Polar Plasmodium falciparum lipids induce lipogenesis in rat adipocytes in vitro

Previous studies have shown that 'toxic malarial antigens' released by Plasmodium yoelii can induce hypoglycaemia in mice and act synergistically with insulin in stimulating lipogenesis in rat adipocytes in vitro. In this study, it was shown that similar bioactivity could be detected in Plasmodium falciparum culture supernatant, and the molecular basis of this activity was further investigated. Boiled spent culture medium from P. falciparum cultures ('BS-Pf') (exclusively released into the culture supernatant when schizonts rupture) acts in synergy with insulin to increase lipogenesis in a rat adipocyte assay by more than 250% (P < 0.001). Control preparations prepared from non-parasitized erythrocytes grown under similar conditions had no effect (P < 0.001). While contamination with mycoplasma has previously been shown to interfere with the interpretation of data obtained with other molecules thought to be released from P. falciparum in culture, including those inducing TNF-alpha and NO production by macrophages, such contamination was unequivocally ruled out here. BS-Pf alone did not stimulate the lipogenesis in short-term assays (less than 4 h), while long-term exposure of rat adipocytes to BS-Pf alone (12-24 h) caused a stimulation of lipogenesis at a level comparable to that observed with insulin. Furthermore, lipogenesis-inducing activity was also detected in the serum of squirrel monkeys infected with different species of malaria parasites (P. vivax, P. falciparum and P. brasilianum). Preliminary biochemical characterization showed that the biological activity was found in the solvent-extracted polar lipid fraction of boiled supernatant of P. falciparum cultures. All the different polar lipid fractions, collected from silica gel column chromatography, showed a comparable lipogenesis-inducing activity. Enzymatic treatment by phospholipase C of the lipid fraction, which co-migrated with the phosphatidylcholine standard, showed that the activity of the fraction was associated with the 1,2-diacylglycerol (1,2-DAG) moieties released from polar lipids. When this exogenous 1,2-DAG was added to the adipocyte cultures (short- and long-term cultures), it induced stimulation of lipogenesis in rat adipocytes, while no lipogenic activity was obtained from bacterial polar lipids and 1,2-DAG isolated from unparasitized erythrocytes. The importance of these findings is discussed with reference to other toxic malarial antigens and also to the potential role of these molecules in the induction of hypoglycaemia in the severe forms of malaria.     

Cis-unsaturated fatty acids modulate the function of the cell-surface cAMP receptor of Dictyostelium discoideum

The binding of cAMP to the chemotactic cAMP receptor in intact Dictyostelium discoideum cells and isolated membranes is strongly inhibited by unsaturated fatty acids. In isolated membranes, cis-unsaturated fatty acids decreased the number of accessible cAMP binding sites, without significantly altering their affinity. Most potent were C₁₈ and C₂₀ cis-poly unsaturated fatty acids, like arachidonic acid, linoleic acid and linolenic acid. Trans-unsaturated fatty acid was less potent than its cis isomer, while saturated fatty acids did not affect the binding of cAMP to receptors at all. Oxidation reactions were not important for the effect of unsaturated fatty acids. When membranes were preincubated with millimolar concentrations of Ca²⁺, the effect of unsaturated fatty acids was strongly diminished. Mg²⁺ was ineffective. Ca²⁺, if presented after the incubation of membranes with unsaturated fatty acids, did not reverse the inhibitory effect. The specificity of the fatty acid effect, and the interference with Ca²⁺, but not Mg²⁺, suggest that the properties of the cAMP receptor are changed as a result of alterations in the lipid bilayer structure of the membrane.     

Unsaturated fatty acids induce non-canonical autophagy

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1^+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.     

Lipidomics of hepatic lipogenesis inhibition by omega 3 fatty acids

We assessed – by a lipidomic approach – the differential incorporation of EPA and DHA into hepatic lipids, after prolonged feeding of rats with fish oil. We also evaluated their effect on lipogenesis and its related enzymes. Rats were administered 100 mg/kg/d fish oil, by oral gavage, for 30 days. The fatty acid profile of total liver lipids was determined by gas–liquid chromatography coupled to mass spectrometry. Individual phospholipid classes and their molecular species were quantified by ESI-MS/MS. Omega 3 fatty acids readily incorporated into hepatic phospholipids, decreased stearoyl-CoA desaturase 16, stearoyl-CoA desaturase, delta 6 desaturase, and delta 5 desaturase activities (calculated as product/substrate ratio) and decreased the “lipogenesis index”, i.e., the proportion of fatty acids endogenously synthesized in the liver and not provided with the diet. Our results show that long-chain omega 3 fatty acids selectively incorporate into hepatic phospholipids, inhibit de novo lipogenesis and change the hepatic fatty acid profile via reduced desaturases' activity in the non-steatotic liver. In addition to corroborating advice to consume adequate amounts of omega 3 fatty acids for overall health, these data contribute mechanistic insights to the clinical observations that provision of omega 3 fatty acids decreases hepatic fat and ameliorates NAFLD prognosis.     

MicroRNA-425 controls lipogenesis and lipolysis in adipocytes

An increasing number of studies have demonstrated that some microRNAs participate in the regulation of growth and development of adipocytes. The present study shows that microRNA-425-5p (miR-425) is a novel strong regulator of adipogenesis and adipolysis in adipocytes. Forced expression of miR-425 in mice promoted body fat accumulation and the development of obesity due to high-fat intake, whereas silencing miR-425 prevented mice from being obese. Mechanically, the expression of miR-425 is controlled by PPARγ during the adipogenesis process in adipocytes. MiR-425 overexpression resulted in a reduction in the proliferation of 3t3-L1 pre-adipocytes but significantly accelerated cellular adipogenic differentiation. Mapk14, a negative regulator of adipogenesis, was predicted and confirmed as a real target gene of miR-425. Moreover, knocking down miR-425 remarkably intensified intracellular lipolysis and promoted lipid oxidation, which is related to the activation of AMPK, a monitor for intracellular energy balance. MiR-425 activated AMPK not only by decreasing cellular ATP concentrations but also by targeting the gene of Cab39, which is an upstream co-activator of AMPK. The findings of the present study suggest that miR-425 could control adipogenesis and adipolysis in adipocytes by simultaneously triggering multidirectional targets.     

Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.     

Decrease by cycloheximide of calcium binding and nonesterified fatty acids in rat-intestinal Golgi-enriched membrane fractions

Rat intestinal Golgi-enriched membrane fractions bind more Ca2+ than do basolateral and microvillus-enriched membrane fractions, and this uptake is reduced by vitamin D-deficiency. The effect of the protein synthesis inhibitor, cycloheximide, on this Ca2+ binding was determined in rat fed a normal, vitamin D-sufficient diet. Cycloheximide, 1.5 mg/kg, rapidly reduced protein synthesis (measured by [3H]leucine incorporation) to 12% of control values within 15 min, but Ca2+ binding diminished gradually to 50% of control values by 60 min. Ca2+ transport across gut sacs was also decreased. The reduction in Ca2+ binding was not due to an alteration in vesicle morphology or to a direct effect of cycloheximide. Nonesterified (free) fatty acids, the probable binding sites for Ca2+ in these membrane fractions, were reduced by cycloheximide to 48% of control values by 60 min. There was no significant change in total lipid phosphate. Cycloheximide may affect the synthesis of proteins necessary for the presence of nonesterified fatty acids in these Golgi membranes.     

Genistein affects lipogenesis and lipolysis in isolated rat adipocytes

Genistein is a phytoestrogen found in several plants eaten by humans and food-producing animals and exerting a wide spectrum of biological activity. In this experiment, the impact of genistein on lipogenesis and lipolysis was studied in isolated rat adipocytes. Incubation of the cells (10⁶ cells/ml in plastic tubes at 37°C with Krebs-Ringer buffer, 90 min) with genistein (0.01, 0.3, 0.6 and 1 mM) clearly restricted (1 nM) [U-¹⁴C]glucose conversion to total lipids in the absence and presence of insulin. When [¹⁴C]acetate was used as the substrate for lipogenesis, genistein (0.01, 0.1 and 1 mM) exerted a similar effect. Thus, the anti-lipogenetic action of genistein may be an effect not only of alteration in glucose transport and metabolism, but this phytoestrogen can also restrict the fatty acids synthesis and/or their estrification. Incubation of adipocytes with estradiol at the same concentrations also resulted in restriction of lipogenesis, but the effect was less marked. Genistein (0.1 and 1 mM) augmented basal lipolysis in adipocytes. This process was strongly restricted by insulin (1 μM) and H-89 (an inhibitor of protein kinase A; 50 μM) and seems to be primarily due to the inhibitory action of the phytoestrogen on cAMP phosphodiesterase in adipocytes. Genistein at the smallest concentration (0.01 mM) augmented epinephrine-stimulated (1 μM) lipolysis but failed to potentiate lipolysis induced by forskolin (1 μM) or dibutyryl-cAMP (1 mM). These results suggest genistein action on the lipolytic pathways before activation of adenylate cyclase. The restriction of lipolysis stimulated by several lipolytic agents – epinephrine, forskolin and dibutyryl-cAMP were observed when adipocytes were incubated with genistein at highest concentrations (0.1 and 1 mM). These results prove the inhibitory action of this phyestrogen on the final steps of the lipolytic cascade, i.e. on protein kinase A or hormone sensitive lipase. Estradiol, added to the incubation medium, did not affect lipolysis. It can be concluded that genistein significantly affects lipogenesis and lipolysis in isolated rat adipocytes.     

Insights into binding of fatty acids by fatty acid binding proteins

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.     

Tumour-promoting phorbol esters increase basal and inhibit insulin-stimulated lipogenesis in rat adipocytes without decreasing insulin binding.

In isolated rat adipocytes, tumour-promoting phorbol esters caused (1) dose-dependent stimulation of lipogenesis in the absence of insulin and (2) inhibition of the lipogenic effect of submaximal concentrations of insulin, but without affecting insulin binding. The possible involvement of protein kinase C in insulin action is discussed.     

Insulin-like stimulation of glucose transport in isolated adipocytes by fatty acids

Glucose transport rate as assessed with the 3-0-Methylglucose method was significantly increased in adipocytes preincubated with aliphatic carboxylic acids. The magnitude of stimulation apparently depended on the chain length of the carboxylic acid, and was highest with palmitic acid (130%). The stimulation was additive to the effect of insulin, and reflected a decrease of km rather than an increase in vmax of the transport rate. The results suggest that fatty acids may modulate the activity of the glucose transporter, providing an insulin-dependent supply of adipose tissue with glycerol-phosphate during lipolysis for reesterification of excess fatty acids. Further, it is suggested that fatty acids mediate the stimulatory effect of catecholamines on glucose transport as observed in isolated fat cells.     

Depot-specific effects of fatty acids on lipid accumulation in children's adipocytes

Circulating concentrations of fatty acids are elevated in obesity, although their effect on regional fat deposition is relatively unexplored. With the increasing prevalence of childhood obesity, we aimed to investigate whether saturated and unsaturated fatty acids lead to differential lipid accumulation (LA) in children's subcutaneous and visceral adipocytes. To examine this, subcutaneous and peri-nephric pre-adipocytes, isolated from fat biopsies from 6 pre-pubertal children, were differentiated in vitro before being exposed to palmitate and/or oleate for 24 h. Lipid accumulation was then quantified by nile red staining. Palmitate significantly increased LA in visceral adipocytes at all doses > or =188 microM (e.g. Palmitate 750 microM: +30.0%[8.2]; p<0.01), whilst only a dose of 375 microM led to a significant, but smaller, increase in LA in subcutaneous adipocytes (Palmitate 375 micro: +13.0%[4.3]; p=0.02). In contrast, oleate significantly increased LA in subcutaneous (Oleate 1000 microM: +36.3%[14.0]; p=0.01), but not visceral (Oleate 1000 microM: +16.2%[9.6]; p=0.25) adipocytes. These data suggest that saturated and unsaturated fatty acids may exert depot-specific effects on lipid accumulation.     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.     

Trans fatty acids induce apoptosis in human endothelial cells.

The present study was designed to investigate the hypothesis that trans fatty acids can induce apoptosis of human umbilical vein endothelial cells (HUVEC). To test this hypothesis apoptosis was measured in HUVEC treated with 0.1, 1.0 or 5.0 mM trans elaidic acid (t-18:1) or linoelaidic acid (t,t-18:2) for 24 hours. For the detection of apoptosis, TdT-mediated dUTP nick end labelling assay (TUNEL), cell binding of annexin V and propidium iodide uptake were measured. Active Caspase-3 and cleaved PARP (poly-ADP-ribose polymerase) were also measured in the cell lysate. Moreover, cellular ability to produce ROS (reactive oxygen species) was measured by DCF fluorescence Both acids studied induce both early (annexin-positive cells) and late stages of apoptosis (cells stained by propidium iodide) in a dose-dependent manner. Also the appearance of TUNEL-positive cells was induced by both trans fatty acids tested, in a dose dependent manner. Both trans acids induce apoptosis through their effect on Caspase-3 activity and on intracellular ROS production. It is worth emphasising that linoelaidic acid proved to be a more potent inducer of apoptosis and ROS production in endothelial cells than elaidic acid. The present studies suggest that trans fatty acids may play a role in damaging and death of vascular endothelial cells in atherosclerosis.     

Insulin and non-esterified fatty acids. Acute regulators of lipogenesis in perfused rat liver.

Livers from fed rats were perfused with whole rat blood and infused with oleate to maintain constant concentrations of serum non-esterified fatty acids over a wide physiological range. Infusion of insulin opposed the antilipogenic effects of increasing concentrations of serum non-esterified fatty acids. Secretion of very-low-density-lipoprotein triacylglycerols was directly proportional to the concentration of serum non-esterified fatty acids and was increased by insulin. The secretion of newly-synthesized fatty acids in very-low-density-lipoprotein triacylglycerols decreased with increasing concentrations of serum non-esterified fatty acid. Insulin opposed this change. Cholesterol biosynthesis was unaffected by alterations in concentration of serum non-esterified fatty acid but was increased by insulin. Equilibrium concentrations of perfusate lactate and glucose were increased by serum non-esterified fatty acids but steady-state rates of hepatic glucose output and lactate uptake were unchanged. Insulin decreased perfusate glucose concentrations and abolished the increase in its concentration that resulted from increases in non-esterified fatty acid concentrations.     

Esterification of exogenous and endogenous fatty acids by rat adipocytes in vitro.

Rat adipocytes were used in vivo to compare the esterification of exogenous fatty acids and fatty acids formed de novo from glucose or acetate. Pure single fatty acids added to the medium were esterified at comparable rates but marked differences were observed when the same acids were supplied as components of a fatty acid mixture of a composition similar to that in the tissue. Fatty acids synthesised de novo from acetate by adipocytes in a medium containing high concentrations of acetate were located predominantly in diacylglycerols. The effect was most marked with adipocytes from older rats and was enhanced by the presence of exogenous long-chain fatty acids. Exogenous oleic acid was esterified predominantly into triacylglycerols at all concentrations of acetate. No such accumulation of endogenously-synthesised fatty acids in diacylglycerols occurred when glucose was the precursor for fatty acid synthesis. The diacylglycerols formed were almost entirely of the sn-1,2-configuration.