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Human adipose tissue gene expression of solute carrier family 19 member 3 (SLC19A3); relation to obesity and weight‐loss

Abstract Objective Adipose tissue is a specialized endocrine organ that is involved in modulating whole‐body energy homeostasis and expresses a specific subset of genes, which may play a role in adipose tissue metabolism. The aim of this study was to search for novel adipose tissue‐specific genes using a tissue panel of RNAseq expression profiles. Methods RNAseq expression profiles from 53 human tissues were downloaded from the GTex database. SLC19A3 expression was analyzed by microarray or real‐time PCR in two sets of paired subcutaneous and omental adipose tissue samples, in two studies with adipose tissue from persons with high or low body mass index (BMI), in adipose tissue from patients who underwent weight loss with a very‐low caloric diet and during preadipocyte‐adipocyte differentiation. Results The RNAseq‐based tissue distribution expression screen identified SLC19A3 (encoding the thiamine transporter 2) as adipose tissue‐specific. SLC19A3 expression was higher in subcutaneous compared with omental adipose tissue in both sample sets (p = 0.043 and p < 0.001). Preadipocyte differentiation towards adipocytes resulted in increased SLC19A3 gene expression (p = 0.018 or less at all‐time points). Subcutaneous adipose tissue expression of SLC19A3 was lower in persons with high BMI in both cohorts (p = 0.008, and p < 0.001) and increased during a weight‐loss intervention (p = 0.006). Conclusion The specific adipose tissue expression pattern of SLC19A3, together with its regulation in obesity and during weight loss, indicate that it plays a key role in adipocyte metabolism.


| INTRODUCTION
The central function of adipose tissue and adipocytes is to control lipid metabolism. 1 In conditions of excess nutrients, the adipocyte store energy in the form of lipids, whereas in the post-prandial period or during starvation, it supplies lipids to other tissues through lipolysis. 2 In response to energy overconsumption, the adipose tissue is expanded through an increase in the number and/or size of adipocytes. 3 Adipose tissue expansion also affects the cellular composition and function of the stromal vascular fraction cells of the adipose tissue. This includes macrophage infiltration and low-grade adipose tissue inflammation, 4 which has been shown to contribute to obesityrelated metabolic comorbidities. 4 Research during the last 3 decades has described many additional specific functions of adipose tissue. Adipose tissue is currently defined as an endocrine organ with secretion of signaling molecules, so-called adipokines, from adipocytes. 5,6 Many of these adipokines play key roles in the regulation of energy homeostasis and contribute to obesity-related metabolic disorders. 7 In addition to the regular white adipose tissue, humans also have smaller amounts of brown adipose tissue. 8 The key feature of brown adipose tissue is the generation of heat via the uncoupling of the respiratory chain 8 . Clearly, also brown adipose tissue expresses a set of unique genes which are related to its unique function, and uncoupling protein 1 (UCP1) is the clearest example of this. 8 The specific functions of the adipocytes and adipose tissue are mediated by proteins that, in many cases, are specifically expressed in the adipocytes. Hence, adipose tissue and adipocytes have distinct expression profiles, and some genes are uniquely expressed by adipocytes. Adipose tissue dysfunction has been linked to several obesity-related comorbidities, and adipocytespecific genes have been shown to play a part also in these comorbidities. 5,6 Analysis of mRNA expression levels has been used to gain insights into the function of genes, and the development of techniques such as microarrays has enabled screening-based approaches to find genes of relevance for many different functions in the human body and different pathological conditions. Microarray-based methods have previously been used to search for genes with a tissue/cell typespecific expression pattern. 9 This methodology is based on the comparison of expression profiles from multiple human tissues and cell types. Our group has used this methodology for the identification of several novel adipocyte/adipose tissue-specific genes, including CIDEA, CIDEC, NQO1, TNMD, SAA1 and ITIH5. [10][11][12][13][14][15] The rationale behind this approach is that genes that are exclusively expressed in a specific tissue most likely have important functions in that tissue.
Dysregulation of genes with key functions in adipose tissue may be linked to obesity-related metabolic dysfunction. RNA sequencing (RNAseq) has now emerged as an advantageous tool for expression profiling, compared to microarrays, since it is not limited by the probe composition of the microarray. 16 RNAseq data from multiple human tissues and cell types can potentially also be used in searches for novel adipose tissue-specific genes.
This study hypothesized that adipose tissue express several unique genes that play key roles in adipose tissue function and that dysregulation of such genes may be linked to obesity-related metabolic dysfunction. This study aimed to use an RNAseq dataset with multiple human tissues to search for novel adipose tissue-specific genes and to characterize the expression pattern of such genes.

| Gene expression dataset and search for adipose tissue-specific genes
Tissue distribution of gene expression was analyzed in a publicly available RNAseq dataset, downloaded from the GTEx portal (Version 7, https://www.gtexportal.org/home/datasets). The RNAseq dataset contains 53 tissues and cell types, and data for each tissue/cell type originate from analysis of 5 to 564 samples, but the replicates of each sample were combined into a mean expression profile for each tissue or cell type. The dataset contains expression profiles from both subcutaneous and omental adipose tissue. Gene expression levels were calculated as transcripts per kilobase million (TPM). To identify genes specifically expressed in subcutaneous adipose tissue, genes that had an expression level (TPM) that was at least 10 standard deviations (SD) higher than the mean TPM of all the other tissues and at least 5-fold higher TPM than the tissue with the second-highest TPM (second tissue) were selected. In these analyses, expression profiles for omental adipose tissue and mammary tissue were excluded due to the high degree of similarities to subcutaneous adipose.
A second publicly available RNAseq dataset (HPA RNA-seq normal tissues, https://www.ncbi.nlm.nih.gov/gene/80704) was also analysed since this dataset contained tissues not included in the first dataset. 17 This dataset contains samples from 95 human individuals representing 27 different tissues (2-7 samples analyses per tissue), and gene expression levels were calculated as reads per kilobase million (RPKM).

| The SibPair parents (SPP) study
The SibPair study was designed to study the genetics of obesity and consists of 154 nuclear families with BMI discordant sib-pairs (BMI difference > 10 Kg/m 2 ). 19 In total, 732 subjects were recruited from all over Sweden and examined at the Obesity Unit, the Sahlgrenska University Hospital in Gothenburg. In the current study, subcutaneous adipose tissue needle biopsies were obtained from the parents in the SibPair study (n = 88) 20 and were used for the analysis of adipose tissue gene expression. This group was also stratified by BMI into individuals being either of normal weight (BMI 18.5-25 Kg/m 2 ; n = 17) or with obesity (BMI > 30 Kg/m 2 ; n = 32).

| Subcutaneous adipose tissue study
Subcutaneous adipose tissue needle biopsies were obtained by needle aspiration of the lower part of the abdomen from a cohort of subjects without diabetes (52 female, 20 male; BMI 26.1 ± 2.9 Kg/ m 2 ) at the Uppsala University Hospital. The adipose tissue was used for mRNA extraction (n = 68), and isolation of adipocytes and stromal vascular fraction (n = 4) and expansion of preadipocytes into adipocytes (n = 8). Subjects with type 1 diabetes, endocrine disorders, cancer or other major illnesses were excluded, as so were those having ongoing medication with systemic glucocorticoids, betablockers and immune-modulating therapies.

| The very low-calorie diet (VLCD) study
As previously described, 10

| Perirenal adipose tissue study
Biopsies of perirenal adipose tissue were obtained from 55 healthy kidney donors at the Sahlgrenska University Hospital in Gothenburg. The samples were screened for UCP1 gene expression, and samples with high UCP1 expression were classified as containing brown adipose tissue (BAT, n = 10). A matched control group (n = 10) was created from the samples displaying no/low UCP1 expression. 21

| Adipose tissue sampling and RNA preparation
Adipose tissue, obtained by needle aspirations or by surgical biopsies, was snap-frozen and stored at −80°C until analysis. Total RNA was prepared using the RNeasy lipid tissue kit (QIAGEN, Chatsworth, CA), or using the phenol-chloroform extraction method of Chomczynski and Sacchi with further purification with RNeasy clean-up columns.

| Microarray analysis
Microarray analysis of paired subcutaneous and omental adipose tissue samples and adipose tissue samples from the SibPair parents study was performed using U133 plus 2.0 microarray (Affymetrix, Santa Clara, CA). Microarray analysis of adipose tissue from the VLCD study was performed using U133A microarray (Affymetrix), and perirenal adipose tissue was analyzed using Gene 1.0 ST microarrays (Affymetrix). All microarray analysis was performed according to the manufacturer's instructions (Affymetrix), and expression data were analyzed using the Robust Multi-array Average algorithm.
SLC19A3 expression was assessed using probe sets 220736_at and 8059538 for U133 microarrays and Gene 1.0 ST microarrays, respectively.

| Real-time PCR
The quantity and quality of the isolated RNA were determined by PPARG (Hs1115513). All samples were run in duplicate, and the data were analysed using 2 −deltaCt using 18S or GUSB as a reference gene.

| Isolation of human adipocytes and stromal vascular cells
Adipocytes and stromal vascular cells were isolated from subcutaneous adipose tissue for preadipocyte differentiation into adipocytes and analyses of SLC19A3 gene and protein expression. The isolation of preadipocytes was performed as previously reported. 22 In brief, adipose tissue was digested with collagenase (1.2 mg/ml, from  The cells were used to determine the SLC19A3 gene and protein expression, and the differentiation rate was confirmed by measuring the expression of the adipogenic markers PPARG and CEBPA.

| Data analysis and statistics
Enrichment analysis was performed using the online David functional annotation tool (version 6.8) using default settings. 23,24 The top 500 genes that positively correlated with SLC19A3 expression in human subcutaneous adipose tissue (SibPair parents, n = 88) were selected, and these 500 genes correspond to 358 unique David IDs. Enrichment analysis was performed with Human Genome U133 plus set as background.
The difference in gene expression in paired subcutaneous and omental adipose tissue biopsies was analyzed using related-samples Wilcoxon signed-rank test. The difference in gene expression in brown adipose tissue and between subjects with differences in BMI was performed using independent sample Mann-Whitney U test or Spearman's rank correlation. Gene and protein expression during adipocyte differentiation and expression during caloric restriction was analyzed by related-samples Friedman's Two-Way Analysis of Variance by rank followed by Wilcoxon signed rank test as post-hoc test. Two-sided p values less than 0.05 were considered statistically significant. Statistical analysis was performed using IBM SPSS statistics 25.

| Ethics approval
The Regional Ethics Committee in Gothenburg, Uppsala and the South Birmingham Ethics Committee approved these studies. All study subjects received written and oral information about the respective study before giving informed consent.

| RNAseq-based search for genes specifically expressed in subcutaneous adipose tissue
The RNAseq based search for adipose tissue specific-genes yielded subcutaneous adipose tissue using the predefined criteria (Table 1).
Classical adipocyte-specific genes such as leptin (LEP), adiponectin (ADIPOQ), Perilipin 1 (PLIN1) and fatty acid binding protein 4 (FABP4) were classified as the top four on the list of genes specifically expressed in subcutaneous adipose tissue (Table 1). Genes previously identified as adipose/adipocyte-specific genes in microarray-based scans, such as CIDEA, 10 CIDEC 11 and TNMD, 13 were also included on the list. A novel finding from this analysis was that the thiamine transporter 2 (THTR2, encoded by the solute carrier family 19 member 3 [SLC19A3] gene) 25 was specifically expressed in adipose tissue and was therefore selected as a focus for further investigation.

| Tissue distribution of thiamine transporters in human tissues
The complete tissue distribution of SLC19A3 expression in the GTEx dataset is shown in Figure 1A. In this dataset, SLC19A3 was highly expressed in visceral and subcutaneous adipose tissue and in mammary tissue. Similar tissue distribution of SLC19A3 mRNA expression was observed also in an additional RNAseq dataset (Supplemental Figure 1). In this dataset, adipose tissue displayed the highest SLC19A3 expression levels, followed by placenta and duodenum.
Higher expression of SLC19A3 in subcutaneous adipose tissue compared with omental adipose tissue was observed in the microarray dataset with paired biopsies from 5 lean women ( Figure 1B

| SLC19A3 expression in adipocytes and during adipocyte differentiation
SLC19A3 mRNA levels were very low in human preadipocytes but increased during the adipocyte differentiation process (p = 0.0014 for differences among repeated differentiation days), and higher

| Adipose tissue expression of SLC19A3 in obesity and during weight-loss
SLC19A3 expression was lower in subcutaneous adipose tissue from subjects with obesity compared with subjects of normal weight  In a weight-loss study (VLCD study) with 24 participants, subcutaneous adipose tissue SLC19A3 mRNA expression was increased during 16 weeks of energy restriction (p = 0.006 after 16 weeks, Figure 4). However, when regular food was gradually re-introduced during the last two weeks of the intervention (between week 16 and 18), the mRNA expression of SLC19A3 was reduced (p = 0.002) almost to the level as before the intervention.

| Function of SLC19A3 in adipose tissue
To gain insights of the potential function of SLC19A3 in adipose tissue, a gene set enrichment analysis was performed. The top 500 genes with a positive correlation to SLC19A3 expression in the subcutaneous adipose tissue in the SibPair parents cohort (n = 88) were used for enrichment analysis. Terms that were enriched in this analysis were dominated by links to the mitochondrion (Table 2). In addition, significantly enriched terms included lipid metabolism and branched-chain amino acid catabolism (Table 2).

| DISCUSSION
This study showed that the thiamine transporter SLC19A3 was specifically expressed in human adipose tissue and that adipose tissue SLC19A3 expression was regulated by obesity, weight-loss and possibly energy intake. Furthermore, an enrichment of genes with mitochondrial function was observed among the genes that correlated with the SLC19A3 adipose tissue expression. Taken together, these results indicate that SLC19A3 plays a role in adipose tissue energy metabolism.
Duodenum has been describes as the main site of thiamin uptake in the body. 26 The gene product of SLC19A3 has been suggested to mediate this uptake due to its high expression in duodenum. 26 Tissue distribution datasets that contain both adipose tissue and duodenum indicated that the expression levels of SLC19A3 were several-fold higher in adipose tissue compared with the duodenum. This tissue distribution pattern does not speak against a key role for SLC19A3 in intestinal thiamine uptake, but it highlights the very high expression which was observed in adipose tissue.

F I G U R E 2
Expression of SLC19A3 during adipocyte differentiation and in isolated adipocytes. Human preadipocytes were differentiated towards adipocytes for 14 days, and SLC19A3 expression was analyzed by real time PCR (A) and western blot (B and C). Data are presented as mean expression and SD, and the experiment was performed with preadipocytes from 8 and 3 donors, respectively. Differentiation of SGBC cells towards adipocytes and analysis of SLC19A3 expression before and after 14 days of differentiation (D). Western blot analysis of SLC19A3 protein expression in isolated adipocytes and stromal vascular fraction cells (SVC) (E and F) Thiamine has its main function as a cofactor of the enzyme pyruvate dehydrogenase, an enzyme active in the citric acid cycle. 27 Hence, decreased thiamin levels can result in reduced mitochondrial activity, impaired oxidative metabolism, and decreased energy production. 27 Skeletal muscle and brown adipose tissue are tissues with high energy demand and an abundance of mitochondria, whereas white adipose tissue has relatively few mitochondria. 28 Functional studies 27  However, the current study indicated that SLC19A3 protein was also expressed in the stromal vascular fraction of adipose tissue.
Ahn et al. 31  Human perirenal adipose tissue was analysed for UCP1 expression and stratified into two groups, samples containing (BAT, n = 10) or not containing (WAT, n = 10) brown adipocytes. Data are presented as a mean expression with SD. ns; not significant