Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.     

CD36 antisense expression in 3T3-F442A preadipocytes

An adipocyte membrane glycoprotein, FAT, homologous to CD36, has been implicated in the binding/transport of long-chain fatty acids. FAT/CD36 was identified by reaction with reactive long chain fatty acids derivatives under conditions where they inhibited FA uptake. Expression of CD36 in fibroblasts lacking the protein led to induction of a saturable high affinity, phloretinsensitive component of oleate uptake. In this report, we have examined the effects of FAT/CD36 antisense expression in 3T3-F442A preadipocyte cells, on FA uptake and cell differentiation. Cells were transfected with pSG5-TAF vector obtained by insertion of antisense coding sequence of FAT/CD36 into the BamH 1 site of pSG5. Four clones were selected based on expression of antisense CD36 mRNA. Levels of CD36 protein were determined by flow cytometry and correlated with rates of oleate uptake. Three clones, TAF13, TAF25, and TAF38 exhibited low CD36 expression and one clone TAF 18 had expression comparable to that of F442A control cells. FA uptake rates in clones TAF13, TAF25 and TAF3 8 were lower than those observed in TAF18. At confluence, adipocyte differentiation could be promoted by addition of insulin and triiodothyronine only in TAF18 cells but not in TAF13, TAF25 or TAF38. Addition of fatty acids to clones TAF13, TAF25 and TAF38 lead to an induction of CD36 expression, an enhancement of FA uptake and better cell differentiation. The data support a role of CD36 in the membrane uptake of long chain FA. CD36 expression and FA uptake appear to be closely linked to preadipocyte differentiation.     

Cholesterol depletion inhibits fatty acid uptake without affecting CD36 or caveolin-1 distribution in adipocytes

Caveolin-1 and CD36 are plasma membrane fatty acid binding proteins that participate in adipocyte fatty acid uptake and metabolism. Both are associated with cholesterol-enriched caveolae/lipid rafts in the plasma membrane that are important for long chain fatty acid uptake. Depletion of plasma membrane cholesterol reversibly inhibited oleate uptake by adipocytes without altering the amount or the cell surface distribution of either caveolin-1 or CD36. Cholesterol levels thus regulate fatty acid uptake by adipocytes via a pathway that does not involve altered cell surface localization of caveolin-1 or CD36.     

In situ labeling of the adipocyte lipid binding protein with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide. Evidence for a role of fatty acid binding proteins in lipid uptake

Differentiating 3T3-L1 cells have been used to investigate the process of fatty acid uptake, its cellular specificity, and the involvement of cytoplasmic carrier proteins. The profile of fatty acid uptake in both differentiated and undifferentiated cells was biphasic, consisting of an initial rapid phase (0-20 s) followed by a second slower phase (60-480 s). In both cell types the initial phase of fatty acid (FA) uptake was temperature-insensitive whereas the rate of uptake during the second phase decreased 4-fold when measurements were made at 4 degrees C. The rate of [9,10-3H]oleate uptake in 3T3-L1 adipocytes was 10-fold greater than in the fibroblastic precursor cells. The acquisition of a differentially expressed cytoplasmic fatty acid binding protein (adipocyte lipid binding protein (ALBP] occurs coincident with the increased ability of these cells to take up FAs. Uptake experiments with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide demonstrated that this photoactivatable FA analogue accumulated intracellularly in a time-, temperature-, and cell-specific fashion. Moreover, when 3T3-L1 adipocytes were presented with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide and then irradiated, a single cytoplasmic 15-kDa protein was labeled. The in situ-labeled 15-kDa protein was identified as ALBP by its ability to be immunoprecipitated with anti-ALBP antisera. Taken together these results indicate that fatty acids traverse the plasma membrane and are bound by ALBP in the cytoplasmic compartment. It is likely that lipid uptake in other cell systems, such as liver, heart, intestine, and nerve tissue, proceeds by a similar process and that this represents a general mechanism for cell-specific FA uptake and utilization.     

Restricted localization of the adipocyte/muscle glucose transporter species to a cell surface-derived vesicle fraction of 3T3-L1 adipocytes. Inhibited lateral mobility of integral plasma membrane proteins in newly inserted membrane areas of differentiated 3T3-L1 cells

The recent demonstration of a large cell surface-derived pool of insulin-sensitive glucose transporters, presumably concentrated in the microvilli of 3T3-L1 adipocytes, induced the assumption that in differentiated adipocytes, newly inserted plasma membrane areas may display restricted lateral mobility, thereby preventing diffusion of integral membrane proteins out of these areas into the adjoining plasma membrane. In order to test this assumption, the cell surface distributions of the two glucose transporter species expressed by 3T3-L1 cells were determined using specific antisera against the HepG2/erythrocyte transporter, GluT1, which is synthesized in both fibroblasts and adipocytes, and the adipocyte/muscle-specific transporter, GluT4, expressed for the first time 3-4 days after induction of adipose conversion. GluT1 was shown to be localized in the plasma membrane of both 3T3-L1 preadipocytes and adipocytes, whereas GluT4 was almost entirely restricted to the low density surface-derived vesicle (LDSV) fraction of 3T3-L1 adipocytes most likely consisting of microvilli-derived vesicles. In contrast to the minor portion of GluT4 found in the adipocyte plasma membrane fraction, equal amounts of the GluT1 protein were detected in both the plasma membrane and the LDSV fractions of adipocytes. Both transporter species were present in the microsomal and the LDSV fractions of adipocytes. The observed distribution of the two transporter species is in accordance with the postulated restriction of the lateral mobility in plasma membrane areas formed by newly inserted transgolgi vesicles of differentiated adipocytes.     

Vanillin induces adipocyte differentiation in 3T3-L1 cells by activating extracellular signal regulated kinase 42/44

AimsTo investigate the effect of vanillin, a dietary component, on adipocyte differentiation and the mechanism involved in the process using 3T3-L1 murine preadipocytes.Main methodsThe effect of vanillin on adipocyte differentiation was detected by Oil Red O analysis. The activation of extracellular signal regulated kinase 42/44 (ERK 42/44), Akt, expression of the key regulator of adipocyte differentiation peroxisome proliferators-activated receptor (PPARγ) and its target gene glucose transporter 4 (GLUT4) were detected by western blotting. Glucose uptake assay was used to determine the insulin sensitivity of adipocytes differentiated by vanillin treatment. To confirm the role of ERK 42/44 and Akt, Oil Red O analysis was performed with cells differentiated in the presence or absence of ERK inhibitor U0126 or Akt kinase 1/2 inhibitor.Key findingsVanillin induced adipocyte differentiation in 3T3-L1 cells in a dose dependent manner and also increased the expression levels of PPARγ and its target gene GLUT4. The adipocytes differentiated by vanillin exhibited insulin sensitivity as demonstrated by a significant increase in glucose uptake. Vanillin treatment activated the phosphorylation of ERK 42/44 during the initial phase of adipocyte differentiation but there was no significant change in the Akt phosphorylation status.SignificanceThe data show that vanillin induces adipocyte differentiation in 3T3-L1 cells by activating ERK42/44 and these adipocytes are insulin sensitive in nature.     

Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, we focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and [35S] methionine incorporation into GAPDH protein increased 5-fold. The increase in GAPDH mRNA and GAPDH biosynthesis could be demonstrated using physiologic concentrations of insulin (0.24 nM), indicating that these effects are mediated through a specific interaction with the insulin receptor. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane

Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism. FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import. In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake. To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs. We demonstrate for the first time that FACS is an integral membrane protein. Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane. Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP. Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells.     

Translocation of fatty acids across the basolateral rat liver plasma membrane is driven by an active potential-sensitive sodium-dependent transport system

In previous studies it was shown that hepatocellular uptake of fatty acids is mediated by a specific fatty acid binding membrane protein. To determine now directly the driving forces for their entry into hepatocytes, the uptake of a representative long chain fatty acid, [3H]oleate, by basolateral rat liver plasma membrane vesicles was examined. Influx of oleate was stimulated by increasing the Na+ concentration of the medium. In the presence of an inwardly directed Na+ gradient (NaSCN, NaNO3, NaCl) oleate was accumulated during the initial uptake phase (20 s) at a concentration of 1.4-1.9-fold that at equilibrium (overshoot). This activation of influx was not observed after replacement of Na+ by Li+, K+, or choline+. Na+-dependent oleate uptake was significantly stimulated by creation of a negative intravesicular potential, either by altering the accompanying anions or by valinomycin-induced K+ diffusion potentials, suggesting an electrogenic transport mechanism. Na+-dependent fatty acid uptake was temperature dependent, with maximal overshoots occurring at 37 degrees C, and revealed saturation kinetics with a Km of 83.1 nM and Vmax of 2.9 nmol X min-1 X mg protein-1. These studies demonstrate that the carrier-mediated hepatocellular uptake of fatty acids represents an active potential-sensitive Na+-fatty acid cotransport system.     

Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents.

Long chain fatty acid transport is selectively up-regulated in adipocytes of Zucker fatty rats, diverting fatty acids from sites of oxidation toward storage in adipose tissue. To determine whether this is a general feature of obesity, we studied [(3)H]oleate uptake by adipocytes and hepatocytes from 1) homozygous male obese (ob), diabetic (db), fat (fat), and tubby (tub) mice and from 2) male Harlan Sprague-Dawley rats fed for 7 weeks a diet containing 55% of calories from fat. V(max) and K(m) were compared with controls of the appropriate background strain (C57BL/6J or C57BLKS) or diet (13% of calories from fat). V(max) for adipocyte fatty acid uptake was increased 5-6-fold in ob, db, fat, and tub mice versus controls (p < 0.001), whereas no differences were seen in the corresponding hepatocytes. Similar changes occurred in fat-fed rats. Of three membrane fatty acid transporters expressed in adipocytes, plasma membrane fatty acid-binding protein mRNA was increased 9-11-fold in ob and db, which lack a competent leptin/leptin receptor system, but was not increased in fat and tub, i.e. in strains with normal leptin signaling capability; fatty acid translocase mRNA was increased 2.2-6.5-fold in tub, ob, and fat adipocytes, but not in db adipocytes; and only marginal changes in fatty acid transport protein 1 mRNA were found in any of the mutant strains. Adipocyte fatty acid uptake is generally increased in murine obesity models, but up-regulation of individual transporters depends on the specific pathophysiology. Leptin may normally down-regulate expression of plasma membrane fatty acid binding protein.     

Dexamethasone regulates the beta-adrenergic receptor subtype expressed by 3T3 L1 preadipocytes and adipocytes

The subtype of the beta-adrenergic receptor expressed in 3T3-L1 preadipocytes and adipocytes differentiated with dexamethasone and methylisobutylxanthine was determined by comparing the affinity of the receptors for epinephrine, norepinephrine, and beta-1 and beta-2 selective antagonist, 8-fold more avidly than adipocyte receptors. In contrast, adipocyte beta-receptors had a 10-fold higher affinity for epinephrine than for norepinephrine and complexed the beta-2 selective agonist zinterol with a 20-fold higher affinity than preadipocyte receptors. Hofstee plots and computer analyses of the binding data revealed that the populations of beta-1 receptors in preadipocytes and beta-2 receptors in adipocytes were nearly homogeneous. Preliminary characterizations of the beta-receptor phenotype in (nondifferentiating) 3T3-C2 cells treated with dexamethasone and methylisobutylxanthine and 3T3-422A adipocytes differentiated with insulin indicated that the expression of beta-2 receptors was not correlated with differentiation, but rather with exposure of the cells to dexamethasone and methylisobutylxanthine. The regulator of beta-receptor subtype was identified as the glucocorticoid analog, dexamethasone, by employing 3T3-L1 adipocytes which were stimulated to differentiate with methylisobutylxanthine and insulin. Detailed binding studies showed that under these conditions the adipocyte receptors retain beta-1 character. Subsequent treatment with 0.5 microM dexamethasone promoted the loss of beta-1 receptors, the appearance of beta-2 receptors, and a net 2- to 3-fold increase in the number of beta-receptors. Dexamethasone effected a complete switch from beta-1 to beta-2 subtype at concentrations as low as 2.5 nM while other steroids were ineffective below a concentration of 10 microM.     

The differentiation-dependent inhibition of SV40 early promoter/enhancer activity in 3T3-L1 cells is mediated through promoter and not enhancer sequences

Using a plasmid bearing chloramphenicol acetyltransferase (CAT) gene controlled by Simian virus 40 (SV40) early promoter/enhancer complex (pA0cat), we analyzed functional enhancer motifs in 3T3-L1 fibroblast and adipocyte cells. Deletion mutant series of pA0 at the enhancer complex showed that gene expression both in fibroblast and adipocyte cells was dependent on a similar set of enhancer motifs. When pA0 was introduced into 3T3-L1 fibroblasts and the cells were induced to differentiate into adipocytes, CAT activity expressed in fibroblasts was suppressed. Experiments with the deletion mutants at the enhancer complex showed that the suppression was not related to any enhancer motif, and CAT activity was observed with a plasmid having only the promoter sequence. When pA0cat was co-transfected with excess of promoter sequence, the suppression in adipocytes was counteracted. This suggested that negativetrans-acting factors of the promoter sequence were responsible for the suppression in adipocytes.Abbreviations CAT chloramphenicol acetyltransferase - CAT the gene encoding CAT - SV40 Simian virus 40 - Asc-P ascorbic acid phosphate     

Palmitate movement across red and white muscle membranes of rainbow trout

We examined the movement of [3H]palmitate across giant sarcolemmal vesicles prepared from red and white muscle of rainbow trout (Oncorhynchus mykiss). Red and white muscle fatty acid carriers have similar affinities for palmitate (apparent Km = 26 +/- 6 and 33 +/- 8 nM, respectively); however, red muscle has a higher maximal uptake compared with white muscle (Vmax = 476 +/- 41 vs. 229 +/- 23 pmol.mg protein-1.s-1, respectively). Phloretin (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 40%, HgCl2 (2.5 mM) inhibited palmitate uptake by 20-30%, and the anion-exchange inhibitor DIDS (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 15 and 30%, respectively. Western blot analysis of red and white muscle vesicles did not detect a mammalian-type fatty acid transporter (FAT); however, preincubation of vesicles with sulfo-N-succinimidyloleate, a specific inhibitor of FAT in rats, reduced palmitate uptake in red and white muscle vesicles by approximately 15 and 25%, respectively. A mammalian-type plasma membrane fatty acid-binding protein was identified in trout muscle using Western blotting, but the protein differed in size between red and white muscle. At low concentrations of free palmitate (2.5 nM), addition of high concentrations (111 microM total) of oleate (18:0) caused approximately 50% reduction in palmitate uptake by red and white muscle vesicles, but high concentrations (100 microM) of octanoate (8:0) caused no inhibition of uptake. Five days of aerobic swimming at approximately 2 body lengths/s and 9 days of chronic cortisol elevation in vivo, both of which stimulate lipid metabolism, had no effect on the rate of palmitate movement in red or white muscle vesicles.     

NOD1 activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes

Chronic inflammation is associated with obesity and insulin resistance; however, the underlying mechanisms are not fully understood. Pattern recognition receptors Toll-like receptors and nucleotide-oligomerization domain-containing proteins play critical roles in innate immune response. Here, we report that activation of nucleotide binding oligomerization domain-containing protein-1 (NOD1) in adipocytes induces proinflammatory response and impairs insulin signaling and insulin-induced glucose uptake. NOD1 and NOD2 mRNA are markedly increased in differentiated murine 3T3-L1 adipocytes and human primary adipocyte culture upon adipocyte conversion. Moreover, NOD1 mRNA is markedly increased only in the fat tissues in diet-induced obese mice, but not in genetically obese ob/ob mice. Stimulation of NOD1 with a synthetic ligand Tri-DAP induces proinflammatory chemokine MCP-1, RANTES, and cytokine TNF-α and MIP-2 (human IL-8 homolog) and IL-6 mRNA expression in 3T3-L1 adipocytes in a time- and dose-dependent manner. Similar proinflammatory profiles are observed in human primary adipocyte culture stimulated with Tri-DAP. Furthermore, NOD1 activation suppresses insulin signaling, as revealed by attenuated tyrosine phosphorylation and increased inhibitory serine phosphorylation, of IRS-1 and attenuated phosphorylation of Akt and downstream target GSK3α/3β, resulting in decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. Together, our results suggest that NOD1 may play an important role in adipose inflammation and insulin resistance in diet-induced obesity.