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. 2008 Dec 15;416(3):347-55.
doi: 10.1042/BJ20080165.

Mfsd2a Encodes a Novel Major Facilitator Superfamily Domain-Containing Protein Highly Induced in Brown Adipose Tissue During Fasting and Adaptive Thermogenesis

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Mfsd2a Encodes a Novel Major Facilitator Superfamily Domain-Containing Protein Highly Induced in Brown Adipose Tissue During Fasting and Adaptive Thermogenesis

Martin Angers et al. Biochem J. .
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Abstract

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 alpha-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 betaAR (beta-adrenergic receptor) agonists. This induction is totally abolished in beta-less (betaAR-deficient) mice. These findings indicate that Mfsd2a is greatly up-regulated in BAT during thermogenesis and that its induction is controlled by the betaAR 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.

Figures

Figure 1
Figure 1. Amino acid sequence alignment of Mfsd2a, Mfsd2b, and MelB
Amino acid sequence alignment of Mfsd2a and Mfsd2b of different species. (-) indicates sequence identity with mouse Mfsd2a; (.) indicates gap. Mouse (m), human (h), Xenopus (x), and zebrafish (z) sequences are compared with those of Na+/melibiose symporter (MelB) of Nostoc punctiforme (n) and Escherichia coli (e). The α-helices indicating the 12 putative transmembrane domains are shaded.
Figure 1
Figure 1. Amino acid sequence alignment of Mfsd2a, Mfsd2b, and MelB
Amino acid sequence alignment of Mfsd2a and Mfsd2b of different species. (-) indicates sequence identity with mouse Mfsd2a; (.) indicates gap. Mouse (m), human (h), Xenopus (x), and zebrafish (z) sequences are compared with those of Na+/melibiose symporter (MelB) of Nostoc punctiforme (n) and Escherichia coli (e). The α-helices indicating the 12 putative transmembrane domains are shaded.
Figure 2
Figure 2. Mfsd2a protein is localized in the ER
Brown fat adipocytes were transfected with pCMV-Mfsd2a-FLAG. Forty-eight hours after transfection, the localization of Mfsd2a-FLAG protein was examined by confocal microscopy with a mouse anti-FLAG M2 antibody and Alexa 488 rabbit anti-mouse IgG. Mitochondria, lysozomes, ER, and nuclei were identified by MitoTracker Red CMX-Ros, LysoTracker Red, ER-targeted protein Red, and DAPI staining, respectively.
Figure 3
Figure 3. Mfsd2a has a wide tissue distribution and its expression oscillates through circadian time
(A) Tissue-specific pattern of expression of Mfsd2a. Total RNA isolated from multiple mouse tissues was examined by Northern blot analysis using a radio-labeled cDNA probe for Mfsd2a. Lower panel shows 18S and 28S rRNA stained with Ethidium Bromide (EtBr). *A combination of subscapular and teres major muscle. (B) Mfsd2a displays an oscillating expression profile. Total RNA from BAT and liver of male mice (n=4) was isolated over a 24 h period at the circadian time (CT) indicated. Subsequently the expression of Mfsd2a mRNA was examined by qRT-PCR analysis. Gene expression was normalized with the corresponding cyclophilin B levels and “double plotted” to display a duplicated 24 h profile. All values are reported as averages ± SE. The black and white bar indicates the light and dark period.
Figure 3
Figure 3. Mfsd2a has a wide tissue distribution and its expression oscillates through circadian time
(A) Tissue-specific pattern of expression of Mfsd2a. Total RNA isolated from multiple mouse tissues was examined by Northern blot analysis using a radio-labeled cDNA probe for Mfsd2a. Lower panel shows 18S and 28S rRNA stained with Ethidium Bromide (EtBr). *A combination of subscapular and teres major muscle. (B) Mfsd2a displays an oscillating expression profile. Total RNA from BAT and liver of male mice (n=4) was isolated over a 24 h period at the circadian time (CT) indicated. Subsequently the expression of Mfsd2a mRNA was examined by qRT-PCR analysis. Gene expression was normalized with the corresponding cyclophilin B levels and “double plotted” to display a duplicated 24 h profile. All values are reported as averages ± SE. The black and white bar indicates the light and dark period.
Figure 4
Figure 4. Mfsd2a expression is induced during adaptive thermogenesis and fasting
(A) Mfsd2a expression is greatly induced during adaptive thermogenesis. Mice (n=4) were subjected to a temperature of 4°C and BAT isolated at the time intervals indicated. The levels of Mfsd2a, Ucp1, and Dio2 mRNA were assessed by qRT-PCR analysis. (B) Total RNA was isolated from the liver, kidney, and BAT (from mice kept at 22°C or 4°C for 8 h). Mfsd2a expression was examined by Northern blot analysis using a radiolabeled cDNA probe. Lower panel shows 18S and 28S rRNA. (C) Induction of Mfsd2a during thermogenesis is partially dependent on the activation of the βAR signaling pathway. WT and β-less mice (n=5) were placed at 4°C or room temperature (22°C). After 6 hrs, BAT was collected, total RNA isolated, and the level of Mfsd2a expression examined by qRT-PCR. (D) Mfsd2a is induced by β-adrenergic agonists. Total RNA was isolated from BAT of WT and β-less mice (n=5) treated with or without β-agonists (Sal, saline; Iso, isoproterenol; CL, CL316243) for 1 hour. The expression of Mfsd2a, Ucp1, and Dio2 were examined by qRT-PCR analysis. (E) Mfsd2a expression is greatly induced in BAT and liver upon fasting (16 h). Total RNA was isolated from non-fasting and fasting WT mice (n=3) and then examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.
Figure 4
Figure 4. Mfsd2a expression is induced during adaptive thermogenesis and fasting
(A) Mfsd2a expression is greatly induced during adaptive thermogenesis. Mice (n=4) were subjected to a temperature of 4°C and BAT isolated at the time intervals indicated. The levels of Mfsd2a, Ucp1, and Dio2 mRNA were assessed by qRT-PCR analysis. (B) Total RNA was isolated from the liver, kidney, and BAT (from mice kept at 22°C or 4°C for 8 h). Mfsd2a expression was examined by Northern blot analysis using a radiolabeled cDNA probe. Lower panel shows 18S and 28S rRNA. (C) Induction of Mfsd2a during thermogenesis is partially dependent on the activation of the βAR signaling pathway. WT and β-less mice (n=5) were placed at 4°C or room temperature (22°C). After 6 hrs, BAT was collected, total RNA isolated, and the level of Mfsd2a expression examined by qRT-PCR. (D) Mfsd2a is induced by β-adrenergic agonists. Total RNA was isolated from BAT of WT and β-less mice (n=5) treated with or without β-agonists (Sal, saline; Iso, isoproterenol; CL, CL316243) for 1 hour. The expression of Mfsd2a, Ucp1, and Dio2 were examined by qRT-PCR analysis. (E) Mfsd2a expression is greatly induced in BAT and liver upon fasting (16 h). Total RNA was isolated from non-fasting and fasting WT mice (n=3) and then examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.
Figure 4
Figure 4. Mfsd2a expression is induced during adaptive thermogenesis and fasting
(A) Mfsd2a expression is greatly induced during adaptive thermogenesis. Mice (n=4) were subjected to a temperature of 4°C and BAT isolated at the time intervals indicated. The levels of Mfsd2a, Ucp1, and Dio2 mRNA were assessed by qRT-PCR analysis. (B) Total RNA was isolated from the liver, kidney, and BAT (from mice kept at 22°C or 4°C for 8 h). Mfsd2a expression was examined by Northern blot analysis using a radiolabeled cDNA probe. Lower panel shows 18S and 28S rRNA. (C) Induction of Mfsd2a during thermogenesis is partially dependent on the activation of the βAR signaling pathway. WT and β-less mice (n=5) were placed at 4°C or room temperature (22°C). After 6 hrs, BAT was collected, total RNA isolated, and the level of Mfsd2a expression examined by qRT-PCR. (D) Mfsd2a is induced by β-adrenergic agonists. Total RNA was isolated from BAT of WT and β-less mice (n=5) treated with or without β-agonists (Sal, saline; Iso, isoproterenol; CL, CL316243) for 1 hour. The expression of Mfsd2a, Ucp1, and Dio2 were examined by qRT-PCR analysis. (E) Mfsd2a expression is greatly induced in BAT and liver upon fasting (16 h). Total RNA was isolated from non-fasting and fasting WT mice (n=3) and then examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.
Figure 4
Figure 4. Mfsd2a expression is induced during adaptive thermogenesis and fasting
(A) Mfsd2a expression is greatly induced during adaptive thermogenesis. Mice (n=4) were subjected to a temperature of 4°C and BAT isolated at the time intervals indicated. The levels of Mfsd2a, Ucp1, and Dio2 mRNA were assessed by qRT-PCR analysis. (B) Total RNA was isolated from the liver, kidney, and BAT (from mice kept at 22°C or 4°C for 8 h). Mfsd2a expression was examined by Northern blot analysis using a radiolabeled cDNA probe. Lower panel shows 18S and 28S rRNA. (C) Induction of Mfsd2a during thermogenesis is partially dependent on the activation of the βAR signaling pathway. WT and β-less mice (n=5) were placed at 4°C or room temperature (22°C). After 6 hrs, BAT was collected, total RNA isolated, and the level of Mfsd2a expression examined by qRT-PCR. (D) Mfsd2a is induced by β-adrenergic agonists. Total RNA was isolated from BAT of WT and β-less mice (n=5) treated with or without β-agonists (Sal, saline; Iso, isoproterenol; CL, CL316243) for 1 hour. The expression of Mfsd2a, Ucp1, and Dio2 were examined by qRT-PCR analysis. (E) Mfsd2a expression is greatly induced in BAT and liver upon fasting (16 h). Total RNA was isolated from non-fasting and fasting WT mice (n=3) and then examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.
Figure 4
Figure 4. Mfsd2a expression is induced during adaptive thermogenesis and fasting
(A) Mfsd2a expression is greatly induced during adaptive thermogenesis. Mice (n=4) were subjected to a temperature of 4°C and BAT isolated at the time intervals indicated. The levels of Mfsd2a, Ucp1, and Dio2 mRNA were assessed by qRT-PCR analysis. (B) Total RNA was isolated from the liver, kidney, and BAT (from mice kept at 22°C or 4°C for 8 h). Mfsd2a expression was examined by Northern blot analysis using a radiolabeled cDNA probe. Lower panel shows 18S and 28S rRNA. (C) Induction of Mfsd2a during thermogenesis is partially dependent on the activation of the βAR signaling pathway. WT and β-less mice (n=5) were placed at 4°C or room temperature (22°C). After 6 hrs, BAT was collected, total RNA isolated, and the level of Mfsd2a expression examined by qRT-PCR. (D) Mfsd2a is induced by β-adrenergic agonists. Total RNA was isolated from BAT of WT and β-less mice (n=5) treated with or without β-agonists (Sal, saline; Iso, isoproterenol; CL, CL316243) for 1 hour. The expression of Mfsd2a, Ucp1, and Dio2 were examined by qRT-PCR analysis. (E) Mfsd2a expression is greatly induced in BAT and liver upon fasting (16 h). Total RNA was isolated from non-fasting and fasting WT mice (n=3) and then examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.
Figure 5
Figure 5. Mfsd2a expression does not change during BAT differentiation and is induced by dibut-cAMP
(A) Mfsd2a expression does not change during BAT differentiation. Pre-adipocytes were grown to confluence (day 0) and induced to differentiate into brown adipocytes as described in Materials and Methods. At the time intervals indicated cells were collected for RNA isolation. Expression of Ucp1 and Mfsd2a mRNA were examined by qRT-PCR analysis. (Exp.) indicates cells were collected at the exponential growth phase. (B) Dibut-cAMP induces Mfsd2a expression in BAT cells. Fully differentiated brown adipocytes were treated with dibut-cAMP or NE and at different time intervals RNA was isolated and Mfsd2a and Ucp1 expression examined by qRT-PCR. (C) Loss of PGC-1α expression does not abolish the induction of Mfsd2a expression by cAMP. Fully differentiated WT and PGC-1α-KO BAT cells were treated with dibut-cAMP. After 6 h of treatment, total RNA was isolated and the expression of Mfsd2a and Ucp1 were examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.
Figure 5
Figure 5. Mfsd2a expression does not change during BAT differentiation and is induced by dibut-cAMP
(A) Mfsd2a expression does not change during BAT differentiation. Pre-adipocytes were grown to confluence (day 0) and induced to differentiate into brown adipocytes as described in Materials and Methods. At the time intervals indicated cells were collected for RNA isolation. Expression of Ucp1 and Mfsd2a mRNA were examined by qRT-PCR analysis. (Exp.) indicates cells were collected at the exponential growth phase. (B) Dibut-cAMP induces Mfsd2a expression in BAT cells. Fully differentiated brown adipocytes were treated with dibut-cAMP or NE and at different time intervals RNA was isolated and Mfsd2a and Ucp1 expression examined by qRT-PCR. (C) Loss of PGC-1α expression does not abolish the induction of Mfsd2a expression by cAMP. Fully differentiated WT and PGC-1α-KO BAT cells were treated with dibut-cAMP. After 6 h of treatment, total RNA was isolated and the expression of Mfsd2a and Ucp1 were examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.
Figure 5
Figure 5. Mfsd2a expression does not change during BAT differentiation and is induced by dibut-cAMP
(A) Mfsd2a expression does not change during BAT differentiation. Pre-adipocytes were grown to confluence (day 0) and induced to differentiate into brown adipocytes as described in Materials and Methods. At the time intervals indicated cells were collected for RNA isolation. Expression of Ucp1 and Mfsd2a mRNA were examined by qRT-PCR analysis. (Exp.) indicates cells were collected at the exponential growth phase. (B) Dibut-cAMP induces Mfsd2a expression in BAT cells. Fully differentiated brown adipocytes were treated with dibut-cAMP or NE and at different time intervals RNA was isolated and Mfsd2a and Ucp1 expression examined by qRT-PCR. (C) Loss of PGC-1α expression does not abolish the induction of Mfsd2a expression by cAMP. Fully differentiated WT and PGC-1α-KO BAT cells were treated with dibut-cAMP. After 6 h of treatment, total RNA was isolated and the expression of Mfsd2a and Ucp1 were examined by qRT-PCR analysis. Relative abundance of mRNA was calculated after normalization to 18S rRNA. *p<0.01, **p<0.05.

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