Taurine Transporter Regulates Adipogenic Differentiation of Human Adipose-Derived Stem Cells through Affecting Wnt/β-catenin Signaling Pathway.

Increased adipocytes are associated with obesity and many human disorders including cancers. To further understand the molecular mechanisms of adipogenesis, transcriptome sequencing was performed to find genes involved in the adipogenic differentiation of human adipose-derived stem cells (hASCs). The mRNA of taurine transporter (TauT, also known as SLC6A6) was found significantly upregulated in hASCs undergoing differentiation. TauT expression was also markedly increased in fat tissues from obese mice induced by high fat diet or genetic mutations (ob/ob and db/db mice). In vitro, downregulation of TauT attenuated effectively the adipogenic differentiation of hASCs, and TauT overexpression promoted the formation of adipocytes. Among the molecules transported by TauT, hypotaurine and β-alanine promoted adipocyte formation, whereas taurine inhibited the process. Moreover, the inhibitory effect of TauT knockdown on hASCs differentiation was largely reversed by hypotaurine and β-alanine through promoting the downregulation of β-catenin. These results indicated that TauT regulate adipocyte formation through transported amino acids and may serve as a target for therapeutic intervention of obesity.

Introduction

In addition to provide thermal insulation and protective padding, and to store excess glucose in the form of triglycerides, adipose tissue has been increasingly recognized as an active endocrine organ. It secretes a number of hormone-like adipokines that play essential roles in energy balance and homeostasis of glucose, lipids, and systemic inflammation, . Noteworthily, the predominantly adipose-produced leptin acts on receptor in the arcuate nucleus of the hypothalamus to inhibit food intake. Dysfunction of leptin or its receptor results in obesity in mice (ob/ob and db/db mice) and human, whereas most of the obese individuals have increased leptin levels and develop leptin resistance .

In contrast to most tissues, adipose tissue exhibits uniquely an almost unlimited capacity to expand. Overweight and obesity, manifested by abnormal or excessive adiposity that are usually defined by body mass index, has been recognized as a global epidemic since the end of last century. Obesity is often associated with altered steroid hormone and adipokines production, insulin resistance, dyslipidemia, and subclinical chronic inflammation, and contributes to cancer initiation, progression, and metastasis. Although the mechanisms linking increased adiposity to malignancy are incompletely understood, it is evident that adipose tissue-derived cytokines and adipose cells in tumors all play important roles in cancer development.

The excessive adiposity in adipose tissue results from increased adipocyte number, adipocyte hypertrophy, and metabolic dysfunction. It has been shown that precursors of adipocytes, often designed as adipose-derived stem cells (ASCs), are capable of differentiating into adipocytes, and presumably play a critical role in the increase and dysfunction of adipocytes in obese individuals To explore the molecular mechanisms involved in adipocyte differentiation, we isolated hASCs from subcutaneous adipose tissues and performed transcriptome sequencing. Taurine transporter (TauT, also known as SLC6A6) was found significantly increased during the early stage of adipogenisis and in fat tissues from obese mice. TauT transported molecules hypotaurine and β-alanine promoted adipogenic differentiation, whereas taurine and TauT knockdown impeded adipogenic differentiation of hASCs in culture. Further analysis revealed that TauT appears required for the downregulation of β-catenin during adipocyte formation. Thus, the taurine transporter plays an important role in adipogenesis, and may serves as a target for therapeutic intervention of obesity.

Materials and Methods

Isolation of human adipose-derived stem cells

This study was approved by the Ethical Review Board of the First affiliated hospital of Soochow University. Human adipose tissue was collected from patients aged 20 to 35 years old. After surgery, the adipose tissues were immediately transported to the laboratory in sterile PBS buffer containing 100 U/ml penicillin and 100 μg/ml streptomycin on ice. After 2x washes, the adipose tissues were minced using sterile scalpels and scissors and then digested with 0.1% type I collagenase in a shaking water bath at 37 °C for 30 min. After digestion, the tissue was filtered with a 70μm-mesh sieve, and the filtrate was centrifuged at 1000 x g for 10 min. Cells obtained from the pellet were cultured with DMEM/F-12 containing 10% FBS. These cells were identified as CD34-/CD73+/CD105+ by flow cytometry. After 3 passages, the cells were frozen in liquid nitrogen for further experiments.

Cell culture and adipogenic differentiation

Human adipose-derived stem cells (hASCs) medium consisted of DMEM/F-12 Medium with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2, 95% air atmosphere. For experiments, cells were seeded and grown to confluence for 2 days. Adipocyte differentiation was induced by the pro-adipogenic components Insulin (10 nM), rosiglitazone (100 nM), 3-isobutyl-1-methylxantine (IBMX, 100 μM) and dexamethasone (Dex, 1 μM) for 3 days. Cells were then switched to adipogenesis progression inducer Insulin (10 nM), rosiglitazone (100 nM), and dexamethasone (Dex, 1 μM) for 2 days. Finally, cells were maintained with Insulin (10 nM).

Plasmid construction and siRNA transfection

To construct the pLVX- TauT plasmid, a fragment of the TauT cDNA (GenBank accession number: NM_003043.5) was amplified and cloned into the lentivirus vector pLVX. TauT siRNAs sequence: siRNA#2 sense: 5-GCUAUGCCUCCGUUGUAAUTT-3, antisense: 5- AUUACAACGGAGGCAUAGCTT-3; siRNA#3 sense: 5-GGAACACACCUCACUGCAT-3, antisense: 5-AUGCAGUGAGGUGUGUUCCTT-3. The TauT ectopic-expression plasmid and siRNAs were transfected into hASCs every 3 days.

Analysis of lipid accumulation

Staining of intracellular lipids by Oil Red O was performed as described previously . To quantify intracellular triglyceride accumulation, cells were washed with PBS twice. In order to obtain quantitative data, 500 μl of isopropyl alcohol was added to the stained culture dish. After 5 minutes, the absorbance of the extract was assayed by a spectrophotometer at 510 nm.

Animal experiments

Twelve-week-old wild type, ob/ob and db/db mice were purchased from Shanghai Laboratory Animal Center. The animals were operated according to the protocol approved by the Institutional Animal Care and Use Committee of the Suzhou Institute of Systems Medicine. Eight-week-old mice were fed with high fat diet for 2 months. Mice were sacrificed and subcutaneous and visceral mouse fats tissues were excised and extracted for total RNA.

RNA extraction and real-time PCR

The culture medium was removed, and the cells were immediately washed with ice-cold PBS. Subsequently, 1 ml of TRIzol reagent was added, and total cellular RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method. Total RNA (1 μg) was used as a template for an MMLV-RT reverse transcriptase reaction, which was performed according to the manufacturer's instructtions. Real-time quantitative reactions were set up in triplicate in a 96-well plate, and each reaction contained 1 μl of cDNA and the SYBR Green PCR mix, to which gene-specific forward and reverse PCR primers were added. Melting curves were analysed to verify the specificity of the RT-PCR reaction and the absence of primer dimer formation. The following primers were used: PPARγ sense: 5-TCGCTGATGCACTGCCTATG-3, antisense: 5-GAGAGGTCCACAGAGCTGATT-3; and FABP4 sense: 5-GGGCCAGGAATTTGACGAAG-3, antisense: 5-CGCATTCCACCACCAGTTTATC-3; β-actin sense: 5-GCGGGAAATCGTGCGTGACATT-3, antisense: 5-GATGGAGTTGAAGGTAGTTTCG-3; human TauT sense: 5-TTTTGTGTCTGGCTTCGCAATTT-3, antisense: 5-TGGGTAGGCAATGAAGGCCAG-3; mouse TauT sense: 5-GCACACGGCCTGAAGATGA-3, antisense: 5-ATTTTTGTAGCAGAGGTACGGG-3. The mRNA levels of the target genes were normalized to β-actin. Each target was measured in triplicate, and data were analyzed using GraphPad Prism 5.

RNAseq

hASCs adipogenic differentiation was induced by the pro-adipogenic components for 3 days. Total RNA of the control and induced samples was extracted using TRIzol reagent. 1 μg total RNA with RIN value above 7 was used for following library preparation. Next generation sequencing library preparations were constructed according to the manufacturer's protocol (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®). The sequences were processed and analyzed by GENEWIZ.

Immunoblotting

The cells were immediately placed on ice and washed with ice-cold PBS. Total protein extract was prepared with RIPA lysis buffer (25 mM Tris-HCl at pH 7.5, 2 mM EDTA, 25 mM NaF and 1% Triton X-100) containing 1 X protease inhibitor mixture (Roche) and 1 X PMSF. The proteins were resolved on 8-12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Merck Millipore). The membranes were blocked with 5% nonfat milk in TBST for 1 hour. Proteins of interest were detected with specific antibodies, blots were scanned using a ChemiDocTM MP imaging system (Bio-Rad).

Reagents and antibodies

TRIzol reagent was purchased from Invitrogen. Fast-start universal SYBR Green master mix was from Roche. The inhibitor TWS119 was from Selleck. The antibodies used for immunoblotting (IB): PPARγ (Cell Signaling, #2443), FABP4 (Cell Signaling, #3544), TauT (Absin, abs140562a), β-catenin (Cell Signaling, #8480), β-actin (Santa Cruz, sc-47778), GAPDH (Proteintech, 60004-1-Ig), α-Tubulin (Santa Cruz, sc-58667).

Statistical Analysis

The data are presented as the means ± SD. All data are representative of at least three independent experiments. The differences between groups were assessed by Student's t-test; all reported differences are *p < 0.05, **p < 0.01, ***p < 0.001 unless otherwise stated.

Results

Upregulation of taurine transporter in adipogenic differentiation

To explore the molecular mechanisms of adipocyte differentiation, we isolated and cultured human adipose-derived stem cells (hASCs) from subcutaneous adipose tissues of donors undergoing plastic surgeries. Transcriptome sequencing was performed to compare the expression of genes in hASCs induced to undergo adipocyte differentiation for 3 days with those of uninduced. The induction changed the gene expression profile markedly (Figure ). While the levels of 216 genes were notably downregulated, the expressions of 133 genes were upregulated more than 2-folds (Table ). Among the elevated genes was SLC6A6, which was ubiquitously expressed in many tissues and encodes a sodium and chloride-ion dependent transporter for taurine and β-alanine, often referred as taurine transporter (TauT). RT-PCR and immunoblotting analysis showed that TauT mRNA and protein levels increased significantly in hASCs after a 3-day induction, which was accompanied by the increase of fatty acid binding protein 4 (FABP4), a well-known marker for adipogenic differentiation (Figure ).

The ob/ob and db/db mice harbor mutations in leptin and its receptor and become profoundly obese. Variations in the leptin receptor have also been associated with obesity in human and with increased susceptibility to Entamoeba histolytica infections. We therefore examined the expression of TauT in fat tissues from the ob/ob and db/db mice. Compared with wild type littermates, both obese mice had significantly increased levels of TauT mRNA (Figure ). Moreover, both subcutaneous and visceral fats from mice fed with high fat diet also expressed elevated levels of TauT mRNA (Figure ). Therefore, TauT might play an important role in adipogenesis and in the formation of obesity.

TauT promoted the adipogenic differentiation of hASCs

To determine whether the increased TauT expression is important in adipogenic differentiation, use was made of siRNA to knockdown TauT in hASCs induced to differentiate into adipocyte. As shown in Figure , transfection of specific siRNA in induced hASCs markedly reduced the levels of TauT mRNA and protein. As such, the expression of adipogenic differentiation marker FABP4 and PPARγ was also downregulated by Day 3 (Figure ). Noteworthily, PPARγ induction appeared relative early in the differentiation and acted as a master transcription factor to induce the expression of downstream targets including FABP4, a marker for later stage of differentiation. Furthermore, the TauT-specific siRNA markedly reduced the formation of adipocytes by Day 9, as measured by Oil Red O staining (Figure ) and triglyceride quantification (Figure ).

We also transfected hASCs cells with TauT-expressing plasmid and induced them toward adipocytes. Compared these transfected with control construct, the induction of FABP4 and PPARγ in TauT-expressing hASCs were markedly enhanced at both mRNA and protein levels by Day 3 (Figure ). Moreover, enforced TauT expression promoted the formation of adipocytes as measured by Oil Red O staining at Day 9 (Figure ) and triglyceride quantification (Figure ). Thus, increased level of TauT appeared promote adipogenesis and is required for differentiation toward adipocytess under these conditions.

Taurine, Hypotaurine and β-alanine modulated the adipogenic differentiation of hASCs

TauT (SLC6A6) is a member of the sodium and chloride-ion dependent transporters family that transports taurine, hypotaurine and β-alanine. The involvement of TauT in adipogenic differentiation prompted us to examine the effects of these amino acids on the process in hASCs. As assessed by RT-qPCR and immunoblotting, administration of hypotaurine to the culture significantly increased the expression of differentiation markers FABP4 and PPARγ by day 3 in induced hASCs (Figure ). The effects were dose-dependent and significant in the range of 5 μM - 5mM. Similar changes of FABP4 and PPARγ were found after the induced cells were treated with β-alanine (Figure ). In contrast, the levels of FABP4 and PPARγ were markedly decreased when the induced hASCs were exposed to taurine (Figure ). The inhibitions were also significant dose-dependently in the range of 50 μM - 5 mM, although taurine as well as hypotaurine and β-alanine did not affect notably the growth of hASCs (data not shown). The effects of these amino acids on induced hASCs were further evaluated by Oil Red O staining and triglyceride quantification. As shown in Figure , hypotaurine and β-alanine treatment led to markedly increase intracellular lipid accumulation by day 9 compared to the control, whereas the addition of taurine resulted in decrease of the Oil Red O staining. Therefore, the substrates of TauT, hypotaurine, taurine and β-alanine all modulate the adipogenic differentiation of hASCs effectively.

The inhibitory effect of TauT knockdown on adipogenic differentiation was reversed by hypotaurine and β-alanine, but not taurine

To further assess whether the role of TauT on adipognesis is related to its ability to transport the amino acids, we knocked down TauT of hASCs in the presence exogenous of hypotaurine, β-alanine, or taurine. As shown in Figure , downregulation of FABP4 and PPARγ in induced hASCs by TauT knockdown were reversed significantly by hypotaurine and β-alanine, but not taurine. We also assessed the formation of adipocytes by Oil Red O staining and triglyceride quantification after the induction was continued for 9 days. While the Oil Red O staining was markedly reduced by TauT knockdown (Figure , panel 3), the presence of hypotaurine and β-alanine significantly alleviated the reduction (Figure , panel 4 & 6). However, addition of taurine to the culture did not rescue the decrease of Oil Red O staining (Figure , panel 5). Similarly, the decrease of total triglyceride by TauT knockdown was rescued to a large extent by hypotaurine and β-alanine, but not taurine (Figure ). These results indicated that the taurine transporter affects adipogenesis at least partially through transporting these amino acids.

TauT regulated the adipogenic differentiation through preventing the downregulation of Wnt/β-catenin signaling

The Wnt/β-catenin signaling pathway plays a critical role in maintaining the stemness of various progenitor cells including hASCs. It has been shown that down -regulation of the pathway is required for the adipogenic differentiation of hASCs and activation of Wnt receptor prevented the differentiation. As shown in Figure , adipogenic differentiation of hASCs led to reduced expression of β-catenin. TauT- specific siRNAs prevented the β-catenin reduction and the expression of differentiation marker (Figure ). Noteworthily, both hypotaurine and β-alanine reduced the level of β-catenin dose-dependently (Figure ), whereas taurine prevented the reduction of β-catenin (Figure ), recapitulating their effects on adipogenesis. Moreover, hypotaurine and β-alanine, but not taurine, abolished to a large extent the increase of β-catenin in TauT-knockdown hASCs induced to differentiate (Figure ). TWS119, an activator of Wnt/β-catenin through inhibiting GSK3β, prevented adipogenic differentiation promoted by hypotaurine and β-alanine (Figure ). Moreover, exposing to TWS119 also abolished the pro-differentiation effect of overexpressing TauT in hASCs (Figure ). Taken together, these results indicated that TauT participated the downregulation of Wnt/β-catenin signaling pathway during adipogenic differentiation, and the effect is at least partially mediated by the amino acids it transports.

Discussion

It has been shown that the CCAAT/Enhancer- binding Protein (C/EBP) family proteins C/EBPβ and δ are among the initially upregulated transcription factors after the progenitor cells were induced to differentiation toward adipocytes. The subsequent increases of C/EBPα and peroxisome proliferator- activated receptor γ (PPARγ) are responsible for the induction of a variety of changes in gene expression during adipocye formation, including the expression of FABP4 and lipoprotein lipase. These transcription factors also act to suppress the Wnt/β-catenin signaling, which is required for adipogenic differentiation. We found in the present study that taurine transporter was markedly increased during adipogenesis, whereas knockdown of TauT impeded adipocyte formation, indicating that the transporter promotes adipogenic differentiation. We also found that TauT expression is elevated in adipose tissues from mice fed with high fat diet or harboring mutations in lepin or leptin receptor. Consistent with the result, it has been shown that TauT-knockout mice exhibited lower body weight and abdominal fat mass than wild-type mice. Since both C/EBP-like family and PPARγ are capable of increasing TauT expression in various cells,, it is conceivable that TauT might act as a positive feedback to amplify the differentiating signal, and serve as a target for intervening adipogenesis. It is worth noting that TauT may transport multiple substrates including taurine, hypotaurine, γ-aminobutyric acid and β-alanine into cells, and meanwhile output ions from the cells. However, only high doses of hypotaurine and β-alanine promoted adipogenesis and reversed the inhibitory effects of TauT knockdown on hASCs differentiation, suggesting that these amino acids are required for adipocyte formation or they can enhance the functional activity of TauT. Interestingly, it was demonstrated recently that taurine supplementation attenuated adipogenesis and inhibited TauT expression in animal experiments. In hASCs, we also found taurine reduced the expression of TauT dose-dependently (data not shown). Thus, it is interesting to know whether hypotaurine and β-alanine could affect TauT expression. It is conceivable that high dose taurine inhibits adipogenic differentiation due to the reduction of cross cell membrane gradient of taurine and reduction of TauT.

Although taurine is not a building block of protein, it is found in millimolar concentration in mammalian tissue such as skeletal and heart muscles, and can be absorbed from dietary and synthesized from cysteine. Interestingly, while the intracellular concentration of taurine is in the ranges of 10-20 mM, its plasma level is only about one 500th. The importance of taurine transportation was also clearly illustrated by the finding that the taurine levels in skeletal and heart muscle of TauT-deficient mice decreased by about 98% compared with that of controls. Noteworthily, in addition to be essential for the function of skeletal muscle, the retina, the cardiovascular and central nervous system, taurine supplemented in drink water had significant anti-obesity effects in animals fed with high fat diet and in genetic obese mice,. Epidemiological studies also found that urinary taurine content was inversely related to BMI and cadiovascular risks. Our finding that high concentration of taurine effectively blocked the adipocyte formation provided an attractive rationale for the anti-obesity activity, and may account for the change of macrophage activity and production of inflammatory cytokines in adipose tissues. This is also consistent with the report that macrophages-derived taurine chloramine inhibited the differentiation of preadipocytes into adipocytes and modulated the expression of adipokines in adipocytes.

In summary, we have started to analyze the differentiation mechanisms of hASCs, a rich source of potential stem cells that could differentiate into many different tissue cells. Our data showed the Taurine transporter played an important role in the differentiation of hASCs into adipocytes. Intriguingly, while TauT substrates hypotaurine and β-alanine promoted adipogenesis, taurine inhibited effectively adipocyte formation. They appeared act in hASCs by promoting and inhibiting β-catenin reduction respectively, which was essential for hASCs differentiation. Thus, TauT is a potential target for preventive or therapeutic intervention of disorders such as obesity and fatty liver. Noteworthily, the taurine transporter expression has also been linked to diabetes, cardiovascular and neurological diseases-. It is conceivable that the different activities of its substrates might provide novel clues to understand and treat these diseases.

Supplementary figures and tables.

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