Slc43a3 is a regulator of free fatty acid flux

Adipocytes take up long chain fatty acids through diffusion and protein mediated transport, whereas fatty acid efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating fatty acid 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 fatty acid efflux, while also increasing fatty acid uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased fatty acid uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate fatty acid flux in adipocytes, functioning as a positive regulator of fatty acid efflux and as a negative regulator of fatty acid uptake. Patient sample collection and RNA preparation : Patients undergoing an elective VSG (vertical sleeve gastrectomy) procedure were recruited and informed consent was obtained from each study participant. Inclusion criteria included men and women older than 21 years of age with a BMI (cid:305) 35.0 kg/m 2 , and meeting criteria by American Diabetes Association standards for pre-diabetes or T2DM. For laparoscopic VSG surgery, a 4.8 mm stapler load was used to divide the greater curvature of the stomach 5 cm from the pylorus and remaining 3 cm from the angularis incisura . 3.5 mm stapler loads were fired thereafter progressing up to the angle of His to complete the VSG. Details of the surgical procedures have been described by Jahansouz et al. Demographic data on sex, age, and T2DM were collected for obese patients at the time of surgery and seven days following bariatric surgery. Weight and height were measured immediately prior to surgery (preop) and during the postoperative visit. Body mass index (BMI) was calculated as weight (kg) divided by height (m 2 ). Additional details as to the molecular changes occurring following bariatric surgery have published by Jahansouz et al. The University and St. Institutional Review Boards approved all investigations and informed consent was obtained from each participant. Abdominal subcutaneous adipose tissue biopsies from patients were processed for tissue analysis.


Introduction
Fatty acids (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 (1)(2)(3). 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 FA for most tissues (4). 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 (5). The rates of cellular FA uptake are also regulated acutely and chronically by hormones (such as insulin) and metabolic status (in case of obesity) (6,7).
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 (8). 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 (7). 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 fatty acid transport proteins (FATPs), long chain fatty acyl coenzyme A synthases (ACSLs) and fatty acid translocase (FAT, also known as CD36), in the protein-facilitated process of FA transport.
However, the mechanisms how fatty acids traverse the plasma membrane to enter the soluble cytoplasm are not yet fully understood (9). Specifically, there is a debate on the rate-limiting step in the overall process of FA uptake and if, and to what extent, one or more membrane-associated proteins could facilitate and/or regulate FA uptake. In contradistinction, fatty acid 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 (10). by guest, on May 8, 2020 www.jlr.org

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To find possible candidates for these mechanisms we performed an in-silico study of all transporter genes fulfilling the following criteria: unknown function, plasma membrane location and significant expression in adipose tissue. One of the transporter gene families that comes into focus is the family of the ATP-binding cassette (ABC) transporters, of which almost half are thought to facilitate the ATP-dependent translocation of lipids or lipid-related compounds (11)(12)(13). Another family of potentially relevant membrane transport proteins is the family of the Solute Carriers (SLC), with over 400 members, most of which are located in the cell membrane (14)(15)(16)(17).
In our present study we investigated the mRNA expression levels of gene candidates from our in-silico study in patients before and after bariatric surgery to test the hypothesis that the expression of proteins linked to fatty acid efflux might be upregulated. Genes that showed a significant change in their expression level were then studied in OP9 murine adipocyte cells and primary adipocytes throughout differentiation. One of the interesting and promising genes identified was the gene for the Slc43a3 solute carrier. The expression levels of Slc43a3 in various mouse adipose depots and during adipocyte differentiation were explored in C57Bl6J mice and OP9 murine adipocyte cells. The functional significance of Slc43a3 in regulating fatty acid transport was studied using overexpression or silencing of Slc43a3 in differentiated OP9 cells. Interestingly, knockdown of Slc43a3 reduced both basal and forskolin-stimulated fatty acid efflux. Moreover, knockdown resulted in an increased uptake of FAs into cells, whereas overexpression of Slc43a resulted in a decreased uptake of FAs. Thus, we conclude that Slc43a3 regulates fatty acid flux in adipocytes, functioning as a positive regulator of fatty acid efflux and as a negative regulator of fatty acid uptake.
Animals-Wildtype C57Bl6/J and CD36 knockout (KO) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and were used for the collection of liver and fat tissues.
Mice were housed in the animal facility at the VA Palo Alto Health Care System on a 12/12 h light/dark cycle. All procedures were in accordance with institution guidelines and approved by Quencher-based free fatty acid efflux assay: The efflux assay was conducted as previously described (10)  BSA in HBSS (1x) at 37°C and 5% CO2. The incubation media were collected and assayed for glycerol content by adding glycerol kinase and glycerol phosphate oxidase in the assay system.
The resulting colorimetric product was quantified at 570 nm using a plate reader (Molecular Devices 5 ME, Sunnyvale, CA, USA). A standard curve was generated for the calculation of the quantity of glycerol released.
Intracellular free fatty acid measurement: Intracellular free fatty acids, both protein bound and unbound, were measured using the ADIFAB2 Fatty Acid Indicator. Differentiated OP9 cells were incubated for 60 minutes with or without 100 nM isoproterenol in 1% BSA in HBSS (1x) at 37°C and 5% CO2. Cells were homogenized in 10 mM Tris, pH 7.4, 1 mM EDTA with a glass homogenizer for more than 10 times. Cell lysates were centrifuged at 14,000 x g for 10 minutes.
The protein bound and unbound free fatty acids were assayed in 50 mM HEPES with 140 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, pH 7.4 with or without 1 M ADIFAB2 indicator, and 5 nM oleic acid:BSA used as standard. The ratio of fluorescence emission at 550 nm and 457 nm was measured, and the amount of aqueous free fatty acids and the fatty acids that bound to ADIFAB were calcualted according to the manufacturer's instructions.
Fatty acid oxidation assay: Cellular fatty acid oxidation (FAO) activity was determined by following the conversion of 3H-palmitic acid into 3H2O using a modification of a previously described procedure (20). OP9 cells were seeded into 12-well plates and transfected with Slc43a3 Pre-designed Silencer Select siRNA or scrambled siRNA control for knockdown. After transfection and differentiation for 5 days following the protocol for OP9 cells (described above),  Statistics: Data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA using Prism 6.02 for Mac OS X (GraphPad Software, Inc., La Jolla, CA, USA).
Differences between groups were considered statistically significant when P < 0.05.

mRNA expression levels of membrane transport proteins in patients before and after bariatric surgery
It is common for patients to show significant improvement in insulin sensitivity one-week post bariatric surgery, before significant weight loss has been observed (23), but coincident with high rates of fatty acid oxidation. In search of genes that are potentially involved in FA transport, particularly any potentially involved in FA efflux, we examined the mRNA expression levels of 22 membrane transport proteins from the ABC transporter family and 33 from the SLC family (see list in Supplemental Table S1), which at the time of study did not have a known ligand, in the adipose tissue of patients post bariatric surgery at this critical time. Table 1 shows the expression levels, determined using branched DNA analysis, of genes that displayed statistically significant differences before and one-week after bariatric surgery. Of the genes examined, eight genes from the SLC family showed significant increases after bariatric surgery and six showed significant decreases. The function of these SLC family members have not yet been determined.

mRNA expression of membrane transporters in OP9 cells and mouse tissues
We then analyzed the mRNA expression of the six plasma membrane proteins in the SLC family that showed substantial basal expression levels and more than 1.5 fold increase after baratric surgery in liver and various adipose depots in mice. For this analysis, four months old male wild type C57Bl6J mice were sacrficed and their liver and fat tissues were collected for analyis of mRNA expression by RT-qPCR. From all analyzed genes Slc43a3 showed the most significant expression in epididymal, subcutaneous and brown adipose tissue ( Figure 1A). Its expression pattern is comparable to genes that are involved in control of lipid homeostasis in adipose tissue, ie. Plin1, Hsl and Fabp4. Further analysis during the differentiation of OP9 from pre-adipocytes to adipocytes showed that expression of Slc43a3 increased significantly between day 0 and day 9 (P<0.01) and between day 3 and day 9 (P<0.05) ( Figure 1B). We also analyzed the gene expression of these specific plasma membrane proteins in primary preadipocytes, which were isolated from wildtype mice, and differentiated for 14 days. As shown in Figure 1C, Slc43a3 showed the most significant changes in expression throughout differentiation: between day 0 and day 3 (P<0.05), between day 3 and day 9 (P<0.001) and between day 0 and day 9 (P<0.001). Slc43a3 mRNA expression is ~ eight fold higher in adipocytes than in the stromal vascular cells isolated from the same fat pad ( Figure 1D), further evidence of its preferential expression in adipocytes.

Effects of Slc43a3 knockdown and overexpression on free fatty acid transport
To characterize the function of Slc43a3, we manipulated levels of www.jlr.org

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When FA influx was examined in control and Slc43a3 siRNA treated cells that had been pretreated with triacsin ( Figure 4B), inhibition of ACSL activity had no effect on FA uptake in control cells. In contrast, Slc43a3 siRNA treated cells displayed increased FA uptake in the absence of triacsin (P<0.001), and inhibition of ACSL activity resulted in a small, but significant (P<0.01), reduction in FA uptake. Thus, a portion of the increased FA influx observed with Slc43a3 knockdown appears to be due to a stimulation of esterification. In contradistinction to FA influx, inhibition of ACSL activity had no effects on FA efflux following lipolytic stimulation in either control or Slc43a3 siRNA treated cells ( Figure 4C). Consequently, treatment with isoproterenol of OP9 cells in which Slc43a3 had been knocked down by siRNA failed to stimulate FA efflux even when FA re-esterification had been inhibited by triacsin, suggesting that an increase in re-esterification does not contribute to the block in FA efflux with Slc43a3 knockdown.
Since knockdown of Slc43a3 appeared to block lipolytic-stimulated FA efflux and reesterification did not appear to contribute to this, FAs would be expected to increase intracellularly following lipolysis in the setting of Slc43a3 knockdown. To assess this directly, we measured intracellular free FAs, both unbound ( Figure 5A) and protein bound ( Figure 5B

Expression of Slc43a3 in CD36 knockout mice
CD36 is known to be important in FA transport and regulation, such that there is decreased FA uptake in adipose tissue of CD36 KO mice. We, therefore, examined expression levels of Slc43a3 in adipose tissues of CD36 KO mice. As shown in Figure 6A, there is a  Figure 6B show that knockdown of CD36 decreases the rate of FA uptake as compared to control, consistent with prior observations (27). Compatible with prior data (Figure 2), knockdown of Slc43a3 again increased the rate of FA uptake compared to control. Interestingly, when both CD36 and Slc43a3 were knocked down, the rate of FA uptake was similar to control, suggesting that the effects of Slc43a3 are mediated independently from CD36.

Effects of Slc43a3 on cAMP and ATP levels
In exploring potential mechanisms through which Slc43a3 might influence cellular homeostasis, we examined changes in cAMP levels with either knockdown or with overexpression of Slc43a3 since Slc43a3 has been reported to be involved in the cellular uptake of extracellular purine nucleobases (28). In general extracellular levels of cAMP are ~ 10 fold overexpressing cells (P<0.01) ( Figure 7C). Therefore, none of the small differences in cAMP appears to provide a mechanistic basis for alterations in FA flux associated with manipulation of Slc43a3 expression. Cellular ATP levels were similar in control, Slc43a3 knockdown, and Slc43a3 overexpressed cells ( Figure 7C). Forskolin and isoproterenol decreased ATP levels similarly in all cells.

Discussion
As a cell with endocrine function, adipocytes actively take up and release FAs under different physiological conditions, contribute to the regulation of lipid homeostasis, and, in turn, participate in whole body metabolism. Dysregulation of FA metabolism is a major factor contributing to the development of disorders such as diabetes, cardiovascular disease and nonalcoholic fatty liver disease (29)(30)(31).
Bariatric surgery has been shown to be the most effective treatment of obesity and obesity related type 2 diabetes (32-34). One of the interesting observations is that by one-week post-surgery, most patients have improved insulin sensitivity without substantial weight loss (23). Studies have shown that in response to the major changes of bariatric surgery, several main regulators of adipose tissue metabolism, such as PPARγ, PPARδ and UCP2, have altered expression during the first week post-surgery, attenuating lipid storage and promoting fatty oxidation (19). Indeed, FA efflux would be expected to be markedly increased at this time since caloric intake is substantially reduced.
In view of these dramatic metabolic changes, our search for potential regulators of FA transport in adipocytes showed that there are many alterations in adipocyte plasma membrane transporters, which currently have unknown functions, during this first week of metabolic transition. One of the transporters that showed increased expression after bariatric surgery was Slc43a3. The SLC43 family of transporters is composed of only three members: two amino acid system L transporters (LAT3 and LAT4) and the orphan transporter EEG1 (embryonic epithelia gene 1) (35)(36)(37). Human LAT3 and LAT4 share ca. 57% amino acid sequence identity, whereas human EEG1 is a distant member of the family with only ca. 27% amino acid sequence identity with the other two members. Mouse Eeg1 was identified as a gene expressed in a cellular model of renal tubulogenesis (38) and the human gene has been assigned as SLC43A3 (39). However, its specific function in adipose tissue and its involvement in lipid metabolism had not yet been investigated prior to the current studies. Slc43a3 was shown to be expressed during embryogenesis in liver and lung and has recently been reported to be involved in the cellular uptake of extracellular purine nucleobases (28).
Our analysis of mRNA expression in normal adult control mice showed that the expression of Slc43a3 in adipose tissue is similar to that in the liver and that it is expressed predominantly in adipocytes as opposed to stromal vascular cells within adipose depots. In vitro study in the murine OP9 adipocyte cell line and in primary mouse preadipocytes showed that However, experiments utilizing triacsin to inhibit ACSL activity, and thus (re)esterification, had no effects on the blockade of lipolytic-stimulated FA efflux in the setting of Slc43a3 knockdown, suggesting that (re)esterification does not contribute to the effects of Slc43a3 on FA efflux.
Nonetheless, triacsin did decrease some of the increased FA uptake observed with Slc43a3 knockdown, consistent with at least a portion of the elevated FA influx in the setting of Slc43a3 knockdown being related to cellular responses that increase esterification. Whatever its precise mechanism of action, we conclude that Slc43a3 regulates fatty acid flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.
Moreover, the demonstration that manipulating a plasma membrane protein can dramatically affect the ability of a cell to export FA provides a new direction for gaining a deeper understanding of the processes mediating FA flux in mammalian cells.   Results are representative of 2 independent experiments, each with n=4-6. * p<0.05.        www.jlr.org