Oleic Acid Uptake Research Articles

Chronic hyperglycemia reduces substrate oxidation and impairs metabolic switching of human myotubes

Skeletal muscle of insulin resistant individuals is characterized by lower fasting lipid oxidation and reduced ability to switch between lipid and glucose oxidation. The purpose of the present study was to examine if chronic hyperglycemia would impair metabolic switching of myotubes. Human myotubes were treated with or without chronic hyperglycemia (20 mmol/l glucose for 4 days), and metabolism of [ 14C]oleic acid (OA) and [ 14C]glucose was studied. Myotubes exposed to chronic hyperglycemia showed a significantly reduced OA uptake and oxidation to CO 2, whereas acid-soluble metabolites were increased compared to normoglycemic cells (5.5 mmol/l glucose). Glucose suppressibility, the ability of acute glucose (5 mmol/l) to suppress lipid oxidation, was 50% in normoglycemic cells and reduced to 21% by hyperglycemia. Adaptability, the capacity to increase lipid oxidation with increasing fatty acid availability, was not affected by hyperglycemia. Glucose uptake and oxidation were reduced by about 40% after hyperglycemia, and oxidation of glucose in presence of mitochondrial uncouplers showed that net and maximal oxidative capacities were significantly reduced. Hyperglycemia also abolished insulin-stimulated glucose uptake. Moreover, ATP concentration was reduced by 25% after hyperglycemia. However, none of the measured mitochondrial genes were downregulated nor was mitochondrial DNA content. Microarray and real-time RT-PCR showed that no genes were significantly regulated by chronic hyperglycemia. Addition of chronic lactate reduced both glucose and OA oxidation to the same extent as hyperglycemia. In conclusion, chronic hyperglycemia reduced substrate oxidation in skeletal muscle cells and impaired metabolic switching. The effect is most likely due to an induced mitochondrial dysfunction.

Trans‐Membrane Uptake and Intracellular Metabolism of Fatty Acids in Atlantic Salmon (Salmo salar L.) Hepatocytes

To elucidate if the trans-membrane uptake of fatty acids is protein-mediated, the uptake of oleic acid (18:1n-9), linoleic acid (18:2n-6), alpha-linolenic acid (18:3n-3), eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) was investigated in vitro in Atlantic salmon (Salmo salar L.) primary hepatocytes. Firstly, optimal fatty acid incubation time and concentration were established for trans-membrane 18:n-9 uptake. Based on saturation kinetics, a 2-h incubation time and 37.5 muM were used for the following experiments. Secondly, in order to identify whether trans-membrane fatty acid uptake in hepatocytes was mainly passive or protein mediated, hepatocytes were pre-incubated with membrane protein inhibitors followed by 2 h of incubation with [1-(14)C] labelled 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Fatty acid uptake into hepatocytes was highest with 20:5n-3 and 22:6n-3 and lowest with 18:1n-9. Phloretin was the most potent fatty acid uptake inhibitor, inhibiting uptake in the following order: 20:5n-3 > 18:3n-3 = 22:6n-3 > 18:2n-6 > 18:1n-9. The uptake of FA in Atlantic salmon hepatocytes seem to be due to both saturable and inhibitable protein mediated uptake, as well as passive uptake processes with more unsaturated and long fatty acids (20:n-3 > 22:6n-3 = 18:3n-3 > 18:2n-6) being more dependent on membrane protein mediated uptake compared to 18:1n-9.

The Lipid Messenger OEA Links Dietary Fat Intake to Satiety

The association between fat consumption and obesity underscores the need to identify physiological signals that control fat intake. Previous studies have shown that feeding stimulates small-intestinal mucosal cells to produce the lipid messenger oleoylethanolamide (OEA) which, when administered as a drug, decreases meal frequency by engaging peroxisome proliferator-activated receptors-alpha (PPAR-alpha). Here, we report that duodenal infusion of fat stimulates OEA mobilization in the proximal small intestine, whereas infusion of protein or carbohydrate does not. OEA production utilizes dietary oleic acid as a substrate and is disrupted in mutant mice lacking the membrane fatty-acid transporter CD36. Targeted disruption of CD36 or PPAR-alpha abrogates the satiety response induced by fat. The results suggest that activation of small-intestinal OEA mobilization, enabled by CD36-mediated uptake of dietary oleic acid, serves as a molecular sensor linking fat ingestion to satiety.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.

Auraptene, a citrus fruit compound, regulates gene expression as a PPARα agonist in HepG2 hepatocytes

Citrus fruit compounds have various activities that improve pathological conditions in many tissues. In this study, we examined the effect of auraptene contained mainly in the peel of citrus on peroxisome proliferator-activated receptor-alpha (PPARalpha) activation. To examine effects of auraptene on the PPARalpha activation in hepatocytes, PPAR ligand assay system was developed using HepG2 hepatocytes, in which the endogenous PPARalpha expression level is very low. In the PPAR ligand assay, the addition of auraptene showed significant effects on the transactivation of GAL4/PPARalpha chimera proteins in a dose-dependent manner. Actually, treatment with auraptene induced the up-regulation of PPAR target genes, such as acyl-CoA oxidase (ACO), carnitine-palmitoyl transferase 1A (CPT1A) and acyl-CoA synthetase (ACS), in PPARalpha-expressing HepG2 hepatocytes. The regulation of gene expression was dependent on PPARalpha because mock-transfected HepG2 hepatocytes showed no regulation. The up-regulation of PPAR target gene expression by auraptene was sufficient to enhance oleic acid uptake into PPARalpha-expressing HepG2 hepatocytes. These results indicate that auraptene acts as a PPARalpha agonist in hepatocytes and that auraptene may improve lipid abnormality through PPARalpha activation in the liver.

Protease inhibition by oleic acid transfer from chronic wound dressings to albumin

High elastase and cathepsin G activities have been observed in chronic wounds to inhibit healing through degradation of growth factors, cytokines, and extracellular matrix proteins. Oleic acid is a non-toxic elastase inhibitor. Cotton wound dressing material was characterized as a transfer carrier for affinity uptake of oleic acid by albumin under conditions mimicking chronic wounds. The mechanism of oleic acid uptake from cotton and binding by albumin was examined with both intact dressings and cotton fiber-designed chromatography. Raman spectra of the albumin–oleic acid complexes under liquid equilibrium conditions revealed fully saturated albumin–oleic acid complexes with a 1:1 weight ratio of albumin:oleic acid. Liquid–solid equilibrium conditions revealed oleic acid transfer from cotton to albumin at 27 mole equivalents of oleic acid per mole albumin. Comparing oleic acid formulated wound dressings for dose dependent ability to lower elastase activity, we found cotton gauze > hydrogel > hydrocolloid. In contrast, the cationic serine protease cathepsin G was inhibited by oleic acid within a narrow range of oleic acid–cotton formulations. 2% albumin was sufficient to transfer quantities of oleic acid necessary to achieve a significant elastase-lowering effect. Oleic acid bound to cotton wound dressings may have promise in the selective lowering of cationic serine protease activity useful in topical application for chronic inflammatory pathogenesis.

Abstracts for Oral Presentation

Abstracts for Oral Presentation

Sustained elevations in NEFA induce cyclooxygenase-2 activity and potentiate THP-1 macrophage foam cell formation

Type 2 diabetes, a major risk factor for atherosclerosis, is associated with a cluster of lipid risk factors, many of which can be mechanistically linked with underlying dysregulated fatty acid metabolism and elevated plasma non-esterified fatty acids (NEFA). Thus, we tested the hypothesis that elevated NEFA dysregulates lipid metabolism at the levels of lipid synthesis and gene expression in THP-1 monocyte derived macrophages (MDM). THP-1 MDM incubated with oleic acid (OA) and a BODIPY-conjugated NEFA, accumulate, respectively, intracellular inclusions that are positive for oil red O and BODIPY-labeling. Parallel studies with [ 14C]OA show dose-dependent accumulation of intracellular 14C-labeled neutral lipid, almost exclusively as triglyceride; the rate of [ 3H]OA uptake increases as THP-1 MDM convert to foam cells. Preincubation of THP-1 MDM with higher concentrations of OA (1.8 mM versus 0.2 mM) was associated with enhanced uptake of Ac-LDL, and increased expression of adipocyte fatty acid binding protein, FAT/CD36, and cyclooxygenase-2 (COX-2); COX-2 mass and activity also increased. These observations suggest a mechanistic link between sustained elevations in albumin-bound NEFA and foam cell formation that may be mediated by enhanced adipogenesis, increased uptake of modified LDL, and upregulated formation of eicosanoids, which may be proinflammatory.

Leukaemia inhibitory factor stimulates glucose transport in isolated cardiomyocytes and induces insulin resistance after chronic exposure

Hypertrophic and failing hearts have increased utilisation of glucose, but also develop insulin resistance and reduced ability to produce ATP. Increased levels of the IL-6-related cytokine leukaemia inhibitory factor (LIF) are found in failing hearts, and we have recently shown that LIF reduces ATP production in isolated cardiomyocytes. In the present study we investigated effects of LIF on glucose metabolism, and how LIF-treated cells respond to insulin stimulation. Cardiomyocytes were isolated from adult Wistar rats by collagen digestion, maintained in culture for 48 h, and then treated with 1 nmol/l LIF. Acute LIF treatment increased deoxyglucose uptake compared with controls, but no additive effect was observed in cardiomyocytes treated with LIF and insulin. The phosphatidylinositol 3-kinase inhibitor wortmannin did not affect LIF-induced glucose uptake. LIF had no effect on AMP-activated protein kinase phosphorylation. Cardiomyocytes treated with LIF for 48 h did not respond to insulin by increasing deoxyglucose uptake and showed a reduced insulin-mediated uptake of oleic acid and formation of complex lipids compared with control cells. Chronic LIF treatment increased gene expression of the suppressor of cytokine signalling (Socs) 3 and reduced expression of solute carrier family 2, member 4 (Slc2a4, previously known as glucose transporter 4 [Glut4]). In line with these observations, chronic LIF treatment reduced insulin-mediated phosphorylation of both Akt/protein kinase B (PKB) and glycogen synthase kinase (GSK)-3. Acute LIF treatment increased glucose uptake in isolated cardiomyocytes by a pathway different from that of insulin. Chronic LIF treatment induced insulin resistance, possibly mediated by altered expression of Socs3 and Slc2a4, and impaired insulin-mediated phosphorylation of GSK-3 and Akt/PKB.

Oleic acid uptake by in vitro English sparrow skeletal muscle

Studies of prolonged avian flight have shown it to require large amounts of energy supplied mainly by free fatty acids (FFA). In the present study, the high levels of plasma ketone bodies found in sparrows (2.58 mmol l(-1)) are supportive of the use of fatty acids for flight. To determine the nature of fatty acid (oleic acid, OA) uptake, various pharmacological agents were used. The uptake of OA was examined using the extensor digitorum communis (EDC) muscle of English sparrows incubated in vitro. Initial studies demonstrated that radiolabeled OA uptake decreased in the presence of increasing unlabeled OA, suggesting that uptake occurred by a facilitative transport process. To further characterize OA uptake, EDC muscles were incubated with either: insulin (2 ng ml(-1)), insulin-like growth factor isoform-1 (IGF-I; 48 ng ml(-1)), 5'-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 2 mmol) or caffeine (5 mmol). Insulin, but not IGF-I, significantly increased OA uptake by avian EDC (P < 0.01). Caffeine and AICAR were ineffective at increasing OA uptake. A specific inhibitor of FFA transport by fatty acid transporters (FAT/CD36), sulfo-N-succinimidyl oleate (SSO; 500 micromoles), significantly decreased OA uptake at 2.5 min. The effectiveness of SSO suggests that a FAT/CD36-like protein is expressed in avian tissues. As uptake of OA was not completely blocked by SSO, it is likely that other mechanisms for FFA movement across membranes, such as diffusion, may be present.

Adsorption Isotherms for Oleic Acid Removal from Ethanol + Water Solutions Using the Strong Anion-Exchange Resin Amberlyst A26 OH

Adsorption isotherms for the removal of oleic acid from ethanol + water solutions using a strong anion-exchange resin (Amberlyst A26 OH) were experimentally determined. The equilibrium data were correlated using the Langmuir model by adjusting the parameters qm/(g of acid·g of dry resin-1), maximum content that can be adsorbed on solid phase, and Kd/(g of acid·g of solvent-1), the equilibrium constant. The Freundlich and Redlich−Petersen models were also used, but the Langmuir model shows the lower average deviations between experimental and calculated results. The behavior and the capacity of the resin to remove the fatty acid from the liquid phase were evaluated at different experimental conditions: the water content in ethanol was varied within the range (0 to 15) mass %, and two equilibrium temperatures were investigated: (298.15 and 313.15) K. It was observed that both variables, water content in ethanol and equilibrium temperature, do not significantly influence the equilibrium behavior. It was also observed that the strong anion-exchange resin (Amberlyst A26 OH) has a good performance in the removal of the fatty acid from the liquid phase. To determine exactly the mechanism for the uptake of oleic acid by the resin, equilibrium experiments were done with different oleic acid concentrations (7, 9, and 11) mass % in solution with water content in ethanol (0.57 ± 0.01) mass % at 298.15 K. In this way the concentration of oleic acid in resin phase was determined, and it was observed than the most important mechanism of uptake is the ion exchange.

Uptake of Micellar Long-Chain Fatty Acid and sn-2-Monoacylglycerol into Human Intestinal Caco-2 Cells Exhibits Characteristics of Protein-Mediated Transport

Long-chain fatty acid and sn-2-monoacylglycerol (2-MG) are the digestive products of dietary triacylglycerol (TG) hydrolysis. Although fatty acid uptake into the enterocyte has been examined widely, less is known about 2-MG uptake, and few studies have mimicked the physiologic conditions present in the postprandial situation. In this study, the cellular uptake of oleic acid and 2-monoolein, presented in taurocholate micellar solution, was examined in human intestinal Caco-2 cells to model the postprandial intestinal milieu. Initial uptake of oleic acid and 2-MG displayed a saturable function of their monomer concentrations, suggesting that fatty acid and 2-MG uptake may be protein-mediated processes at low unbound concentrations of lipid. The initial rate of oleate uptake was faster and the apparent Km was lower than values for 2-MG. Unlabeled oleic acid and, to a lesser extent, unlabeled 2-MG, inhibited the uptakes of both [3H]oleic acid and [3H]2-monoolein, suggesting competitive uptake. The nonphysiologic isomer sn-1-MG had effects similar to 2-MG, whereas the intermediate digestive product, diacylglycerol (DG), did not inhibit either oleate or 2-monoolein uptake. These results suggest that in the postprandial state, fatty acid and 2-MG derived from dietary TG are transported into the enterocyte, at least in part, via a protein-mediated pathway that is shared by both lipids, but not by the intermediate digestive product, DG.

Control of Microthrix parvicella in Activated Sludge Plants by Dosage of Polyaluminium Salts: Possible Mechanisms

AbstractAn effective control of the filamentous bacterium Microthrix parvicella in activated sludge wastewater treatment plants can take place by dosage of polyaluminium chloride, PAX‐14. The mechanism how it works is largely unknown. The purpose of this study was to investigate in detail the mechanism by which PAX‐14 controls M. parvicella. After addition of PAX‐14, a reduction in substrate uptake, measured as uptake of radiolabelled oleic acid and trioleic acid under anaerobic conditions was observed by microautoradiography, indicating an effect on the physiology of M. parvicella or on the ability to transport substrate into the cell. Hydrophobic surface properties of the bacterium were not altered, but a significant reduction of exoenzyme activity was observed after PAX‐14 addition. This was measured as bulk enzyme activity as well as surface‐associated enzyme activity for esterase and lipase. New lipase production was not inhibited, indicating a need for a continuous addition of PAX‐14 for an efficient control of M. parvicella. Furthermore, since PAX‐14 is an efficient flocculating agent, it improved formation of dense flocs, so M. parvicella was embedded into floc material, making access to substrate difficult due to increased diffusional resistance. In conclusion, there seemed to be several reasons why PAX‐14 could control M. parvicella: The physiology and particularly the lipase production were partly inhibited, the accessibility of substrate was affected, and a better flocculation occurred.

Insulin and leptin do not affect fatty acid uptake and metabolism in human placental choriocarcinoma (BeWo) cells

Placental transport of long chain polyunsaturated fatty acids is important for fetal growth and development. In order to examine the effects of leptin and insulin on fatty acid uptake by the placenta, placental choriocarcinoma (BeWo) cells were used. BeWo cells were incubated for 5 h at 37 °C in the absence or presence of different concentrations of insulin (0.6, 60, and 100 ng) or leptin (10 ng) with 200 μM of various radiolabeled fatty acids (docosahexaenoic acid, arachidonic acid, eicosapentaenoic acid, and oleic acid, mixed with 1:1 bovine serum albumin (fat free). After incubation, the uptake and distribution of these fatty acids into different cellular lipid fractions were determined. The uptakes of oleic, eicosapentaenoic, arachidonic, and docosahexaenoic acids were 15.36±4.1, 19.95±3.6, 28.56±8.1, and 62.25±9.5 nmol/mg of protein, respectively, in BeWo cells. Incubation of these cells with insulin (0.6 or 60 ng/ml) or leptin (10 ng/ml) did not significantly alter uptake of any of these fatty acids ( P > 0.5 ). Insulin or leptin also did not affect beta oxidation of fatty acids in these cells. In contrast, leptin (10 ng/ml) and insulin (0.60 ng/ml)) stimulated the uptake of oleic acid (7.4±2.3 nmol/mg protein) in human adipose cells, SGBS cells by 1.28- and 2.48-fold ( P < 0.05 ), respectively. The distribution of fatty acids in different cellular lipid fractions was also not affected by these hormones. Our data indicate that unlike adipose tissue, fatty acid uptake and metabolism in placental trophoblasts is not regulated by insulin or leptin.

Differential Utilization of Saturated Palmitate and Unsaturated Oleate

We recently described a primarily reduced palmitate oxidation in myotubes established from type 2 diabetic subjects, whereas triacylglycerol (TAG) accumulation seemed to be adaptive. However, it is still uncertain whether these changes are similar for saturated and unsaturated fatty acids and whether high concentrations of glucose and/or insulin may change this picture. Studies of palmitic acid and oleic acid metabolism in human myotubes established from control and type 2 diabetic subjects under conditions of acute high concentrations of insulin and/or glucose may solve these questions. Total oleic acid and palmitic acid uptake in myotubes was increased during acute insulin stimulation (P < 0.01) but not under acute, high-glucose concentrations, and no differences were found between the groups. Type 2 diabetic myotubes expressed a reduced palmitic acid oxidation to carbon dioxide (P </= 0.04), whereas oleic acid oxidation showed no differences between myotubes from both groups. High glucose concentrations decreased oleic acid oxidation (P </= 0.03). Lipid distribution was not different in diabetic and control myotubes when palmitic acid and oleic acid incorporation into cellular lipids was compared. Myotubes that were exposed to palmitic acid showed an increased palmitic acid incorporation into diacylglycerol (DAG) and TAG compared with myotubes that were exposed to oleic acid (P < 0.05) expressing an increased intracellular free fatty acid (FFA) level (P < 0.05). Lipid distribution was not affected by high glucose, whereas insulin increased FFAs, DAG, and TAG (P < 0.05). De novo lipid synthesis from glucose in both diabetic and control myotubes was of the same magnitude independent of glucose and insulin concentrations. These results indicate that palmitic acid and oleic acid are utilized in the same pattern in diabetic and control myotubes even though palmitic acid oxidation is primarily reduced in diabetic cells. Palmitic acid and oleic acid are handled differently by myotubes: Palmitic acid seems to accumulate as DAG and TAG, whereas oleic acid accumulates as intracellular FFAs. These observations indicate that oleic acid is preferable as fatty acid as it accumulates to a lesser extent as DAG and TAG than palmitic acid. Neither acute hyperglycemia nor de novo lipid synthesis from glucose seems central to the TAG accumulation in obesity or type 2 diabetes.

식이 지방산이 흰쥐 뇌조직 Subcellular Fractions내 Oleic Acid(ω9) 조성에 미치는 영향

ω3계 지방산이 부족한 SO군과 P/M/S 및 ω6/ω3 비율이 적절한 MO군으로 생후 9주까지 사육한 흰쥐의 적혈구와 뇌조직의 시냅토솜, 미토콘드리아 및 마이크로솜 내 oleic acid 조성 비율은 생후 3주에는 두 군간에 유의한 차이를 보이지 않고 일정한 수준으로 유지되는 것으로 나타났으나 생후 9주에는 MO군에서 SO군보다 높게 나타났다. 실험식이 내 oleic acid 조성은 SO군이 MO군보다 매우 낮으나, SO군의 모유에서 oleic acid 조성비율이 식이에 비해 크게 증가된것으로 나타나, 모유가 뇌 성장ㆍ발달기간중의 뇌조직내 oleic acid의 주요 급원이 될 수 있음을 나타내주고 있다. 18:0에서 oleic acid의 de novo 합성 정도를 나타내는 간접지표인 Δ-9 desaturation index는 생후 3주에는 실험군간(시냅토솜 예외)에 유의한 차이를 보이지 않았으나, 생후 9주에는 식이내 oleic acid가 풍부한 MO군의 시냅토솜에서 높게 나타났다. 한편 생후 9주 마이크로솜 분획에서 oleic acid 수준은 실험군 간에 차이를 보이지 않았으나, Δ-9 desaturation index는 SO군에서 MO군보다 높아 뇌조직에서 oleic acid가 생합성될 수 있음을 간접적으로 설명해주고 있다. 따라서 흰쥐 뇌조직의 oleic acid는 식이와 모유 등의 이미 합성된 oleic acid가 뇌로 우선적으로 유입되는 부분과 뇌조직 자체에서 합성되는 부분에 의한다고 생각된다. 그러나 식이중 oleic acid가 부족하면 뇌 세포분획내의 oleic acid 수준이 유의하게 감소하였으므로, 식이중 적절한 수준의 oleic acid가 정상적인 뇌발달에 필수적임을 다시 한번 강조하게 한다.

Long-chain fatty acid uptake is upregulated in omental adipocytes from patients undergoing bariatric surgery for obesity

To determine the impact of obesity on adipocyte cell size and long-chain fatty acid (LCFA) uptake kinetics in human subjects undergoing laparoscopic abdominal surgery. A total of 10 obese patients (BMI 49.8+/-11.9 (s.d.) kg/m(2)) undergoing laparoscopic bariatric surgery, and 10 nonobese subjects (BMI 24.2+/-2.3 kg/m(2)) undergoing other clinically indicated laparoscopic abdominal surgical procedures. Cell size distribution and [(3)H]oleic acid uptake kinetics were studied in adipocytes isolated from omental fat biopsies obtained during surgery. Adipocyte surface area (SA) was calculated from the measured cell diameters. Plasma leptin and insulin concentrations were measured by RIA in fasting blood samples obtained on the morning of surgery. The mean SA of obese adipocytes (41 508+/-5381 mu(2)/cell) was increased 2.4-fold compared to that of nonobese adipocytes (16 928+/-6529 mu(2)/cell; P<0.01). LCFA uptake in each group was the sum of saturable and nonsaturable components. Both the V(max) of the saturable component (21.3+/-6.3 vs 5.1+/-1.9 pmol/s/50,000 cells) and the rate constant k of the nonsaturable component (0.015+/-0.002 vs 0.0066+/-0.0023 ml/s/50 000 cells) were increased (P<0.001) in obese adipocytes compared with nonobese controls. When expressed relative to cell size, V(max)/mu(2) SA was greater in obese than nonobese adipocytes (P<0.05), whereas k/mu(2) SA did not differ between the groups. The data support the concepts that (1) adipocyte LCFA uptake consists of distinct facilitated (saturable) and diffusive processes; (2) increased saturable LCFA uptake in obese adipocytes is not simply a consequence of increased cell size, but rather reflects upregulation of a facilitated transport process; and (3) the permeability of adipocyte plasma membranes to LCFA is not appreciably altered by obesity, and increased nonsaturable uptake in obese adipocytes principally reflects an increase in cell SA. Regulation of saturable LCFA uptake by adipocytes may be an important control point for body adiposity.

Lipid micelles stimulate the secretion of triglyceride-enriched apolipoprotein B48-containing lipoproteins by Caco-2 cells.

Intestinal triglyceride-rich lipoproteins (TRL) are synthesized from dietary lipids. This study was designed to evaluate the effects of lipid micelles, mimicking post-digestive duodenal micelles, on the fate of apolipoprotein B (apoB)48-containing lipoproteins by Caco-2 cells. Such micelles, consisting of oleic acid (OA), taurocholate, 2-monooleoylglycerol (2-MO), cholesterol (Chol), and L-alpha-lysophospatidylcholine, were the most efficient inducers of OA uptake and esterification. The efficiency of TG and apoB48 secretion increased specifically as a function of cell differentiation. PAGE analysis of secreted lipoproteins separated by sequential ultracentrifugation after [35S] labeling revealed differences in the secretion of apoB100- and apoB48-containing lipoproteins. In absence of micelles, apoB48 was secreted mostly in "HDL-like" particles, as observed in enterocytes in vivo. Micelle application increased 2.7-fold the secretion of apoB, resulting in 53 times more apoB48 being recovered as TG-enriched lipoproteins at d < 1.006 g/ml. Electron microscopy revealed the presence of lipid droplets in the secretory pathway and the accumulation of newly synthesized TG in cytoplasmic lipid droplets, as in enterocytes in vivo. We showed that these droplets could be used for secretion. However, apoB48 preferentially bound to newly synthesized TG in the presence of micelles, accounting in part for the functional advantage of apoB editing in the intestine. While Caco-2 cells express both apoB isoforms, our results show that the apical supply of complex lipid micelles favors the physiological route of apoB48-containing TG-enriched lipoproteins.

Chronic hyperglycaemia promotes lipogenesis and triacylglycerol accumulation in human skeletal muscle cells.

The present study was conducted to evaluate the effect of hyperglycaemia in itself on glucose and lipid metabolism in human skeletal muscle cells. Satellite cells were isolated from biopsy samples from the vastus lateralis muscle and differentiated into multinucleated myotubes in cultures. Metabolism studies were performed using isotopes ([3H]deoxyglucose, [14C]glucose, [14C]oleic acid and [14C]palmitic acid), and mRNA and protein levels were analysed by real-time RT-PCR and western blotting respectively. Exposure of myotubes to 20 mmol/l glucose for 4 days reduced insulin-stimulated glucose uptake and glycogen synthesis to 57+/-5% (p<0.0001) and 56+/-5% (p<0.0001) of normoglycaemic (5.5 mmol/l glucose) controls respectively. Basal glucose uptake and glycogen synthesis were both reduced, whereas glucose oxidation was unaltered. Total cell content of glycogen and expression of GLUT1 and GLUT4 mRNA were not affected. There was a significant increase in the incorporation of glucose into cellular NEFA (88+/-17% increase, p=0.006), triacylglycerol (44+/-21% increase, p=0.04) and cholesterol ester (89+/-36% increase, p=0.02) in hyperglycaemic myotubes compared with controls. Diacylglycerol tended to be increased though not significantly, and phospholipid formation were unchanged. Relative to controls, total cell content of triacylglycerol was increased by 25+/-7% (p=0.02) and acyl-CoA:1,2-diacylglycerol acyltransferase 1 activity was increased by 34+/-4% (p=0.004), whereas acyl-CoA:1,2-diacylglycerol acyltransferase 1 mRNA expression was unchanged. Total cellular uptake of palmitic acid was reduced by 18+/-3% (p=0.006) in hyperglycaemic cells compared with controls, while uptake of oleic acid was unchanged. Oxidation of palmitic acid or oleic acid was not affected by hyperglycaemia. Chronic hyperglycaemia increased triacylglycerol accumulation and the incorporation of carbohydrate into triacylglycerol (i.e. de novo lipogenesis) concomitantly with a reduced insulin-stimulated glucose uptake and glycogen synthesis. Enhanced acyl-CoA:1,2-diacylglycerol acyltransferase 1 activity supported the increased triacylglycerol synthesis during hyperglycaemia.

Long-chain fatty acid uptake into adipocytes depends on lipid raft function.

This study investigates the role of lipid rafts and caveolae, a subclass of lipid raft microdomains, in the binding and uptake of long-chain fatty acids (LCFA) by 3T3-L1 cells during differentiation. Disruption of lipid rafts by beta-cyclodextrin (betaCD) or selective inhibition of caveolae by overexpression of a dominant-negative mutant of caveolin-3 (Cav(DGV)) resulted in disassembly of caveolae structures at the cell surface, as assessed by electron microscopy. While in 3T3-L1 fibroblasts, which express few caveolae, Cav(DGV) or betaCD had no effect on LCFA uptake, in 3T3-L1 adipocytes the same treatments decreased the level of [(3)H]oleic acid uptake by up to 55 +/- 8 and 49 +/- 7%, respectively. In contrast, cholesterol loading of 3T3-L1 adipocytes resulted in a 4-fold increase in the extent of caveolin-1 expression and a 1.7-fold increase in the level of LCFA uptake. Both the inhibitory and enhancing effects of these treatments were constantly increasing with the [(3)H]oleic acid incubation time up to 5 min. Incubation of 3T3-L1 adipocytes with [(3)H]stearate followed by isolation of a caveolin-1 positive detergent-resistant membrane (DRM) fraction revealed that [(3)H]stearate binds to caveolae. Fatty acid translocase (FAT/CD36) was found to be present in this DRM fraction as well. Our data thus strongly indicate a critical involvement of lipid rafts in the binding and uptake of LCFA into 3T3-L1 adipocytes. Furthermore, our findings suggest that caveolae play a pivotal role in lipid raft-dependent LCFA uptake. This transport mechanism is induced in conjunction with cell differentiation and might be mediated by FAT/CD36.