Common mechanisms of monoacylglycerol and fatty acid uptake by human intestinal Caco-2 cells

2001 ◽  
Vol 281 (4) ◽  
pp. C1106-C1117 ◽  
Author(s):  
Shiu-Ying Ho ◽  
Judith Storch
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Monomer Concentration ◽  
Protein S ◽  
Fatty Acid Uptake ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Fatty Acid Binding ◽  
Bound Lipids ◽  
Kinetics Of

Free fatty acids (FFA) and sn-2-monoacylglycerol ( sn-2-MG), the two hydrolysis products of dietary triacylglycerol, are absorbed from the lumen into polarized enterocytes that line the small intestine. Intensive studies regarding FFA transport across the brush-border membrane of the enterocyte are available; however, little is known about sn-2-MG transport. We therefore studied the kinetics of sn-2-MG transport, compared with those of long-chain FFA (LCFA), by human intestinal Caco-2 cells. To mimic postprandial luminal and plasma environments, we examined the uptake of taurocholate-mixed lipids and albumin-bound lipids at the apical (AP) and basolateral (BL) surfaces of Caco-2 cells, respectively. The results demonstrate that the uptake of sn-2-monoolein at both the AP and BL membranes appears to be a saturable function of the monomer concentration of sn-2-monoolein. Furthermore, trypsin preincubation inhibits sn-2-monoolein uptake at both AP and BL poles of cells. These results suggest that sn-2-monoolein uptake may be a protein-mediated process. Competition studies also support a protein-mediated mechanism and indicate that LCFA and LCMG may compete through the same membrane protein(s) at the AP surface of Caco-2 cells. The plasma membrane fatty acid-binding protein (FABPpm) is known to be expressed in Caco-2, and here we demonstrate that fatty acid transport protein (FATP) is also expressed. These putative plasma membrane LCFA transporters may be involved in the uptake of sn-2-monoolein into Caco-2 cells.

Effect of surface and intracellular pH on hepatocellular fatty acid uptake

1996 ◽  
Vol 271 (6) ◽  
pp. G1067-G1073
Author(s):  
C. Elsing ◽  
A. Kassner ◽  
W. Stremmel
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Intracellular Ph ◽  
Outer Surface ◽  
Fatty Acid Uptake ◽  
Proton Gradient ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Acid Transport

Fatty acids enter hepatocytes, at least in part, by a carrier-mediated uptake mechanism. The importance of driving forces for fatty acid uptake is still controversial. To evaluate possible driving mechanisms for fatty acid transport across plasma membranes, we examined the role of transmembrane proton gradients on fatty acid influx in primary cultured rat hepatocytes. After hepatocytes were loaded with SNARF-1 acetoxymethyl ester, changes in intracellular pH (pHi) under different experimental conditions were measured and recorded by confocal laser scanning microscopy. Fatty acid transport was increased by 45% during cellular alkalosis, achieved by adding 20 mM NH4Cl to the medium, and a concomitant paracellular acidification was observed. Fatty acid uptake was decreased by 30% during cellular acidosis after withdrawal of NH4Cl from the medium. Cellular acidosis activates the Na+/H+ antiporter to export excessive protons to the outer cell surface. Inhibition of Na+/H+ antiporter activity by amiloride diminishes pHi recovery and thereby accumulation of protons at the outer surface of the plasma membrane. Under these conditions, fatty acid uptake was further inhibited by 57% of control conditions. This suggests stimulation of fatty acid influx by an inwardly directed proton gradient. The accelerating effect of protons at the outer surface of the plasma membrane was confirmed by studies in which pH of the medium was varied at constant pHi. Significantly higher fatty acid influx rates were observed at low buffer pH. Recorded differences in fatty acid uptake appeared to be independent of changes in membrane potential, because BaCl2 did not influence initial uptake velocity during cellular alkalosis and paracellular acidosis. Moreover, addition of oleate-albumin mixtures to the NH4Cl incubation buffer did not change the observed intracellular alkalinization. In contrast, after cells were acid loaded, addition of oleate-albumin solutions to the recovery buffer increased pHi recovery rates from 0.21 +/- 0.02 to 0.36 +/- 0.05 pH units/min (P < 0.05), indicating that fatty acids further stimulate Na+/H+ antiporter activity during pHi recovery from an acid load. It is concluded that carrier-mediated uptake of fatty acids in hepatocytes follows an inwardly directed transmembrane proton gradient and is stimulated by the presence of H+ at the outer surface of the plasma membrane.

Sex differences in hepatic fatty acid uptake reflect a greater affinity of the transport system in females

1992 ◽  
Vol 263 (3) ◽  
pp. G380-G385 ◽  
Author(s):  
D. Sorrentino ◽  
S. L. Zhou ◽  
E. Kokkotou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Transport System ◽  
Fatty Acid Uptake ◽  
Surface Expression ◽  
Female Rats ◽  
Acid Uptake ◽  
Fatty Acid Binding ◽  
Male And Female Rats

In this study, we examined the hypothesis that the reported sex difference in hepatic free fatty acid (FFA) uptake involves the putative FFA transport system, the plasma membrane fatty acid binding protein (FABPpm). In hepatocytes isolated from both male and female rats, initial [3H]oleate uptake velocity reflected transmembrane influx and not subsequent metabolism and was a saturable function of the unbound oleate concentration. Although Vmax values were similar (61 +/- 2 vs. 65 +/- 5 pmol.min-1.5 x 10(4) cells-1 for females and males, respectively), the apparent Km was significantly smaller in females (40 +/- 4 vs. 90 +/- 11 nM; P less than 0.05), reflecting faster influx velocities in female cells over a range of unbound oleate concentrations. The oleate efflux rate constant was also greater in females (0.280 +/- 0.014 vs. 0.198 +/- 0.020 min-1; P less than 0.05) despite their greater hepatic content of cytosolic FABP. Finally, despite the greater rates of transmembrane FFA flux in female hepatocytes, the surface expression of FABPpm was virtually identical in the two sexes (2.5 +/- 0.5 vs. 2.4 +/- 0.4 microgram/10(6) cells). Collectively, these data indicate that at FFA-to-albumin ratios occurring in vivo the plasma membrane of female hepatocytes transports oleate bidirectionally at a greater rate than that of male hepatocytes. A sex-related difference in the functional affinity of FABPpm for FFA appears the most likely explanation for the greater oleate uptake in females.

Insulin induces the translocation of the fatty acid transporter FAT/CD36 to the plasma membrane

2002 ◽  
Vol 282 (2) ◽  
pp. E491-E495 ◽  
Author(s):  
Joost J. F. P. Luiken ◽  
David J. Dyck ◽  
Xiao-Xia Han ◽  
Narendra N. Tandon ◽  
Yoga Arumugam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Muscle Contraction ◽  
Kinase Inhibitor ◽  
Fatty Acid Uptake ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Hindlimb Muscles ◽  
Fatty Acid Transporter ◽  
Acid Transporter

It is well known that muscle contraction and insulin can independently translocate GLUT-4 from an intracellular depot to the plasma membrane. Recently, we have shown that the fatty acid transporter FAT/CD36 is translocated from an intracellular depot to the plasma membrane by muscle contraction (<30 min) (Bonen et al. J Biol Chem 275: 14501–14508, 2000). In the present study, we examined whether insulin also induced the translocation of FAT/CD36 in rat skeletal muscle. In studies in perfused rat hindlimb muscles, we observed that insulin increased fatty acid uptake by +51%. Insulin increased the rate of palmitate incorporation into triacylglycerols, diacylglycerols, and phospholipids ( P < 0.05) while reducing muscle palmitate oxidation ( P < 0.05). Perfusing rat hindlimb muscles with insulin increased plasma membrane FAT/CD36 by +48% ( P < 0.05), whereas concomitantly the intracellular FAT/CD36 depot was reduced by 68% ( P < 0.05). These insulin-induced effects on FAT/CD36 translocation were inhibited by the phosphatidylinositol 3-kinase inhibitor LY-294002. Thus these studies have shown for the first time that insulin can induce the translocation of FAT/CD36 from an intracellular depot to the plasma membrane.This reveals a previously unknown level of regulation of fatty acid transport by insulin and may well have important consequences in furthering our understanding of the relation between fatty acid metabolism and insulin resistance.

scholarly journals New insights into long-chain fatty acid uptake by heart muscle: a crucial role for fatty acid translocase/CD36

2002 ◽  
Vol 367 (3) ◽  
pp. 561-570 ◽  
Author(s):  
Joep F.F. BRINKMANN ◽  
Nada A. ABUMRAD ◽  
Azeddine IBRAHIMI ◽  
Ger J. vanderVUSSE ◽  
Jan F.C. GLATZ
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Heart Muscle ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Membrane Fatty Acid ◽  
Acid Transport ◽  
Fatty Acid Binding ◽  
Fatty Acid Translocase ◽  
Long Chain

Long-chain fatty acids are an important source of energy for several cell types, in particular for the heart muscle cell. Three different proteins, fatty acid translocase (FAT)/CD36, fatty acid transport protein and plasma membrane fatty acid binding protein, have been identified as possible membrane fatty acid transporters. Much information has been accumulated recently about the fatty acid transporting function of FAT/CD36. Several experimental models to study the influence of altered FAT/CD36 expression on fatty acid homoeostasis have been identified or developed, and underscore the importance of FAT/CD36 for adequate fatty acid transport. These models include the FAT/CD36 null mouse, the spontaneously hypertensive rat and FAT/CD36-deficient humans. The fatty acid transporting role of FAT/CD36 is further demonstrated in mice overexpressing muscle-specific FAT/CD36, and in transgenic mice generated using a genetic-rescue approach. In addition, a wealth of information has been gathered about the mechanisms that regulate FAT/CD36 gene expression and the presence of functional FAT/CD36 on the plasma membrane. Available data also indicate that FAT/CD36 may have an important role in the aetiology of cardiac disease, especially cardiac hypertrophy and diabetic cardiomyopathy. This review discusses our current knowledge of the three candidate fatty acid transporters, the metabolic consequences of alterations in FAT/CD36 levels in different models, and the mechanisms that have been identified for FAT/CD36 regulation.

Protein-mediated fatty acid uptake: regulation by contraction, AMP-activated protein kinase, and endocrine signals

2007 ◽  
Vol 32 (5) ◽  
pp. 865-873 ◽  
Author(s):  
James G. Nickerson ◽  
Iman Momken ◽  
Carley R. Benton ◽  
James Lally ◽  
Graham P. Holloway ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Protein Kinase ◽  
Acid Oxidation ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Acid Transport ◽  
Fatty Acid Binding ◽  
Mitochondrial Fatty Acid ◽  
Amp Activated Protein Kinase

Fatty acid transport into heart and skeletal muscle occurs largely through a highly regulated protein-mediated mechanism involving a number of fatty acid transporters. Chronically altered muscle activity (chronic muscle stimulation, denervation) alters fatty acid transport by altering the expression of fatty acid transporters and (or) their subcellular location. Chronic exposure to leptin downregulates while insulin upregulates fatty acid transport by altering concomitantly the expression of fatty acid transporters. Fatty acid transport can also be regulated within minutes, by muscle contraction, AMP-activated protein kinase activation, leptin, and insulin, through induction of the translocation of fatty acid translocase (FAT)/CD36 from its intracellular depot to the plasma membrane. In insulin-resistant muscle, a permanent relocation of FAT/CD36 to the sarcolemma appears to account for the excess accretion of intracellular lipids that interfere with insulin signaling. Recent work has also shown that FAT/ CD36, but not plasma membrane associated fatty acid binding protein, is involved, along with carnitine palmitoyltransferase, in regulating mitochondrial fatty acid oxidation. Finally, studies in FAT/CD36 null mice indicate that this transporter has a key role in regulating fatty acid metabolism in muscle.

Fatty acid transport and metabolism in HepG2 cells

2006 ◽  
Vol 290 (3) ◽  
pp. G528-G534 ◽  
Author(s):  
Wen Guo ◽  
Nasi Huang ◽  
Jun Cai ◽  
Weisheng Xie ◽  
James A. Hamilton
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Hepg2 Cells ◽  
Fatty Acid Uptake ◽  
Metabolic Inhibitors ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Triacsin C ◽  
Membrane Bound

The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pHin), indicating diffusion of fatty acids across the plasma membrane. pHin returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pHin drop after the addition of oleate but greatly suppressed pHin recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pHin recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pHin decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pHin drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.

FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C2C12 muscle cells

2011 ◽  
Vol 301 (5) ◽  
pp. E785-E796 ◽  
Author(s):  
Margarete Digel ◽  
Simone Staffer ◽  
Friedrich Ehehalt ◽  
Wolfgang Stremmel ◽  
Robert Ehehalt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Enzymatic Activity ◽  
Muscle Cells ◽  
Fatty Acid Uptake ◽  
Fatty Acid Transport ◽  
Synthetase Activity ◽  
Acid Uptake ◽  
Acid Transport ◽  
Short Term

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C2C12 muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C2C12 cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.

Changes in fatty acid transport and transporters are related to the severity of insulin deficiency

2002 ◽  
Vol 283 (3) ◽  
pp. E612-E621 ◽  
Author(s):  
Joost J. F. P. Luiken ◽  
Yoga Arumugam ◽  
Rhonda C. Bell ◽  
Jorge Calles-Escandon ◽  
Narendra N. Tandon ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Acid Transport ◽  
Fatty Acid Binding ◽  
Fatty Acid Transporter ◽  
Acid Transporter

We have examined the effects of streptozotocin (STZ)-induced diabetes (moderate and severe) on fatty acid transport and fatty acid transporter (FAT/CD36) and plasma membrane-bound fatty acid binding protein (FABPpm) expression, at the mRNA and protein level, as well as their plasmalemmal localization. These studies have shown that, with STZ-induced diabetes, 1) fatty acid transport across the plasma membrane is increased in heart, skeletal muscle, and adipose tissue and is reduced in liver; 2) changes in fatty acid transport are generally not associated with changes in fatty acid transporter mRNAs, except in the heart; 3) increases in fatty acid transport in heart and skeletal muscle occurred with concomitant increases in plasma membrane FAT/CD36, whereas in contrast, the increase and decrease in fatty acid transport in adipose tissue and liver, respectively, were accompanied by concomitant increments and reductions in plasma membrane FABPpm; and finally, 4) the increases in plasma membrane transporters (FAT/CD36 in heart and skeletal muscle; FABPpm in adipose tissue) were attributable to their increased expression, whereas in liver, the reduced plasma membrane FABPpm appeared to be due to its relocation within the cell in the face of slightly increased expression. Taken together, STZ-induced changes in fatty acid uptake demonstrate a complex and tissue-specific pattern, involving different fatty acid transporters in different tissues, in combination with different underlying mechanisms to alter their surface abundance.

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