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FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure.

Hino S, Sakamoto A, Nagaoka K, Anan K, Wang Y, Mimasu S, Umehara T, Yokoyama S, Kosai K, Nakao M - Nat Commun (2012)

Bottom Line: The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor.We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration.In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, the Global Center of Excellence 'Cell Fate Regulation Research and Education Unit', Kumamoto University, 860-0811, Japan. s-hino@kumamoto-u.ac.jp

ABSTRACT
Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration. In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function. Our data suggest a novel mechanism where LSD1 regulates cellular energy balance through coupling with cellular FAD biosynthesis.

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Inhibition of cellular FAD synthesis blocks LSD1-mediated repression of energy-expenditure genes.(a) Biosynthesis pathway of FAD in mammalian cells. Flavin mononucleotide (FMN). (b) Effect of the disruption of FAD synthesis on LSD1-target genes. RFK- (blue bars) or FADS- (white bars) knockdown 3T3-L1 cells were induced to differentiate for 24 h, followed by RNA extraction and quantitative RT–PCR. Values are shown as the fold difference against control siRNA-introduced samples (black bars). *P<0.05, **P<0.01 versus control siRNAs by Student's t-test. (c) Venn diagram of the probe sets induced by LSD1-KD and RFK-KD. (d) Unidirectional effects of RFK-KD on LSD1-target genes. (e) Effect of wild-type or FAD-binding mutant-type LSD1 on promoter activity. GAL4-fused LSD1-expressing plasmid (0.1 or 0.5 μg) was transfected into 293T cells, together with the GAL4x5-containing luciferase reporter construct. **P<0.01 versus GAL4 mock by Student's t-test. (f) Effect of RFK-KD on LSD1-mediated transcriptional repression. Control (black bars) or RFK (blue bars) siRNA-introduced 3T3-L1 cells were transfected with indicated GAL4 plasmids 48 h before the luciferase measurement. *P<0.05 between indicated conditions by Student's t-test. (g) Effect of lumiflavin treatment on LSD1-mediated transcriptional repression. 3T3-L1 cells were exposed to vehicle (black bars) or 50 μM lumiflavin (red bars) 48 h before the luciferase measurement. (h) Effect of lumiflavin on endogenous LSD1-target genes. Differentiating 3T3-L1 cells were exposed to vehicle (black bars), 25 μM (orange bars) or 50 μM (red bars) lumiflavin for 24 h and were subjected to RNA analyses. (i) Increase of FAD concentration during adipogenic differentiation of 3T3-L1 cells. **P<0.01 versus day 0 by Student's t-test. (j) Increase of FAD concentration after 24-hour palmitate exposure in mature 3T3-L1 adipocytes (day 7). Values are normalized to the protein concentration. *P<0.05 versus control by Student's t-test. All histogram values are means±s.d. of three independent samples.
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f6: Inhibition of cellular FAD synthesis blocks LSD1-mediated repression of energy-expenditure genes.(a) Biosynthesis pathway of FAD in mammalian cells. Flavin mononucleotide (FMN). (b) Effect of the disruption of FAD synthesis on LSD1-target genes. RFK- (blue bars) or FADS- (white bars) knockdown 3T3-L1 cells were induced to differentiate for 24 h, followed by RNA extraction and quantitative RT–PCR. Values are shown as the fold difference against control siRNA-introduced samples (black bars). *P<0.05, **P<0.01 versus control siRNAs by Student's t-test. (c) Venn diagram of the probe sets induced by LSD1-KD and RFK-KD. (d) Unidirectional effects of RFK-KD on LSD1-target genes. (e) Effect of wild-type or FAD-binding mutant-type LSD1 on promoter activity. GAL4-fused LSD1-expressing plasmid (0.1 or 0.5 μg) was transfected into 293T cells, together with the GAL4x5-containing luciferase reporter construct. **P<0.01 versus GAL4 mock by Student's t-test. (f) Effect of RFK-KD on LSD1-mediated transcriptional repression. Control (black bars) or RFK (blue bars) siRNA-introduced 3T3-L1 cells were transfected with indicated GAL4 plasmids 48 h before the luciferase measurement. *P<0.05 between indicated conditions by Student's t-test. (g) Effect of lumiflavin treatment on LSD1-mediated transcriptional repression. 3T3-L1 cells were exposed to vehicle (black bars) or 50 μM lumiflavin (red bars) 48 h before the luciferase measurement. (h) Effect of lumiflavin on endogenous LSD1-target genes. Differentiating 3T3-L1 cells were exposed to vehicle (black bars), 25 μM (orange bars) or 50 μM (red bars) lumiflavin for 24 h and were subjected to RNA analyses. (i) Increase of FAD concentration during adipogenic differentiation of 3T3-L1 cells. **P<0.01 versus day 0 by Student's t-test. (j) Increase of FAD concentration after 24-hour palmitate exposure in mature 3T3-L1 adipocytes (day 7). Values are normalized to the protein concentration. *P<0.05 versus control by Student's t-test. All histogram values are means±s.d. of three independent samples.

Mentions: To investigate the biological importance of FAD-dependent LSD1 activities, we examined whether cellular FAD synthesis affects the expression of the LSD1-target genes involved in energy metabolism. The biosynthetic pathway from riboflavin to FAD is composed of two enzymes, riboflavin kinase (RFK) and FAD synthetase (FADS)33 (Fig. 6a). The siRNA-mediated knockdown of these two genes resulted in a mild reduction of the cellular FAD content in 3T3-L1 cells whereas RFK-KD showing the stronger effect, as assessed by two different methods (Supplementary Fig. S5a,b). Expression of most LSD1-target genes was increased by RFK-KD while FADS-KD did not affect PGC-1α expression, in agreement with their effects on FAD production (Fig. 6b; Supplementary Fig. S5c). These results imply that RFK is the rate-limiting enzyme in the FAD biosynthetic process. It might be analogous to the case of the NAD+ (nicotinamide adenine dinucleotide) synthetic pathway in which Nampt, the first enzyme of the process, strongly affects the cellular NAD+ pool34. To elucidate the substantial overlap in the target genes, expression microarray analysis was performed using LSD1-KD and RFK-KD cells (Fig. 6c). A total of 132 genes were commonly induced more than twofold compared with the control under both knockdown conditions. In addition, as we focused on the probe sets upregulated by LSD1-KD, we found significant enrichment of the probe sets that were similarly upregulated by RFK-KD (P=3.5×10−47 by χ2 test), compared with those oppositely regulated (Fig. 6d).


FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure.

Hino S, Sakamoto A, Nagaoka K, Anan K, Wang Y, Mimasu S, Umehara T, Yokoyama S, Kosai K, Nakao M - Nat Commun (2012)

Inhibition of cellular FAD synthesis blocks LSD1-mediated repression of energy-expenditure genes.(a) Biosynthesis pathway of FAD in mammalian cells. Flavin mononucleotide (FMN). (b) Effect of the disruption of FAD synthesis on LSD1-target genes. RFK- (blue bars) or FADS- (white bars) knockdown 3T3-L1 cells were induced to differentiate for 24 h, followed by RNA extraction and quantitative RT–PCR. Values are shown as the fold difference against control siRNA-introduced samples (black bars). *P<0.05, **P<0.01 versus control siRNAs by Student's t-test. (c) Venn diagram of the probe sets induced by LSD1-KD and RFK-KD. (d) Unidirectional effects of RFK-KD on LSD1-target genes. (e) Effect of wild-type or FAD-binding mutant-type LSD1 on promoter activity. GAL4-fused LSD1-expressing plasmid (0.1 or 0.5 μg) was transfected into 293T cells, together with the GAL4x5-containing luciferase reporter construct. **P<0.01 versus GAL4 mock by Student's t-test. (f) Effect of RFK-KD on LSD1-mediated transcriptional repression. Control (black bars) or RFK (blue bars) siRNA-introduced 3T3-L1 cells were transfected with indicated GAL4 plasmids 48 h before the luciferase measurement. *P<0.05 between indicated conditions by Student's t-test. (g) Effect of lumiflavin treatment on LSD1-mediated transcriptional repression. 3T3-L1 cells were exposed to vehicle (black bars) or 50 μM lumiflavin (red bars) 48 h before the luciferase measurement. (h) Effect of lumiflavin on endogenous LSD1-target genes. Differentiating 3T3-L1 cells were exposed to vehicle (black bars), 25 μM (orange bars) or 50 μM (red bars) lumiflavin for 24 h and were subjected to RNA analyses. (i) Increase of FAD concentration during adipogenic differentiation of 3T3-L1 cells. **P<0.01 versus day 0 by Student's t-test. (j) Increase of FAD concentration after 24-hour palmitate exposure in mature 3T3-L1 adipocytes (day 7). Values are normalized to the protein concentration. *P<0.05 versus control by Student's t-test. All histogram values are means±s.d. of three independent samples.
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f6: Inhibition of cellular FAD synthesis blocks LSD1-mediated repression of energy-expenditure genes.(a) Biosynthesis pathway of FAD in mammalian cells. Flavin mononucleotide (FMN). (b) Effect of the disruption of FAD synthesis on LSD1-target genes. RFK- (blue bars) or FADS- (white bars) knockdown 3T3-L1 cells were induced to differentiate for 24 h, followed by RNA extraction and quantitative RT–PCR. Values are shown as the fold difference against control siRNA-introduced samples (black bars). *P<0.05, **P<0.01 versus control siRNAs by Student's t-test. (c) Venn diagram of the probe sets induced by LSD1-KD and RFK-KD. (d) Unidirectional effects of RFK-KD on LSD1-target genes. (e) Effect of wild-type or FAD-binding mutant-type LSD1 on promoter activity. GAL4-fused LSD1-expressing plasmid (0.1 or 0.5 μg) was transfected into 293T cells, together with the GAL4x5-containing luciferase reporter construct. **P<0.01 versus GAL4 mock by Student's t-test. (f) Effect of RFK-KD on LSD1-mediated transcriptional repression. Control (black bars) or RFK (blue bars) siRNA-introduced 3T3-L1 cells were transfected with indicated GAL4 plasmids 48 h before the luciferase measurement. *P<0.05 between indicated conditions by Student's t-test. (g) Effect of lumiflavin treatment on LSD1-mediated transcriptional repression. 3T3-L1 cells were exposed to vehicle (black bars) or 50 μM lumiflavin (red bars) 48 h before the luciferase measurement. (h) Effect of lumiflavin on endogenous LSD1-target genes. Differentiating 3T3-L1 cells were exposed to vehicle (black bars), 25 μM (orange bars) or 50 μM (red bars) lumiflavin for 24 h and were subjected to RNA analyses. (i) Increase of FAD concentration during adipogenic differentiation of 3T3-L1 cells. **P<0.01 versus day 0 by Student's t-test. (j) Increase of FAD concentration after 24-hour palmitate exposure in mature 3T3-L1 adipocytes (day 7). Values are normalized to the protein concentration. *P<0.05 versus control by Student's t-test. All histogram values are means±s.d. of three independent samples.
Mentions: To investigate the biological importance of FAD-dependent LSD1 activities, we examined whether cellular FAD synthesis affects the expression of the LSD1-target genes involved in energy metabolism. The biosynthetic pathway from riboflavin to FAD is composed of two enzymes, riboflavin kinase (RFK) and FAD synthetase (FADS)33 (Fig. 6a). The siRNA-mediated knockdown of these two genes resulted in a mild reduction of the cellular FAD content in 3T3-L1 cells whereas RFK-KD showing the stronger effect, as assessed by two different methods (Supplementary Fig. S5a,b). Expression of most LSD1-target genes was increased by RFK-KD while FADS-KD did not affect PGC-1α expression, in agreement with their effects on FAD production (Fig. 6b; Supplementary Fig. S5c). These results imply that RFK is the rate-limiting enzyme in the FAD biosynthetic process. It might be analogous to the case of the NAD+ (nicotinamide adenine dinucleotide) synthetic pathway in which Nampt, the first enzyme of the process, strongly affects the cellular NAD+ pool34. To elucidate the substantial overlap in the target genes, expression microarray analysis was performed using LSD1-KD and RFK-KD cells (Fig. 6c). A total of 132 genes were commonly induced more than twofold compared with the control under both knockdown conditions. In addition, as we focused on the probe sets upregulated by LSD1-KD, we found significant enrichment of the probe sets that were similarly upregulated by RFK-KD (P=3.5×10−47 by χ2 test), compared with those oppositely regulated (Fig. 6d).

Bottom Line: The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor.We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration.In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, the Global Center of Excellence 'Cell Fate Regulation Research and Education Unit', Kumamoto University, 860-0811, Japan. s-hino@kumamoto-u.ac.jp

ABSTRACT
Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration. In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function. Our data suggest a novel mechanism where LSD1 regulates cellular energy balance through coupling with cellular FAD biosynthesis.

Show MeSH
Related in: MedlinePlus