B-cell translocation gene 2 enhances fibroblast growth factor 21 production by inducing Kruppel-like factor 15

Fibroblast growth factor 21 (FGF21) is a hormone that is vital for the regulation of metabolic homeostasis. In the present study, we report that Kruppel-like factor 15 (KLF15) is a novel mediator of b-cell translocation gene 2 (BTG2)-induced FGF21 biosynthesis. The expression levels of hepatic Fgf21, Btg2, and Klf15, and the production of serum FGF21 increased significantly in fasted and forskolin (FSK)-treated mice. The overexpression of Btg2 using an adenoviral delivery system elevated FGF21 production by upregulating Klf15 transcription. Interaction studies indicated that BTG2 was co-immunoprecipitated with KLF15 and recruited by the Fgf21 promoter. The disruption of hepatic Btg2 and Klf15 genes markedly attenuated the induction of Fgf21 expression and FGF21 biosynthesis in fasted mice. Similarly, the FSK-mediated induction of Fgf21 promoter activity was strikingly ablated by silencing of Btg2 and Klf15. Taken together, these findings suggest that KLF15 and BTG2 are mediators of fasting-induced hepatic FGF21 expression. Therefore, targeting BTG2 and KLF15 might be a therapeutically important strategy for combat metabolic dysfunction.

expression in other tissues, including the kidney, pancreas, muscle, and heart 12 . KLF15 expression is upregulated in diverse tissues by glucagon and glucocorticoids during starvation or under diabetic conditions, whereas feeding and insulin downregulate KLF15 expression 12,13 . KLF15 also regulates the transcription of target genes involved in diverse physiological processes, including fibrosis, cardiovascular disease, and the immune response. It is associated with metabolic dysfunction, including cardiac hypertrophy, obesity, inflammation, and diabetes 12,14 . Gluconeogenic signals are known to modulate FGF21 production via the activation of glucocorticoid receptor (GR) and PPARα in mice subjected to prolonged fasting or starvation 15,16 . We have previously shown that BTG2 is induced under glucagon and fasting condition 7,11 . Moreover, KLF15 is known to be upregulated by glucagon and downregulated by insulin 12,13 . However, the connection of BTG2 and KLF15 in the regulation of FGF21 production is largely unknown.
In the current study, we demonstrate that BTG2 is a key modulator of FGF21 production and reveal a novel molecular mechanism that links KLF15 to the control of FGF21 biosynthesis during starvation.

Gluconeogenic signals elevate FGF21 gene expression and biosynthesis. Previous studies have
showed that gluconeogenic signals modulate FGF21 production via the GR and PPARα in mice during prolonged fasting and/or starvation 15,16 . Based on these findings, we examined the physiological connection between gluconeogenic modulators and hepatic FGF21 metabolism. Notably, the expression of Fgf21 was significantly elevated under prolonged fasting, along with the induction of Btg2 and Klf15 expression when compared with that of the fed state (Fig. 1a). Consistent with the increase in Fgf21, Btg2, and Klf15 mRNA expression, the corresponding protein levels were markedly elevated in the livers of the fasted mice (Fig. 1b). In addition, serum FGF21 concentrations was also increased in the fasted mice compared to the fed state (Fig. 1c). Furthermore, we examined the critical effect of gluconeogenic signaling on the regulation of Fgf21 expression and the secretion of FGF21 in the liver. FSK treatment enhanced the protein and mRNA levels of FGF21, BTG2, and KLF15 (Fig. 1d,e). Likewise, FSK challenge also increased the serum FGF21 concentration relative to that of the control groups (Fig. 1f). Collectively, these findings suggested a potential link between BTG2 and FGF21 biosynthesis in response to gluconeogenic signals.
BTG2 elevates FGF21 production via the induction of KLF15. We have attempted to investigate the critical role of BTG2 as a key modulator of FGF21 biosynthesis using an adenoviral delivery system expressing Btg2 (Ad-Btg2) or a control green fluorescent protein (Ad-GFP) in mouse livers. Ad-Btg2 was successfully delivered to the livers of wild-type (WT) mice via tail vein injection. The expression levels of Klf15 and Fgf21 were significantly higher in the Ad-Btg2-infected mice than in the Ad-GFP control mice (Fig. 2a,b). As expected, Ad-Btg2 significantly increased the serum FGF21 level compared to that in the Ad-GFP control mice (Fig. 2c). Next, to determine whether BTG2-mediated induction of FGF21 expression and production can be modulated by KLF15, we assessed the effect of Klf15 on the regulation of FGF21 gene expression and biosynthesis using adenoviral-mediated overexpression of Btg2 (Ad-Btg2) and knockdown of Klf15 (Ad-shKlf15) both in vivo and in vitro. Ad-Btg2 effectively enhanced Klf15 and Fgf21 mRNA levels, while this phenomenon was markedly negated by silencing of Klf15 in mouse livers and hepatocytes (Fig. 2d,f). Similarly, the production of FGF21 increased by Ad-Btg2 was remarkably decreased in Klf15 knockdown hepatocytes and mouse livers (Fig. 2e,g). Interestingly, Ad-shKlf15 slightly reduced basal Fgf21 expression in the hepatocytes and mice relative to the Ad-GFP control group, but not the production of FGF21 ( Fig. 2d-g). To further confirm the transcriptional activity of Fgf21 by FSK treatment, Btg2, Klf15, we investigated Fgf21 promoter activity in hepatocytes. Notably, FSK exposure or transiently expressed Btg2 significantly elevated the promoter activity of Fgf21, whereas this stimulation was strikingly blunted in Klf15 knockdown groups compared to the control groups (Fig. 2h). Overall, these results demonstrate that BTG2 acts as a major modulator of FGF21 gene expression and biosynthesis by depending on KLF15 both in vivo and in vitro.
Gluconeogenic signal-stimulated FGF21 production depends on BTG2. We further investigated the pivotal role of BTG2 in fasting-mediated FGF21 metabolism using lentiviral-mediated knockdown of Btg2 (shBtg2) in mouse livers. The expression of Btg2 was successfully attenuated in the mouse livers. The elevation of Btg2, Klf15, and Fgf21 mRNA and protein levels during fasting were markedly alleviated by endogenous Btg2 knockdown (Fig. 3a,b). As anticipated, the increase of serum FGF21 concentration induced by fasting was strikingly reduced in the Btg2 knockdown mice (Fig. 3c). Moreover, basal FGF21 expression was weakly attenuated in Btg2-silenced mice compared to the control group, but not the secretion of FGF21 (Fig. 3a-c). Next, we further verified whether BTG2 modulates the transcriptional activity of Fgf21 in hepatocytes. As shown in Fig. 3d, Fgf21 www.nature.com/scientificreports www.nature.com/scientificreports/ promoter activity was enhanced by FSK exposure, and this stimulatory effect of FSK was markedly diminished when Btg2 was silenced. Taken together, these findings suggest that BTG2 plays an important role in modulating FGF21 production during fasting.
KLF15 is required for fasting-induced FGF21 expression and biosynthesis. We explored the possible effects of KLF15 on FGF21 gene regulation and production in response to gluconeogenic stimuli in mouse livers using Ad-Klf15. As shown in Fig. 4a,b, qPCR and Western blot analysis showed the successful overexpression of Klf15 in mouse livers. Ad-Klf15 significantly elevated Fgf21 gene expression (Fig. 4a,b), and consequently increased serum FGF21 levels compared with those in the control groups (Fig. 4c). We further investigated the direct effect of KLF15 on fasting-induced FGF21 gene expression and biosynthesis using Ad-shKlf15. The expression of Klf15 in the liver of mice was successfully decreased by Ad-shKlf15 delivery. The increase in Fgf21 gene expression induced by fasting was markedly attenuated by Klf15 silencing (Fig. 4d,e). Moreover, the fasting-induced serum FGF21 concentration was dramatically diminished by the knockdown of endogenous Klf15 (Fig. 4f). Notably, basal Fgf21 expression was slightly reduced in the Klf15-silenced mice relative to the control group, but not the production of FGF21 (Fig. 4d-f). Collectively, these observations strongly suggest that gluconeogenic stimuli guide FGF21 gene expression and production and are partially dependent on KLF15.
BTG2 physically interacts with KLF15 and controls KLF15 occupancy on the Fgf21 promoter. To further elucidate whether BTG2 and KLF15 in the regulation of Fgf21 gene transcription via physically interaction, we carried out co-immunoprecipitation (Co-IP) assays using lysates from fasted and fed mouse livers. Endogenous BTG2 interacted strongly with KLF15 protein in the fasted mice compared to the fed mice (Fig. 5a). We identified the KLF15-binding site on the Fgf21 promoter using in silico analysis. Our reporter gene assay indicated that Btg2 and Klf15 alone increased Fgf21 promoter activity, whereas this stimulation was completely negated in the KLF15-binding site-mutated Fgf21 promoter compared to the control group (Fig. 5b). To further confirm that BTG2 affects the recruitment of KLF15 protein to the Fgf21 promoter, we carried out using a chromatin immunoprecipitation (ChIP) assay with an anti-KLF15 antibody in the mouse livers. As expected, endogenous KLF15 occupancy of the proximal (Pro) region was significantly enhanced by fasting and/or Ad-Btg2 compared to that in the control groups. However, this effect was totally lost in the nonspecific distal (Dis) region of the Fgf21 promoter (Fig. 5c). Overall, these results strongly suggest that BTG2 associates with KLF15 and enhances Fgf21 transcription by recruiting KLF15. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In the current study, we demonstrated that fasting and/or FSK treatment remarkably induced hepatic FGF21 gene transcription, and BTG2 elevated FGF21 biosynthesis via the upregulation of KLF15 expression. Conversely, the stimulatory effect of fasting or FSK exposure on hepatic FGF21 gene expression and production was prominently attenuated by the disruption of Btg2 and Klf15 expression in mouse livers and hepatocytes. Therefore, we propose that the fasting-BTG2-KLF15 signaling network may represent a novel molecular mechanism underlying the modulation of hepatic FGF21 gene expression and its biosynthesis.
Several previous reports have shown that gluconeogenic signals modulate hepatic FGF21 gene expression and biosynthesis in rodents [15][16][17] . Our previous study revealed that the elevation of BTG2 by glucagon increased hepcidin levels and gluconeogenesis in mouse livers 11,18 . However, the potent effect of BTG2 in modulating hepatic FGF21 gene expression and FGF21 biosynthesis remains largely unexplored. The results of the current study demonstrate that enhanced BTG2 levels induced by fasting or FSK challenge modulate hepatic FGF21 production by upregulating the expression of Klf15. Fasting and FSK treatment significantly increase the expression of Btg2, Klf15, and Fgf21 in mouse livers and significantly elevate the concentration of serum FGF21 (Fig. 1). Ad-Btg2 significantly increased hepatic FGF21 gene expression and production by stimulating KLF15 expression (Fig. 2), whereas this stimulation was markedly attenuated in Btg2 knockdown cells and mice (Fig. 3). Our findings suggest that BTG2 plays a crucial role in modulating the fasting-mediated induction of hepatic FGF21 gene expression and biosynthesis via the induction of KLF15.
KLF15 modulates the transcription of several target genes that are involved in various physiological processes, such as fibrosis, cardiac hypertrophy, obesity, inflammation, insulin resistance, and diabetes [12][13][14]19,20 . Based on these findings, we investigated the molecular mechanism underlying fasting-stimulated Fgf21 gene transcription by the BTG2-KLF15 pathway both in vivo and in vitro. First, the increase in FGF21 gene expression induced by fasting was markedly diminished by silencing of Klf15 relative to that in the control groups (Fig. 4). Second, BTG2 strongly interacted with KLF15 in the liver lysates from fasted mice compared to the lysates from fed mice. Third, the endogenous KLF15 occupancy of the proximal region of the Fgf21 promoter was effectively enhanced by fasting and Ad-Btg2 transduction; and the transcriptional activity of Fgf21 was significantly increased by Btg2 and Klf15 in the hepatocytes (Fig. 5). Collectively, our results reveal a connection between Fgf21 transcription and the BTG2-KLF15 axis. However, we cannot rule out the possibility that the detailed molecular mechanism that connects FGF21 gene expression and the BTG2-KLF15 signaling network depends on unknown mechanisms such as an association with transcriptional co-activators and competition with co-repressors, microRNAs, and protein stability to modulate hepatic FGF21 gene transcription.
In conclusion, the present study demonstrate that FGF21 is a novel target of BTG2, and that BTG2 promotes FGF21 biosynthesis by upregulating the expression of Klf15 during fasting or FSK exposure. These findings suggest that the elevation of BTG2 by gluconeogenic signals modulates hepatic FGF21 homeostasis by inducing the expression of Klf15. Therefore, as depicted in Fig. 6, a molecular mechanism involving hepatic FGF21 homeostasis in response to BTG2-KLF15 signaling may provide the basis for the development of novel therapeutic agents for the treatment of metabolic disorders. www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Animals. We used 8-week-old male C57BL/6 mice (Samtako, Osan, Republic of Korea) for the experiments, as described below 11 . For the fasting and feeding experiments, we fed the mice ad libitum, and then fasted them for 24 h. For the forskolin (FSK) stimulation experiments, we injected the mice intraperitoneally with FSK (Sigma-Aldrich, St. Louis, MO, USA) at a dose rate of 5 mg/kg of body weight and left them for 6 h. For the Btg2 and Klf15 overexpression experiments, we injected wild-type (WT) mice with adenoviral vectors expressing Btg2 and Klf15 (1 × 10 9 plaque-forming units, pfu) via their tail veins for 7 days. For the disruption of the Btg2 and Klf15 genes, we injected WT mice with lentivirus short hairpin RNA (shRNA) targeting Btg2 (shBtg2, a single dose of 1 × 10 9 transducing units, TU/mL) and with Ad-shKlf15 (1 × 10 9 pfu) through their tail veins for 7 days. At the end of the specified experiments or challenge periods, we euthanized the mice with CO 2 , and harvested liver tissues and blood samples. All animal experiments and protocols were approved and performed by the Institutional Animal Care and Use Committee (IACUC) of the Kyungpook National University according to the rules and guidelines of the National Institutes of Health (NIH).
Measurement of serum FGF21. We collected the blood samples immediately, and determined serum