This study describes the identification of Mfsd2a (major facilitator superfamily domain-containing protein 2a), a novel mammalian major facilitator superfamily domain-containing protein, and an additional closely related protein, Mfsd2b. Most intron/exon junctions are conserved between the two genes, suggesting that they are derived from a common ancestor. Mfsd2a and Mfsd2b share a 12 transmembrane α-helical domain structure that bears greatest similarity to that of the bacterial Na+/melibiose symporters. Confocal microscopy demonstrated that Mfsd2a localizes to the endoplasmic reticulum. Mfsd2a is expressed in many tissues and is highly induced in liver and BAT (brown adipose tissue) during fasting. Mfsd2a displays an oscillatory expression profile in BAT and liver, consistent with a circadian rhythm. Although the basal level of Mfsd2a expression is relatively low in mouse BAT, it is greatly induced during cold-induced thermogenesis and after treatment with βAR (β-adrenergic receptor) agonists. This induction is totally abolished in β-less (βAR-deficient) mice. These findings indicate that Mfsd2a is greatly up-regulated in BAT during thermogenesis and that its induction is controlled by the βAR signalling pathway. The observed induction of Mfsd2a expression in cultured BAT cells by dibutyryl-cAMP is in agreement with this conclusion. The present study suggests that Mfsd2a plays a role in adaptive thermogenesis.
- β-adrenergic receptor (βAR)
- brown adipose tissue (BAT)
Energy homoeostasis can be defined as the maintenance of the delicate balance between energy intake and energy expenditure. BAT (brown adipose tissue) and WAT (white adipose tissue) play important but opposite roles in energy homoeostasis [1,2]. The major and most efficient way to store excess energy is in the form of TAGs (triacylglycerols), which are primarily stored in WAT . This energy can be released during times of energy deprivation. TAGs are then hydrolysed into free fatty acids, which are then mobilized into peripheral tissues where they can be directly oxidized to generate energy for cellular and organ functions, physical activity and adaptive thermogenesis. Cold-induced thermogenesis involves increased thermal energy output in skeletal muscle and BAT [4–7]. BAT converts the energy stored in TAG into heat by uncoupling ATP production from respiration, a process known as non-shivering thermogenesis.
The β3-adrenergic signalling pathway has been demonstrated to play a key role in the control of adaptive thermogenesis in rodents; however, this process appears to be less important in humans [4,5,8]. In response to cold exposure, the sympathetic nervous system releases NE [adrenaline (norepinephrine)] which interacts with βARs (β-adrenergic receptors) expressed on the surface of brown adipocytes. This activation elevates intracellular cAMP levels, increases the rate of lipolysis and induces the expression of multiple genes, including Ucp1 (uncoupling protein 1) and Dio2 (deiodinase 2) [6,7,9]. Oxidation of free fatty acids ultimately leads to the creation of a proton electrochemical gradient [4,5,8]. Under β-adrenergic stimulation, Ucp1 uncouples this energy from the synthesis of ATP and instead promotes its conversion into heat.
In addition to βAR, a number of different transcription factors and co-factors, including CREB (cAMP-response-element-binding protein), NRF (nuclear respiratory factor) 1, NRF2 and several nuclear receptors, have been implicated in the regulation of gene expression during adaptive thermogenesis [4,10–13]. Previous studies have shown that PGC-1α [PPAR (peroxisome-proliferator-activated receptor)γ-co-activator 1α] plays a critical role in the regulation of gene expression during thermogenesis [13,14].
In the present study, we describe the identification of a novel member of the major facilitator domain-containing family, referred to as Mfsd2a (major facilitator superfamily domain-containing protein 2a). Sequence comparison identified an additional related protein, not reported previously, designated Mfsd2b. Both Mfsd2a and Mfsd2b exhibit amino-acid sequence and structural similarities to bacterial permeases and symporter proteins, suggesting that they are members of the major facilitator superfamily. We examined the regulation of Mfsd2a during brown fat differentiation, adaptive thermogenesis and fasting. Our results indicate that Mfsd2a is greatly up-regulated in BAT during thermogenesis and that its induction is controlled by the βAR signalling pathway. This is supported by the induction of Mfsd2a expression in cultured BAT cells by dibutyryl-cAMP. These results are consistent with the conclusion that activation of the βAR signalling pathway plays a major role in the induction of Mfsd2a expression during adaptive thermogenesis.
MATERIALS AND METHODS
C57BL/6 RORαsg/sgRORγ−/− (where ROR is retinoid-related orphan receptor) DKO (double knockout) mice, deficient in the expression of RORα and RORγ, and β-less (βAR-deficient) AKR/J mice, which are mice deficient in the expression of all three βARs, have been described previously [15,16]. Mice were individually housed at 22 °C and maintained on a constant 12 h light/12 h dark cycle, with the light cycle beginning at 06:00 h. In some experiments, mice were exposed to cold (4 °C) for up to 8 h or fasted for 16 h, starting at the onset of the dark cycle. Mice were fed on a diet of NIH-A31 chow and water ad libitum. In experiments with β-agonist treatment, mice were injected intraperitoneally with 100 mg/kg isoproterenol (I5627 Sigma), 1 mg/kg CL316243 (C5976; Sigma) or 0.9% NaCl (control). During fasting, mice had unlimited access to water. All animal protocols followed the guidelines outlined by the NIH (National Institutes of Health) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the NIEHS (National Institute of Environmental Health Sciences) and Harvard Medical School.
The mouse Mfsd2a gene sequence was submitted to the GenBank® nucleotide sequence database (accession number AY880264). The genomic structure was deduced from the alignment of GenBank® nucleotide sequence AY880264 with the mouse genomic DNA sequence (GenBank® nucleotide sequence database accession number AC125518.3) containing the entire Mfsd2a gene. Mfsd2a was amplified by PCR from a cDNA clone (IMAGE consortium clone 5707105; Open Biosystems) with the forward primer 5′-TTGAATTCCCCGCGGGTCATGGCCAAAG-3′ and the reverse primer 5′-TTGGATCCGAGAATACTGGCCAGCTCTG-3′ containing an EcoRI and a BamHI restriction site respectively (underlined). The PCR product was cloned into the EcoRI and BamHI restriction sites of the pCMV-3×FLAG-14 expression vector (Sigma), resulting in a FLAG tag being inserted in-frame at the C-terminus of Mfsd2a. The DNA sequence was verified by restriction-enzyme analysis and DNA sequencing.
BAT cell culture
Immortalized WT (wild-type) and PGC-1α KO (knockout) brown fat adipocytes have been described previously  and were grown in pre-adipocyte medium [DMEM (Dulbecco's modifed Eagle's medium), 20% (v/v) FBS (fetal bovine serum), 100units/ml penicillin and 100μg/ml streptomycin]. When cells reached confluence, the medium was replaced with adipocyte medium [20 mM Hepes (pH 7.4), DMEM, 10% (v/v) FBS, 20 nM insulin and 1 nM T3 (3,3′,5-tri-iodothyronine)] containing 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone and 0.125 mM indomethacin in order to promote differentiation. After 2 days of incubation at 37 °C, the medium was switched to adipocyte medium only for a further 5 days. The medium was renewed every other day. Fully differentiated BAT cells (7 days after induction) were treated with 0.5 mM dibutyryl-cAMP or 5 μM NE for various time points before analysis.
Tissues, including abdominal WAT and interscapular BAT, were promptly isolated and kept overnight at 4 °C in RNAlater® solution (Ambion). The following day, the tissues were removed from the RNAlater® solution and stored at −80 °C until use. Tissues were homogenized using a Polytron PT 3000 homogenizer (Brinkmann Instruments) in 2 ml of RLT solution (Qiagen) containing 20μM 2-mercaptoethanol. The homogenate was then loaded into a QIAschredder™ column (Qiagen) and centrifuged at 17000 g for 3 min. The supernatant was mixed with one volume of 70% (v/v) ethanol [50% (v/v) for liver] and RNA was isolated using the RNeasy® Mini or Midi kit (Qiagen) following the manufacturer's instructions. Total RNA from cells was isolated with TRIReagent® (Molecular Research Center) according to the manufacturer's instructions.
Northern blot analysis
Total RNA (15 μg) was separated on a 1.2% (w/v) agarose/5% (v/v) formaldehyde gel in 1× MOPS buffer, transferred on to a nylon membrane (Sigma) and subsequently UV cross-linked. The membranes were hybridized to 32P-labelled cDNA probes (10000 Bq) specific for Mfsd2a. Hybridizations were performed at 68 °C for at least 3 h. The membranes were then washed twice with 2× SSC (1× SSC is 0.15 M NaCl/0.015 M sodium citrate) and 0.1% SDS for 20 min at 22 °C, and subsequently with 0.1× SSC and 0.1% SDS for 15 min at 50 °C. Autoradiography was carried out at −70 °C using Hyperfilm (Amersham Biosciences).
Total RNA isolated from BAT from WT and RORα/RORγ DKO mice was amplified using Agilent Low RNA Input Fluorescent Linear Amplification Kit following the manufacturer's instructions. Gene-expression analyses were conducted by the NMG (NIEHS Microarray Group) on Agilent mouse 20000-oligo chips as described previously . The results discussed in this publication have been deposited in NCBI Gene Expression Omnibus at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=bfohfqeyyqikipyaccession#GSE9969.
qRT–PCR (quantitative real-time–PCR)
Total RNA was first reverse-transcribed into cDNA using a High Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's instructions. qRT–PCR was then carried out in triplicate with a 7300 Real Time PCR system (Applied Biosystems) using either the TaqMan® Universal PCR Master Mix (Applied Biosystems) or the RT2 Real-Time™ SYBR Green/ROX PCR Master Mix (Superarray). cDNA (50 ng) was used in each reaction to quantify the various gene-expression levels, whereas 25 pg/reaction were used to measure 18S rRNA levels. Briefly, cDNA was denatured for 10 min at 95 °C, followed by 40 cycles consisting of 15 s at 95 °C and 60 s at 60 °C. The average Ct (threshold cycle value) from triplicate PCRs for every gene was normalized against the Ct obtained from the 18S transcript or cyclophilin B, which served as internal controls. The r2 values of each appropriate standard curves scored above 0.99. Pre-designed Assays-on-Demand™ primers and probe sets were purchased from Applied Biosystems, whereas all other primers and probes were designed in-house using Primer Express 2.0 software (Applied Biosystems) and synthesized by Sigma-Genosys. The sequences of the primers and probes are shown in Supplementary Table S1 (http://www.BiochemJ.org/bj/416/bj4160347add.htm).
Brown fat adipocytes or COS-1 cells (1000 cells/cm2) were plated in glass-bottomed tissue-culture dishes (MatTek) and transfected 24 h later with pCMV-Mfsd2a-3×FLAG-14 (0.2 μg) using either FuGENE™ 6 (Roche) according to the manufacturer's instructions. Cells were co-transfected with pDsRed2-ER (Clontech) or stained with either MitoTracker Red CMX-Ros or LysoTracker Red (Molecular Probes) to identify the ER (endoplasmic reticulum), mitochondria and lysosomes respectively. After 48 h, cells were fixed in 4% (w/v) paraformaldehyde for 20 min and then permeabilized with 0.3% Triton X-100. After a 15 min incubation at room temperature (22 °C) in SuperBlock® Blocking Buffer (Pierce), cells were incubated for 3 h at room temperature with a mouse monoclonal anti-FLAG M2 antibody (1:20000 dilution; Sigma), followed by an incubation for 30 min at room temperature with an Alexa Fluor® 488-conjugated goat anti-mouse IgG antibody (1:1000 dilution; Molecular Probes). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma). Fluorescence was observed using a Zeiss LSM 510 NLO confocal microscope (Zeiss).
The qRT–PCR data were analysed by Student's t test. qRT–PCR expression levels are presented as means±S.E.M., with *P<0.01 and **P<0.05 where indicated.
Identification of Mfsd2a
The nuclear receptors RORα and RORγ are both expressed in BAT . To obtain insights into the function of these receptors in this tissue, we compared the gene-expression profiles of WT and RORα/RORγ DKO mice by microarray analysis. This comparison identified a novel gene that was expressed at significantly higher levels in BAT from RORα/RORγ DKO mice. The gene encoded a 59 kDa protein not reported previously. The nucleotide and amino-acid sequences are shown in Supplementary Figure S1A (http://www.BiochemJ.org/bj/416/bj4160347add.htm). The analysis of both the amino-acid sequence and the domain structure indicated that this protein is related to the Na+/melibiose symporter and other related bacterial carbohydrate transporters amd permeases (Figure 1), suggesting that it is a member of the major facilitator/sugar transporter family [18–21]. We refer to this protein as Mfsd2a.
Comparison of the mouse Mfsd2a protein sequence with protein sequences in GenBank® (Blastn) identified the human homologue (GenBank® Entrez Gene database accession number BAD38634). The amino-acid sequence of Mfsd2a is highly conserved among species; mouse and human Mfsd2a proteins are ∼85% identical (Figure 1). In addition, Mfsd2a showed high sequence homology with a sequence of another previously unreported protein closely related to Mfsd2a (GenBank® Entrez Gene database accession numbers AK172423 and NP_001073942 for the mouse and human homologues respectively). This protein, referred to as Mfsd2b, consists of 494 amino acids and exhibits a 59% similarity and a 42% identity with Mfsd2a (Figure 1). Additional sequence analysis showed that Mfsd2a exhibits a 68% identity and 80% similarity to the amino-acid sequence of an unpublished Xenopus laevis protein (GenBank® Entrez Gene database accession number AAI23089) and a 61% and 64% identity at the amino-acid level with two previously unpublished zebrafish (Danio rerio) proteins (GenBank® Entrez Gene database accession numbers NP_001007452 and CAK04287), suggesting that they represent the Xenopus and zebrafish homologues of Mfsd2a (Figure 1). Sequence comparison revealed that both Mfsd2a and Mfsd2b are only distantly related to other mammalian transporters. The highest amino-acid sequence similarity was found between Mfsd2a/Mfsd2b and the Na+/melibiose symporter MelB [18–20] from several different bacteria, including those of the cyanobacterium Nostoc punctiforme (GenBank® Entrez Gene database accession number ZP_00109128; 47% similarity), the proteobacterium Escherichia coli (GenBank® Entrez Gene database accession number ZP_00721556; 43% similarity) and several related carbohydrate transporters (Figure 1). The phylogenetic relationship between these proteins is shown in Supplementary Figure S1B. On the basis of their sequence similarities, it was concluded that the Xenopus and zebrafish proteins are more closely related to Mfsd2a than to Mfsd2b.
Alignment of mRNA and genomic sequences revealed the genomic structure of the mouse Mfsd2a gene (Supplementary Figure S1C). The Mfsd2a gene spans approx. 14.3 kb and consists of 14 exons and 13 introns, whereas the Mfsd2b gene consists of 13 exons and 12 introns. All intron–exon junctions are conserved between the Mfsd2a and Mfsd2b genes, except those associated with intron 6. In addition, Mfsd2a contains one extra intron (intron 9). This similarity in genomic structure suggests that Mfsd2a and Mfsd2b are derived from a common ancestral gene. The mouse and human Mfsd2a genes map on to chromosomes 4D2.2 and 1p33 respectively, whereas the mouse and human Mfsd2b genes map on to 12A1.1 and 2p23 respectively.
Putative TMD (transmembrane domain) structure
Although Mfsd2a exhibits only 43–47% amino-acid sequence similarity with the Na+/melibiose symporter MelB and related bacterial carbohydrate transporters, prediction analysis of the transmembrane helix domain structure of the Mfsd2 proteins (http://www.cbs.dtu.dk/services/TMHMM/, http://www.predictprotein.org/ and http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=toppred) indicated that their domain structures are strongly conserved. Previous topological studies of MelB have demonstrated that it contains 12 α-helical TMDs [18–20,22,23]. On the basis of our topological analysis and comparison with MelB, it was predicted that Mfsd2a contains 12 putative α-helices (Figure 1), with each α-helix containing at least 17 amino acids, in agreement with the concept that they function as TMDs. The putative secondary structures of Mfsd2a and MelB proteins are compared in Figure 1. The similarities between the secondary structures of Mfsd2a and MelB support the conclusion that Mfsd2a also contains 12 TMDs.
As predicted by the similarities in their genome structure and amino-acid sequence, the α-helices of Mfsd2a align with those of Mfsd2b (Figure 1). This analysis supports the assumption that both Mfsd2a and Mfsd2b function as membrane proteins and are members of the major facilitator superfamily. Several regions within Mfsd2a and Mfsd2b are highly conserved between Mfsd2a and Mfsd2b and also between various species, suggesting they might have a functional role. These residues include: GRLMPW (overlaps with TMD 3), VPYSALTMF (in TMD 4), ERDSATAYRMT (in the loop between TMDs 4 and 5), GFLFTSLA (in TMD 7), LTRFGKKT (between TMDs 8 and 9), LLPWSMLPDVIDDF (in the loop between TMDs 10 and 11), YVFFTK (in TMD 11) and LGVSTLSLDFA (between TMDs 11 and 12).
Subcellular localization of Mfsd2a
Since the domain structure predicted that Mfsd2a is a membrane-associated protein, we determined what membrane it was associated with. To examine the subcellular localization of Mfsd2a, BAT cells were transfected with pCMV-Mfsd2a-FLAG encoding Mfsd2a fused to a 3×FLAG tag at its C-terminus. The localization of Mfsd2a–FLAG was subsequently examined by confocal microscopy and compared with that of markers for mitochondria, lysosomes and the ER. As shown in Figure 2, Mfsd2a colocalized with the ER marker rather than with markers for mitochondria and lysosomes. In addition, little Mfsd2a appeared to be localized to the plasma membrane. Similar results were obtained with COS-1 cells (results not shown). These results suggest that under the conditions used, Mfsd2a is largely associated with the ER. Consistent with this, Mfsd2a contains four putative ER-localization signals (KKXX, Supplementary Figure S1A) , one of which is conserved between human and mouse Mfsd2a.
Tissue-specific pattern of Mfsd2a mRNA expression
To examine the tissue-specific expression of Mfsd2a, Northern blot analysis was performed using total RNA isolated from multiple adult mouse tissues (Figure 3A). The Mfsd2a cDNA probe hybridized to an mRNA of approx. 2.2 kb in size. Mfsd2a mRNA was expressed in many tissues; with the highest level of expression observed in brain, intestine, kidney, liver, lung, mammary gland and prostate. Compared with Mfsd2a, Mfsd2b was expressed in few tissues and at relatively low levels (see Supplementary Figures S2A and S2B at http://www.BiochemJ.org/bj/416/bj4160347add.htm). Highest expression of Mfsd2b was detected in spleen, followed by lung and testis.
As mentioned above, Mfsd2a was originally identified by gene-expression profiling as a gene which is up-regulated in BAT from RORα/RORγ DKO mice (see Materials and Methods section for details). To validate this observation, Mfsd2a expression was compared between BAT from WT and RORα/RORγ DKO mice. As shown in Supplementary Figure S2C, Mfsd2a was expressed in BAT from RORα/RORγ DKO mice at a >15-fold higher level compared with that of WT mice.
Mfsd2a follows a circadian expression pattern
Examination of the level of Mfsd2a mRNA expression over a period of 24 h showed that Mfsd2a expression displayed an oscillatory expression profile in both BAT and liver (Figure 3B). The expression of Mfsd2a steadily increased during the light cycle, reached its zenith at approx. CT12 (circadian time 12), and then rapidly decreased during the early phase of the dark cycle. The periodicity was identical for BAT and liver. These results show that Mfsd2a exhibits an oscillatory expression pattern consistent with a circadian rhythm.
Induction of Mfsd2a expression during adaptive thermogenesis and fasting
To gain further insight into the function of Mfsd2a in BAT, we examined whether it might have a role in adaptive thermogenesis. Therefore WT mice were exposed to an ambient temperature of 4 °C and, at different times during cold exposure, BAT mRNA was isolated and Mfsd2a expression was examined by qRT–PCR. As shown in Figure 4A, Mfsd2a mRNA expression was dramatically induced in a time-dependent manner in BAT during cold exposure, similar to Dio2 and Ucp1 expression, genes known to be induced during thermogenesis [6,7,9]. This dramatic induction of Mfsd2a was confirmed by Northern blot analysis (Figure 4B). These results demonstrate that increased Mfsd2a expression in BAT is associated with adaptive thermogenesis, suggesting a function for Mfsd2a in this process. Cold exposure did not change significantly the expression of Mfsd2a in liver, kidney and skeletal muscle, or that of Mfsd2b in BAT (results not shown).
Previous studies have demonstrated that activation of βARs plays a key role in driving adaptive thermogenesis in BAT [4,10]. Therefore we compared the induction of Mfsd2a expression during thermogenesis between WT and β-less mice. qRT–PCR analysis showed that, as observed for Ucp1 and Dio2 expression, the induction of Mfsd2a was greatly reduced in BAT from β-less mice upon exposure to cold (Figure 4C). These results suggest that activation of βARs plays a critical role in the regulation of Mfsd2a expression during adaptive thermogenesis. To obtain further support for this concept, mice were treated with the βAR pan-agonist isoproterenol or the β3-specific agonist CL316243 and their effect on Mfsd2a expression was examined. As shown in Figure 4(D), both isoproterenol and CL316243 significantly increased the expression of Mfsd2a. Mfsd2a expression was increased to a greater extent than that of Ucp1 and Dio2. These results support the hypothesis that the induction of Mfsd2a during adaptive thermogenesis is dependent on the activation of the β-adrenergic signalling pathway.
A number of genes that are induced during adaptive thermogenesis have been reported also to be up-regulated during fasting, another process that involves the release of energy from stored fatty acids or carbohydrates [25,26]. Therefore we were interested in determining whether Mfsd2a expression was also enhanced during fasting. As shown in Figure 4(E), the expression of Mfsd2a was highly induced in both BAT and liver after fasting.
Regulation of Mfsd2a expression in cultured brown adipocytes
To obtain further insights into the role of Mfsd2a in brown adipocytes, we examined whether Mfsd2a expression was altered during differentiation of cultured BAT cells. As shown in Figure 5(A), Mfsd2a was expressed at relatively low levels in pre-adipocytes, and its expression levels remained unchanged during their differentiation into brown adipocytes. In contrast, the brown adipocyte marker Ucp1 was highly induced during brown adipocyte differentiation [7,14].
The results in Figures 4(C) and 4(D) showed that the induction of Mfsd2a expression during thermogenesis was dependent on the activation of βAR. Activation of the βAR signalling pathway results in an increase in cAMP levels and activation of PKA (protein kinase A) that subsequently leads to the phosphorylation and activation of several transcriptional mediators, and the induction of several thermogenic marker genes. The latter can be reproduced in cultured differentiated BAT cells by the addition of dibutyryl-cAMP [4,8,10]. We therefore examined the effect of cAMP on Mfsd2a expression. As shown in Figure 5(B), treatment of fully differentiated BAT cells with dibutyryl-cAMP induced expression of Mfsd2a in a time-dependent manner as reported previously for Ucp1 . Mfsd2a expression was induced 10-fold 2 h after the addition of dibutyryl-cAMP. Treatment of BAT cells with NE (Figure 5B) also enhanced Mfsd2a mRNA expression. Addition of dibutyryl-cAMP to pre-adipocytes did not induce Mfsd2a expression (results not shown).
Previous studies have shown that approx. 32% of the genes induced by cAMP in BAT cells, including Ucp1, are dependent on PGC-1α expression . As shown in Figure 5(C), the induction of Mfsd2a in PGC-1α−/− BAT cells by cAMP was not significantly different from that in WT cells. These results suggest that the induction of Mfsd2a is independent of PGC-1α expression.
In the present study, we describe the identification of a novel subfamily of major facilitator domain-containing proteins (Mfsd2) not reported previously. Murine Mfsd2a was identified as a gene which is up-regulated in BAT of mice deficient in the expression of the nuclear receptors RORα and RORγ. Sequence analysis discovered a closely related protein, referred to as Mfsd2b, that exhibited an ∼60% amino-acid similarity to Mfsd2a. Their highly conserved genomic structure suggests that Mfsd2a and Mfsd2b belong to the same subfamily and are derived from the duplication of an ancestral gene. In addition, sequence comparison identified a Xenopus and two zebrafish homologues of Mfsd2 that, based on sequence similarity, were more closely related to Mfsd2a than to Mfsd2b. The Mfsd2 proteins exhibited a 43–47% sequence similarity with the Na+/melibiose symporter MelB and related carbohydrate transporters [18–20].
The structure of MelB has been extensively studied [18,19,22,23,27]. These topological analyses demonstrated the presence of 12 α-helical transmembrane-spanning segments. Secondary structure analysis of Mfsd2 proteins predicted the presence of 12 putative α-helical TMDs (Figure 1). Comparison of the sequence with that of MelB supported the conclusion that these proteins probably contain 12 TMDs. The latter is in agreement with the observations showing that many members of the major facilitator/permease superfamily contain 12 TMDs . However, based on their sequence homology, Mfsd2 proteins are only distantly related to other mammalian major facilitator domain-containing proteins, including members of the GLUT family, as the Mfsd2 proteins do not contain any of the conserved sequences characteristic of the GLUT family [28,29].
A recent study has shown that the cytoplasmic loop between helices 4 and 5 of MelB is functionally important . Mutation of the amino acids Arg141 or Glu142 within this loop greatly impairs the Na+/sugar translocation; however, these mutants retain their sugar-binding capacity. Interestingly, these two amino acids are conserved between MelB and Mfsd2 proteins. In addition, several of the amino acids that are essential for cation binding and recognition in MelB, including Asp55, Asn58 and Asp59, are conserved in Mfsd2 proteins. These similarities further support the hypothesis that Mfsd2 proteins might function as cation/carbohydrate symporters. Although many carbohydrate transporters localize to the plasma membrane and Mfsd2a was found to be largely associated with the ER, the trafficking of several transporters from intracellular compartments to the plasma membrane is under strict control. For example, insulin stimulates the translocation of GLUT4 to the plasma membrane, whereas activation of βAR promotes accumulation into intracellular compartments [30,31]. A good antibody is needed to determine what the distribution of endogenous Mfsd2a is and whether the subcellular localization of Mfsd2a is regulated in a similar manner.
Mfsd2a was found to be expressed in many tissues. In BAT and liver, Mfsd2a displayed an oscillatory expression profile consistent with a circadian rhythm (Figure 3C). Mfsd2a mRNA expression reached its highest level at the end of the light cycle at CT12 and then rapidly decreased. We reported previously that in BAT, RORα1 and RORγ1 also exhibit an oscillatory expression profile . Interestingly, expression of Mfsd2a decreases at a time when the expression of RORα1 and RORγ1 increases. This suggested that RORs might function as repressors of Mfsd2a expression. Consistent with this concept is the observation that Mfsd2a is up-regulated in BAT from RORα/RORγ DKO mice (Figure 3B).
Because Mfsd2a was identified as a gene up-regulated in BAT from RORα/RORγ DKO mice, we were particularly interested in its expression and function in this tissue. Mfsd2a is expressed at relatively low levels in BAT and in cultured pre-adipocytes. The expression of Mfsd2a remains unchanged during differentiation of pre-adipocytes into fully differentiated BAT cells (Figure 5A), suggesting that Mfsd2a does not play a major role during differentiation. However, our results demonstrated that Mfsd2a was highly induced in BAT during cold-induced thermogenesis, suggesting that Mfsd2a has a function in BAT during adaptive thermogenesis. Previous studies have shown that cold-induced thermogenesis is dependent on β-adrenergic stimulation [5,15]. The induction of genes normally induced during thermogenesis, including Ucp1 and Dio2 [4–7,9,14], is greatly diminished in β-less mice. Our results show that Mfsd2a expression is not greatly increased upon cold exposure in BAT from β-less mice, suggesting that activation of the β-adrenergic signalling pathway plays a major role in the induction of Mfsd2a expression during adaptive thermogenesis. This is further supported by results showing that treatment with βAR agonists induced Mfsd2a expression in WT mice, but not in β-less mice.
Activation of the βAR leads to an increase in cAMP levels and activation of PKA followed by phosphorylation and activation of various transcription factors that subsequently enhance the transcription of thermogenic genes [4,8,10]. This action is mimicked in cultured differentiated BAT cells by treatment with dibutyryl-cAMP or NE, an activator of βAR. Our results demonstrated that treatment of BAT cells with dibutyryl-cAMP or cAMP-inducing agents greatly induced the expression of Mfsd2a. These observations support the hypothesis that the regulation of Mfsd2a expression during thermogenesis is at least in part dependent on the activation of βAR and PKA signalling. The precise molecular mechanisms involved in the induction of gene expression during adaptive thermogenesis are not yet fully understood. The nuclear receptors PPARs, T3R (tri-iodothyronine receptor) and ERRα (estrogen-related receptor α), PGC-1α, and the transcription factors NRF1/2 and CREB have all been implicated in the regulation of various genes during thermogenesis [4,8,10,12,32]. Activation of PKA mediates the activation of CREB, which in turn enhances the transcription of a number of genes, including PGC-1α and Dio2. PGC-1α functions as a co-activator for a number of nuclear receptors, including PPARs and ERRα, as well as NRF1. A recent study showed that PGC-1α is required for 32% of the genes induced in BAT by cAMP . The present results show that the induction of Mfsd2a is independent of PGC-1α. Future studies have to be performed in order to determine the precise molecular mechanism that regulates Mfsd2a transcription.
Glucose uptake is markedly enhanced in BAT during cold exposure and is dependent on the activation of the βAR pathway [33,34]. It is interesting to note that the induction of Mfsd2a during thermogenesis resembles that of the glucose transporter GLUT1 (SLC2A1). As for Mfsd2a, GLUT1 expression is also induced in BAT in vitro and in vivo by βAR stimulation [31,33]. In contrast, GLUT4 expression is down-regulated after treatment with βAR agonists. These observations suggest distinct regulation and roles of different sugar transporters in thermogenesis. It is believed that GLUT1 is responsible for a large portion of the increase in glucose uptake observed during cold exposure. Whether Mfsd2a contributes to the increased uptake of sugars during adaptive thermogenesis needs to be determined. Identification of the molecules transported by Mfsd2a will be necessary to understand the physiological functions of Mfsd2a.
In summary, in the present study we describe a new subfamily of major facilitator proteins that is closely related to the bacterial Na+/melibiose symporter MelB. We provide evidence showing that in BAT Mfsd2a is dramatically induced during adaptive thermogenesis cold exposure, suggesting that Mfsd2a plays a specific role in BAT during adaptive thermogenesis. We demonstrate that activation of the βAR signalling pathway, but not PGC-1α, plays a major role in this induction.
We thank Dr John Pritchard and Dr Jennifer Perry for their valuable comments on the manuscript, Laura Miller for her assistance with the mice and Dr Xiying Wu for his assistance with the circadian rhythm analysis. This research was supported by the Intramural Research Program of the NIEHS (National Institute of Environmental Health Sciences), NIH (National Institutes of Health) (Z01-ES-101586 awarded to A. M. J.) and by a CNRU (Clinical Nutrition Research Unit) Center Grant 1P30 DK072476 entitled ‘Nutritional Programming: Environmental and Molecular Interactions’ sponsored by the NIDDK (National Institute of Diabetes and Digestive and Kidney Disease) (awarded to J. M. G.).
Abbreviations: βAR, β-adrenergic receptor; β-less, βAR-deficient; BAT, brown adipose tissue; CREB, cAMP-response-element-binding protein; Ct, threshold cycle value; CT12, circadian time 12; Dio2, deiodinase 2; DKO, double knockout; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; ERRα, estrogen-related receptor α; FBS, fetal bovine serum; Mfsd2 etc., major facilitator superfamily domain-containing protein 2 etc; NE, adrenaline (norepinephrine); NRF, nuclear respiratory factor; PKA, protein kinase A; PPAR, peroxisome-proliferator-activated receptor; PGC-1α, PPARγ-co-activator 1α; qRT–PCR, quantitative real-time–PCR; ROR, retinoid-related orphan receptor; TAG, triacylglycerol; TMD, transmembrane domain; Ucp1, uncoupling protein 1; WAT, white adipose tissue; WT, wild-type
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