained with oleic acid was 1.5 times higher in deficient animals, while the plateau value for arachidonic acid was 1.7 times higher. An experiment in which deficient animals were allowed access to a control diet for 12 or 24 h prior to the labeling experiment suggested that the higher levels of radioactivity found in brain lipids of deficient animals was not due to an isotope dilution effect. Such animals still displayed the labeling pattern of deficient animals with arachidonic acid, while the results with oleic acid varied somewhat. Our results suggest that essential fatty acid deficiency does not alter the ability of the brain to take up the fatty acids studied. However, the fatty acids, especially arachidonic, are retained in the brain to a greater extent in the deficient animals.     

Effect of essential fatty acid deficiency on the epidermal sphingolipids of the rat

The epidermal sphingolipids from rats maintained on either a rat stock diet or a fat-free diet have been analyzed. Thin-layer chromatographic analyses have revealed glucosylceramides, acylglucosylceramides and four fractions of ceramides, one of which proved to be an acylceramide. The relative amounts of the glucosylceramides, acylglucosylceramides and acylceramides were increased in the essential fatty acid-deficient epidermis while one ceramide fraction was diminished. The other two ceramide fractions remained unchanged. The acylceramides and acylglucosylceramides from normal rat epidermis both contained long-chain omega-hydroxy acids in amide linkage to sphingosine bases and high proportions of linoleic acid in ester linkage. The linoleate, which is known to be crucial for the formation and maintenance of the epidermal water barrier, was replaced by oleate in the essential fatty acid-deficient rats.     

The effect of essential fatty acid deficiency on hepatic bile salt sulphotransferase in rats.

Hepatic bile salt sulphotransferase (BSS) activity and the contents of unconjugated oestradiol-17 beta (E2) and conjugated oestrone (cE1) in liver tissue was significantly lower in young essential fatty acid (EFA) deficient female rats than in female control rats. No corresponding differences were found between male EFA deficient and control rats. A significant sex difference, with higher values in females, was found for BSS activity and E2 and cE1 contents in control rats but not in EFA deficient rats. The decrease in hepatic BSS activity in female rats caused by EFA deficiency may be mediated via a decreased estrogenic action on the liver.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the ?EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F^?, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in ?EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in ?EFA membranes than in +EFA. Total Na⁺, K⁺ (Mg²⁺)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent K_m and V_max. 5′-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Phorbol esters inhibit alpha1 adrenergic stimulation of glycogenolysis in isolated rat hepatocytes

Tumor promoting phorbol esters can stimulate Ca⁺⁺-phospholipid-dependent protein kinase. It has been suggested that this enzyme may mediate the effects of calcium-dependent hormones. In this paper the effects of phorbol 12-myristate 13-acetate (TPA) on isolated rat hepatocyte metabolism were studied. Phorbol esters completely blocked alpha₁-adrenergic stimulation of glycogenolysis. This effect is quite specific for alpha₁-adrenergic actions, as the stimulations of glycogenolysis by vasopressin, angiotensin II, ionophore A-23187 and glucagon were unaffected by TPA. The potencies of the different phorbol esters used in this study suggests that the inhibitory effects of these agents may be due to activation of protein kinase C. The effect of phorbol esters on alpha₁-adrenergic actions seems to occur at an early step of the alpha₁-adrenergic action. TPA (10^?11–10^?6M) was unable to stimulate glycogenolysis. Urea synthesis, which is stimulated by vasopressin and alpha₁-adrenergic agents, was not stimulated by phorbol ester, neither alone nor in combination with the Ca⁺⁺ ionophore A-23187.     

Effect of essential fatty acid deficiency on release of triglycerides by the perfused rat liver

Studies are reported on release of triglycerides during perfusion of livers of male Sprague-Dawley rats fed a fat-free diet or diets containing hydrogenated coconut oil or corn oil. Perfusions were carried out with Krebs-Ringer bicarbonate buffer containing albumin with and without infusion of oleate or linoleate. Infusion with sodium oleate or linoleate caused an accumulation of triglycerides in the livers of the corn oil-fed animals and stimulated the release of triglycerides into the perfusing medium. In similar experiments with essential fatty acid-deficient animals, which were fed fat-free diets or diets containing hydrogenated coconut oil, there was no increase in secretion of triglycerides into the perfusate, and the amount of triglyceride which accumulated in the liver was greater than in the livers of the control (corn oil-fed) animals. Tracer experiments with oleate-1-(14)C or linoleate-1-(14)C also showed that with livers of essential fatty acid-deficient animals, secretion of triglyceride into the perfusate was not stimulated by infusion of fatty acids into the perfusing medium. It is concluded that impairment of the secretion of triglycerides is a factor in the accumulation of fat in the livers of essential fatty acid-deficient animals.     

The influence of hypothyroidism on the adrenergic stimulation of glycogenolysis in perfused rat liver

The ability of noradrenaline (1 microM), phenylephrine (10 microM), and isoproterenol (1 microM) to stimulate glycogenolysis in euthyroid and hypothyroid perfused rat livers was investigated. It was found that hypothyroidism severely impaired alpha-receptor-mediated (noradrenaline, phenylephrine) glucose release. The initial Ca2+ efflux and K+ influx induced by these agonists in the euthyroid control group were almost totally absent in the hypothyroid group, while glycogen phosphorylase a activity in the hypothyroid rat livers was markedly lower than in the controls after infusing noradrenaline for 1 min. Diminished CA2+ efflux (and possibly diminished K+ influx) is likely to play a role in the large impairment in the action of noradrenaline or phenylephrine on glycogenolysis in the perfused hypothyroid rat liver. After prolonged stimulation (15 min) with noradrenaline, however, the phosphorylase a activity in the hypothyroid and euthyroid groups did not differ significantly. This was accompanied by Ca2+ influx in the hypothyroid livers, probably facilitated by a beta-adrenergic effect of noradrenaline in this group. Hypothyroidism potentiated the effect of isoproterenol on glycogenolysis. The glucose 6-phosphate content in the hypothyroid rat livers was markedly higher than in the euthyroid group after stimulation by noradrenaline or isoproterenol.     

Effects of essential fatty acid deficiency on mitochondria and peroxisomes in rat hepatocytes with special reference to a partially hydrogenated fish oil diet

Feeding male rats a high cal% partially hydrogenated fish oil diet induced morphological and biochemical changes in hepatocytes at the mitochondrial and peroxisomal level. At the mitochondrial level, formation of megamitochondria was related to the development of an essential fatty acid deficiency, as measured by a high 20:3/20:4 fatty acid ratio. These mitochondrial changes were fully prevented by adding linoleic acid to the partially hydrogenated fish oil diet. The megamitochondria revealed a normal specific content of respiratory chain pigments, normal specific respiratory rates and a normal energy coupling. At the peroxisomal level, feeding of the partially hydrogenated fish oil diet caused a considerable proliferation, which was unrelated to essential fatty acid deficiency. The total number of peroxisomes increased 1.9-fold, and 2.6-fold in the presence of added linoleic acid. Essential fatty acid deficiency seemed to result in an inhibition of peroxisomal biogenesis. It was concluded that the induction of megamitochondria by partially hydrogenated fish oil was fully attributable to essential fatty acid deficiency, whereas peroxisomal proliferation must be attributed to other factors in the diet.     

Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups

Dietary lipid quantity and quality have recently been shown to affect serum leptin levels in adult rats. Moreover, suckling pups from dams fed a high fat diet had increased serum leptin levels. The aim of the present study was to analyze the influence of essential fatty acid (EFA) deficiency on serum leptin levels in dams and their pups during the suckling period. For the last 10 days of gestation and throughout lactation, pregnant rats were fed a control or an EFA-deficient (EFAD) diet. The levels of leptin and EFA in the serum of the dams and pups were analyzed 1, 2, and 3 weeks after delivery. In parallel, serum levels of glucose and corticosterone were analyzed in the pups. Low serum leptin levels were found in the control lactating dams during the entire lactation period compared with the age-matched nonlactating animals. The leptin concentrations in the lactating dams fed the EFAD diet were lower compared with those fed the control diet. The serum leptin levels of suckling pups from dams on the EFAD diet were markedly decreased compared with controls (P < 0.05). The reduced serum leptin levels could not be explained by nutritional restriction as evaluated by serum levels of glucose and corticosterone. These results indicate the importance of the EFA composition of the maternal diet for serum leptin levels in both dams and pups. EFA deficiency in lactating dams may cause long-term effects on the pups through dysregulation of leptin and leptin-dependent functions. -- Korotkova, M., B. Gabrielsson, L. A. Hanson, and B. Strandvik. Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups. J. Lipid Res. 2001. 42: 359--365.     

Changes in fatty acid composition of peripheral nerve myelin in essential fatty acid deficiency

Severe essential fatty acid deficiency (EFAD) was induced by feeding weanling rats a diet free of essential fatty acids 8 months after weaning. The fatty acid compositions of phospholipids and glycosphingolipids in peripheral nerve myelin were compared in rats with and without EFAD. With the deficient diet, 20:3ω9 was found in the major myelin phospholipids. The level of 18:1 was increased and the levels of 18:2ω6, 20:4ω6, and 22:4ω6 were decreased. Both sphingomyelin and cerebroside showed higher proportion of 24:1 and lower proportions of 24:0 in EFA-deficient rats than in control rats. The fatty acid chain elongating system in myelin cerebroside was also depressed by EFAD. A two- to sevenfold increase of the ratio 20:4ω6 to 20:3ω6 was found in myelin phospholipids of regenerated nerve from rats fed control diet. However, this ratio was suppressed by EFAD diet. The biochemical index (20:3ω9/20:4ω6) for EFAD was not affected by crush injury. These results suggest that dietary EFAD in postweaning rats can induce fatty acid alterations in peripheral nerve myelin without resulting in detectable changes in function or structure and that myelin lipids may be sequestered and reused during nerve degeneration and regeneration.     

The role of fatty acid binding protein on the metabolism of fatty acids in isolated rat hepatocytes

In isolated rat hepatocytes flavaspidic acid, a competitor with free fatty acids for the fatty-acid-binding-protein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of CO₂ and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acid-activating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake.     

Stimulatory effect of the intestinal peptide PHI on glycogenolysis and gluconeogenesis in isolated rat hepatocytes.

The newly isolated peptide PHI provoked a dose-dependent stimulation of glycogenolysis and gluconeogenesis in isolated rat hepatocytes; at 1 microM-PHI, both processes were increased 1.6-fold as compared with basal values. These PHI-mediated effects were accompanied by the activation of glycogen phosphorylase and the inactivation of pyruvate kinase. PHI (1 microM) also caused a 2-fold increase in hepatocyte cyclic AMP.