Slc43a3 is a regulator of free fatty acid flux

Abstract

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake. Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake. long chain fatty acyl-CoA synthetase branched DNA embryonic epithelia gene 1 FA binding protein FA oxidation FA transport protein amino acid system L transporter long chain FA oleic acid solute carrier vertical sleeve gastrectomy FAs play important roles in a variety of cellular functions, such as the production and storage of energy, synthesis of phospholipids, glycolipids, and cellular signaling messengers, modification of proteins for targeting to cellular membranes, and regulation of gene expression (1Berg, J. M., J. L., Tymoczko, and L., Stryer, . 2002. Biochemistry. 5th edition. W. H. Freeman, New York.Google Scholar, 2Glatz J.F. Challenges in fatty acid and lipid physiology.Front. Physiol. 2011; 2: 45Crossref PubMed Scopus (10) Google Scholar, 3Das U.N. Essential fatty acids: biochemistry, physiology and pathology.Biotechnol. J. 2006; 1: 420-439Crossref PubMed Scopus (477) Google Scholar). Because most cells, with the exception of adipocytes, have a very limited capacity for storing FAs, in the form of triglycerides (or for de novo synthesis of FA), circulating plasma FAs are the most important source of FAs for most tissues (4Xu S. Jay A. Brunaldi K. Huang N. Hamilton J.A. CD36 enhances fatty acid uptake by increasing the rate of intracellular esterification but not transport across the plasma membrane.Biochemistry. 2013; 52: 7254-7261Crossref PubMed Scopus (75) Google Scholar). Short and medium chain FAs have high plasma membrane permeability and can diffuse freely and easily through the plasma membranes of cells. Long chain FAs (LCFAs), however, have much lower water solubility and otherwise bind to albumin to facilitate large volume fluxes. Different rates of FA uptake are observed among various cell types, with metabolically active cells having increased rates of FA uptake (5Knittle J.L. Hirsch J. Effect of chain length on rates of uptake of free fatty acids during in vitro incubations of rat adipose tissue.J. Lipid Res. 1965; 6: 565-571Abstract Full Text PDF PubMed Google Scholar). The rates of cellular FA uptake are also regulated acutely and chronically by hormones (such as insulin) and metabolic status (in case of obesity) (6Berk P.D. Zhou S.L. Kiang C.L. Stump D. Bradbury M. Isola L.M. Uptake of long chain free fatty acids is selectively up-regulated in adipocytes of Zucker rats with genetic obesity and non-insulin-dependent diabetes mellitus.J. Biol. Chem. 1997; 272: 8830-8835Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Bonen A. Benton C.R. Campbell S.E. Chabowski A. Clarke D.C. Han X.X. Glatz J.F. Luiken J.J. Plasmalemmal fatty acid transport is regulated in heart and skeletal muscle by contraction, insulin and leptin, and in obesity and diabetes.Acta Physiol. Scand. 2003; 178: 347-356Crossref PubMed Scopus (52) Google Scholar). In addition to the bidirectional flip-flop model for transporting FAs across the plasma membrane, studies have demonstrated the importance of specific LCFA transport systems in metabolically active tissues, such as intestine, heart, adipose tissue, and the liver (8Stahl A. A current review of fatty acid transport proteins (SLC27).Pflugers Arch. 2004; 447: 722-727Crossref PubMed Scopus (235) Google Scholar). This transport appears to involve membrane proteins that can mediate FA uptake via a rapid, saturable, substrate-specific, and hormonally related mechanism. Further studies have established that LCFA uptake into heart and muscle is regulated acutely by contraction, insulin, and leptin and chronically in conditions like obesity and diabetes (7Bonen A. Benton C.R. Campbell S.E. Chabowski A. Clarke D.C. Han X.X. Glatz J.F. Luiken J.J. Plasmalemmal fatty acid transport is regulated in heart and skeletal muscle by contraction, insulin and leptin, and in obesity and diabetes.Acta Physiol. Scand. 2003; 178: 347-356Crossref PubMed Scopus (52) Google Scholar). In adipocytes, permeation of LCFAs across the plasma membrane relies on a high-affinity low-capacity protein-facilitated transport system. Studies have demonstrated the importance of several different proteins, such as FA transport proteins (FATPs), long chain fatty acyl-CoA synthetases (ACSLs), and FA translocase (also known as CD36), in the protein-facilitated process of FA transport. However, the mechanisms of how FAs traverse the plasma membrane to enter the soluble cytoplasm are not yet fully understood (9Glatz J.F. Luiken J.J. From fat to FAT (CD36/SR-B2): understanding the regulation of cellular fatty acid uptake.Biochimie. 2017; 136: 21-26Crossref PubMed Scopus (131) Google Scholar). Specifically, there is a debate on the rate-limiting step in the overall process of FA uptake and whether, and to what extent, one or more membrane-associated proteins could facilitate and/or regulate FA uptake. In contradistinction, FA efflux from adipocytes is attributed to diffusion without any proteins being associated with facilitating the process, though we previously reported evidence to suggest the possibility of a protein-facilitated process (10Henkin A.H. Ortegon A.M. Cho S. Shen W.J. Falcon A. Kraemer A. for protein-mediated fatty acid efflux by Physiol. PubMed Scopus Google Scholar). To for we in of transporter the plasma membrane and significant expression in adipose One of the transporter gene that into is the family of the of are to facilitate the of or de of transporters in lipid transport and 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, M. The transporter Lipid Res. Full Text Full Text PDF PubMed Google Scholar, D.C. the to Biol. PubMed Scopus Google Scholar). family of membrane transport proteins is the family of the solute with most of are in the cell membrane A. and PubMed Google Scholar, transporters as PubMed Scopus Google Scholar, The of membrane transporters in and 2013; PubMed Scopus Google Scholar, A. D. A.M. for on solute Full Text Full Text PDF PubMed Scopus Google Scholar). In we the expression levels of gene from in in patients before and after bariatric surgery to the that the expression of proteins to FA efflux that showed a significant in expression were in OP9 murine adipocyte cells and primary adipocytes One of the and the gene for the Slc43a3 solute The expression levels of Slc43a3 in various adipose and during adipocyte differentiation were in and OP9 murine adipocyte cells. The of Slc43a3 in regulating FA transport using overexpression or of Slc43a3 in differentiated OP9 cells. of Slc43a3 reduced both basal and forskolin-stimulated FA resulted in increased uptake of FAs into cells, whereas overexpression of resulted in a decreased uptake of we that Slc43a3 FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake. were from the from from FA uptake from FA from were from and CD36 were from the and were for the of liver and fat were in the the on a were in with and by the and of the vertical sleeve gastrectomy were and from and of with a and by for or a to the of the from the and from the were up to the of to the of the have by H. N. A. A. K. acids from after bariatric PubMed Scopus Google Scholar). on and were for obese patients the of surgery and bariatric surgery. and were to surgery and during the as by as to the changes bariatric surgery have by H. S. N. K. of adipose lipid by expression and function of after bariatric J. PubMed Scopus Google Scholar). The of and and from This in with the of subcutaneous adipose tissue from patients were for tissue of fat were from and with and of adipose tissue were using to the levels were using from are that a branched DNA for for the of gene expression The with to the and of samples are to the the are with and the to both the and the to the the is through of the and to the addition of a that is to the of is using a This system a and for members of the transporter family and 33 members from the family with with for on the for with that have and The are with specific to the gene to a the by the by a 1 with the DNA The were by a 1 with the DNA DNA to the and for 1 the were by for with The were and on the OP9 cells from were to in medium and OP9 cells were with and 1 on and with 1 insulin on were on and were by of adipose tissue from by and in medium and preadipocytes were differentiated as and on and Slc43a3 into the and of the via Slc43a3 were from siRNA from and as a negative OP9 cells were for with Slc43a3 siRNA and were in and of siRNA or overexpression with a were for the medium with medium and and to the differentiation the cells were for FA efflux and uptake as as and OP9 cells and were and in in a or using the from in a the in to and to for with the as using system using The of specific by the of to the Slc43a3, and the for OP9 cells were into a with Slc43a3 siRNA or siRNA a and for to the differentiation a FA uptake to the the medium from the and with medium and The for 1 the a One of the in and were to and in a for a for using a The efflux as previously (10Henkin A.H. Ortegon A.M. Cho S. Shen W.J. Falcon A. Kraemer A. for protein-mediated fatty acid efflux by Physiol. PubMed Scopus Google with OP9 cells were into a with Slc43a3 siRNA or siRNA a and for to the differentiation for OP9 cells. The cells were with of the lipid to in for the with of with and by were and the were for of the of were with and 1 The were for in a a OP9 cells were into and with Slc43a3 siRNA or siRNA for and Slc43a3 expression and as for a the cells were for to the differentiation for OP9 cells The cells were with of acid of from to in medium for the with of with were to the cells. were with and 1 or for from and in a A to the OP9 cells were for with or without 1 or in in and The were and for by and in the system. The using a A for the of the of free FAs, both protein and were using the FA OP9 cells were for with or without in in and were in and 1 with a for more were for The protein and free FAs were in with and 1 with or without 1 and oleic acid were as The of and and the of free FAs and the FAs that to were to the FA oxidation by the of acid into using a modification of a previously H. J. Shen W.J. M. A. S. J. of on expression of involved in lipid in liver of to of and Google Scholar). OP9 cells were into and with Slc43a3 siRNA or siRNA for and differentiation for the for OP9 cells cells were with or without in the of acid with 1 the with addition of of for The the and in a with cellular protein A to and OP9 cells were with or without 1 or in in and The were after and and the cells were using to of and levels were in cell as as the by using the to the The of production to the of A to cellular OP9 cells were for with or without 1 or in in and were and cellular levels were by the from the cells were in to and to the cell in a OP9 cells were with or without for 1 to were in a 1 1 and were for in were by acid and were rates of in cell were using of cell in the of or in and in a volume of The by the addition of the cell and by the addition of 1 as previously J. acyl-CoA synthetase expression in liver of via signaling Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). and were and the in a The in a that but cell as a negative fatty were using a previously reported W. J.J. A.M. of Chem. Biol. PubMed Scopus Google with OP9 cells were in and with The and The with of were 1 with and The with water and with of A of the for with are expressed as were by using for were considered significant is for patients to significant in insulin 1 bariatric before significant observed A. A. M. A. of PubMed Scopus Google but with high rates of In of that are involved in FA transport, any involved in FA efflux, we the expression levels of membrane transport proteins from the transporter family and 33 from the family in the of not have a known in the adipose tissue of patients bariatric surgery 1 the expression using of that significant before and 1 after bariatric surgery. the from the family showed significant after bariatric surgery and showed significant The function of family members have not yet expression levels of membrane transport proteins in patients before and after bariatric surgery significant transporter family transporter family in a the expression of the plasma membrane proteins in the family that showed basal expression levels and more after bariatric surgery in liver and various adipose in were and liver and fat tissues were for of expression by From Slc43a3 showed the most significant expression in and adipose tissue expression is to that are involved in of lipid in adipose tissue, and Further during the differentiation of OP9 from preadipocytes to adipocytes showed that expression of Slc43a3 increased and and and also the gene expression of specific plasma membrane proteins in primary were from and differentiated for 14 in Slc43a3 showed the most significant changes in expression and and and and Slc43a3 expression is in adipocytes in the cells from the fat evidence of expression in To the function of Slc43a3, we levels of Slc43a3 in OP9 cells by using siRNA or overexpression and the cells after of more of lipid in OP9 cells after differentiation with of Slc43a3, whereas there a significant in lipid with overexpression of Slc43a3 The expression levels of Slc43a3 in and cells were by and Slc43a3 siRNA decreased Slc43a3 expression with siRNA whereas of cells with increased Slc43a3 expression with The expression of the regulator for not significant changes with or overexpression of however, there a for expression to reduced with overexpression of In contrast, several involved in lipid droplet and were increased Slc43a3 and decreased Slc43a3 that overexpression of Slc43a3 have with adipocyte However, significant changes in levels were observed with or overexpression of Slc43a3 in or In contrast, the expression levels of several involved in and were increased Slc43a3 and decreased Slc43a3 FA uptake and efflux were in cells Slc43a3 or in of FA uptake showed that of Slc43a3 increased the rate of FA uptake whereas overexpression of Slc43a3 decreased the rate of FA uptake The of Slc43a3 on FA efflux also of Slc43a3 decreased the rates of FA efflux with cells basal conditions and FA efflux cells were with to and FA efflux FA flux were with a we to the efflux using cells with acid in and of OP9 cells with or increased both FA and In and to that observed using of OP9 cells in Slc43a3 by siRNA with or to FA efflux however, both of of Slc43a3 resulted in basal though FA and efflux, however, the of differentiation observed with overexpression of In of the increased expression levels of several involved in esterification observed with of Slc43a3, we the of to the increased FA and the decreased FA efflux with of Slc43a3 by acyl-CoA synthetase esterification the of FAs to fatty through the of the family of a fatty acid and via protein Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). in as using and as increased in OP9 cells in Slc43a3 with the increased expression of involved in However, we were not to any in cellular fatty in and Slc43a3 cells not of OP9 cells with resulted in of in both and Slc43a3 cells, though not as in Slc43a3 cells using as FA in and Slc43a3 cells that with of on FA uptake in cells. In contrast, Slc43a3 cells increased FA uptake in the of and of resulted in a but significant in FA uptake. a of the increased FA observed with Slc43a3 appears to to a of In to FA of on FA efflux in or Slc43a3 cells with of OP9 cells in Slc43a3 by siRNA to FA efflux FA by that in not to the in FA efflux with Slc43a3 Because of Slc43a3 to FA efflux, and not to to FAs to in the of Slc43a3 To we intracellular free FAs, both and protein in and Slc43a3 OP9 cells basal conditions and of with of cells with resulted in a significant in intracellular free FAs without a in protein free FAs, as previously reported A.M. acid transport in adipocytes by intracellular free fatty acid Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google most of the FAs efflux from the is important to that the of intracellular FAs in adipocytes are protein by FA binding proteins such as and A. proteins and 1997; PubMed Scopus Google and a very is of Slc43a3 cells with also resulted in a significant in intracellular free FAs to however, in to cells, there a significant in protein free FAs with a in FA efflux and the of the free FAs by To the in intracellular free FAs a metabolic we the rates of fatty oxidation in and Slc43a3 OP9 cells basal conditions and of with in and Slc43a3 OP9 cells, though there a for in Slc43a3 cells. increased to a in and Slc43a3 cells. CD36 is known to important in FA transport and such that there is decreased FA uptake in adipose tissue of CD36 expression levels of Slc43a3 in adipose tissues of CD36 in there is a significant of expression of Slc43a3 with in both the subcutaneous and adipose tissue of CD36 To we the expression of both Slc43a3 and CD36 in OP9 cells. OP9 cells were with Slc43a3 CD36 both Slc43a3 and CD36 siRNA the or with in that of CD36 the rate of FA uptake as with with M. uptake and of long chain fatty acids in muscle and adipose tissues of CD36 Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with of Slc43a3 increased the rate of FA uptake with both CD36 and Slc43a3 were the rate of FA uptake to that the of Slc43a3 are from In potential mechanisms through Slc43a3 cellular we changes in levels with or with overexpression of Slc43a3 Slc43a3 reported to involved in the cellular uptake of J. K. J. K. H. H. of as transporter involved in in PubMed Scopus Google Scholar). In levels of are intracellular the basal of among from to the changes in are as changes to the basal in levels were in cells Slc43a3 in cells or in cells with of Slc43a3 with and for however, there were among the cells to levels in and cells both and levels were increased by and in Slc43a3 cells with and Slc43a3 cells Therefore, of the in appears to a for in FA flux associated with of Slc43a3 levels were in Slc43a3 and Slc43a3 cells and decreased levels in cells. a cell with adipocytes take up and FAs different to the regulation of lipid in in of FA is a to the of such as and fatty liver obesity not a PubMed Scopus Google Scholar, and metabolic is adipose tissue the PubMed Scopus Google Scholar, Lipid and liver liver and fatty acid J. Physiol. Physiol. 2006; PubMed Scopus Google Scholar). surgery to the most of obesity and obesity related diabetes S. J. W.J. K. for the of and the 2013; PubMed Scopus Google Scholar, M. S. surgery and are there of PubMed Scopus Google Scholar, J. D. A. surgery to or of diabetes and significant of and PubMed Scopus Google Scholar). One of the is that by 1 most patients have insulin without A. A. M. A. of PubMed Scopus Google Scholar). Studies have that in to the changes of bariatric several of adipose tissue such as and have expression during the lipid storage and fatty oxidation H. S. N. K. of adipose lipid by expression and function of after bariatric J. PubMed Scopus Google Scholar). FA efflux to increased is In of metabolic for potential of FA transport in adipocytes showed that there are in adipocyte plasma membrane have functions, during of metabolic One of the transporters that showed increased expression after bariatric surgery The family of transporters is of amino acid system L transporters and and the transporter embryonic epithelia gene 1 S. A. M. J. of a amino acid transporter with system L Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. N. S. of a system L amino acid transporter from amino acid Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. and of a gene up-regulated in PubMed Scopus Google Scholar). and amino acid whereas is a of the family with amino acid with the as a gene expressed in a cellular model of A. D. a transporter expressed during with embryonic transporter expression during J. Physiol. Physiol. PubMed Google and the gene as S. D. M. The system amino acid transporters and the 2013; PubMed Scopus Google Scholar). However, specific function in adipose tissue and in lipid not yet to the current Slc43a3 to expressed during in liver and and reported to involved in the cellular uptake of J. K. J. K. H. H. of as transporter involved in in PubMed Scopus Google Scholar). of expression in showed that the expression of Slc43a3 in adipose tissue is to that in the liver and that is expressed in adipocytes as to cells adipose In vitro in the murine OP9 adipocyte cell and in primary preadipocytes showed that Slc43a3 expression is induced during the differentiation of of Slc43a3 resulted in increased rates of FA uptake and increased lipid in cells. of Slc43a3 decreased the rates of FA efflux with cells basal conditions and FA efflux cells were with or to and FA the efflux of by or not by of Slc43a3, the of Slc43a3 were specific for FA efflux and were observed or FAs were This FA and efflux is to a that but not efflux (10Henkin A.H. Ortegon A.M. Cho S. Shen W.J. Falcon A. Kraemer A. for protein-mediated fatty acid efflux by Physiol. PubMed Scopus Google that Slc43a3 have the of the of Slc43a3 in OP9 cells resulted in decreased rates of FA uptake and lipid in the cells with FA and however, Slc43a3 overexpression also resulted in reduced expression of and that overexpression have with adipocyte Slc43a3 appears to FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake. Studies to to potential mechanisms Slc43a3 rates of FA flux showed that of Slc43a3 resulted in a in forskolin-stimulated intracellular but changes in or changes were to the FA efflux, in of the efflux observed with or of Slc43a3 resulted in in forskolin-stimulated but changes in intracellular changes were to the in FA the overexpression of Slc43a3 any significant on cellular In of Slc43a3 not the expression of a of such as and known to involved in cellular FA and lipid we showed that the of Slc43a3 on FA uptake are of CD36 The through Slc43a3 on FA flux to and is the of the one a primary function of Slc43a3 to facilitate FA of intracellular free FA showed that of Slc43a3 cells resulted in a significant in intracellular free FAs to however, in to cells, there a significant in protein free is that adipocytes very high levels of such as and A. proteins and 1997; PubMed Scopus Google are of binding and the of intracellular This of intracellular free FAs can considered to with a of FA efflux in the of Slc43a3 and with the of the free FAs by the in intracellular free FAs not associated with any significant changes in the is that Slc43a3 is not involved in FA but the function of proteins involved in facilitating FA flux or the diffusion or flip-flop of FAs across the plasma In to we can that involved in the of the cell during FA functioning as a or of to the intracellular changes associated with the flux of FA Slc43a3 or to increased FA in This possibility is with the of the in expression of involved in and with the in Slc43a3 However, to and on the of FA efflux in the of Slc43a3 that not to the of Slc43a3 on FA of the increased FA uptake observed with Slc43a3 with a of the FA in the of Slc43a3 being related to cellular that of we that Slc43a3 FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake. the that a plasma membrane protein can the of a cell to FA a for a understanding of the FA flux in cells. The Long and for of cellular fatty with

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