Growth hormone promotes hepatic gluconeogenesis by enhancing BTG2–YY1 signaling pathway

Growth hormone (GH) is one of the critical factors in maintaining glucose metabolism. B-cell translocation gene 2 (BTG2) and yin yang 1 (YY1) are key regulators of diverse metabolic processes. In this study, we investigated the link between GH and BTG2–YY1 signaling pathway in glucose metabolism. GH treatment elevated the expression of hepatic Btg2 and Yy1 in primary mouse hepatocytes and mouse livers. Glucose production in primary mouse hepatocytes and serum blood glucose levels were increased during GH exposure. Overexpression of hepatic Btg2 and Yy1 induced key gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6 phosphatase (G6PC) as well as glucose production in primary mouse hepatocytes, whereas this phenomenon was markedly diminished by knockdown of Btg2 and Yy1. Here, we identified the YY1-binding site on the Pck1 and G6pc gene promoters using reporter assays and point mutation analysis. The regulation of hepatic gluconeogenic genes induced by GH treatment was clearly linked with YY1 recruitment on gluconeogenic gene promoters. Overall, this study demonstrates that BTG2 and YY1 are novel regulators of GH-dependent regulation of hepatic gluconeogenic genes and glucose production. BTG2 and YY1 may be crucial therapeutic targets to intervene in metabolic dysfunction in response to the GH-dependent signaling pathway.

www.nature.com/scientificreports/ B-cell translocation gene 2 (BTG2) is an anti-proliferative gene and it is downregulated in many human cancers 15,16 . Previous reports showed that BTG2 is induced by growth factors in several cell types 17,18 . BTG2 is highly expressed in the liver and it can also be detected in various other tissues. Our previous study demonstrated that BTG2 acts as a crucial coactivator of CREB to regulate hepatic gluconeogenesis in hepatocytes 19 and as a positive regulator of hepatic gluconeogenesis via the induction of Nur77 in diabetic mouse model 20 . Yin Yang 1 (YY1) is a member of the polycomb protein family and functions as a transcription factor. It is predominantly expressed in diverse tissues and involved in the regulation of multiple target genes via chromatin modification [21][22][23] . Lu et al. demonstrated that YY1 promotes gluconeogenesis through glucocorticoid receptor in the livers of mice 24 . It has also been shown that YY1 represses insulin/insulin-like growth factor (IGF)-signaling activation and skeletal muscle-specific YY1 knockout mice improved glucose tolerance and insulin-signaling activation 25 . However, the critical role of BTG2 and YY1 in regulating the GH-dependent hepatic glucose metabolism remains unexplored.
In this study, we demonstrated that GH treatment significantly increased hepatic gluconeogenesis via the induction of BTG2 and YY1 gene expression. Moreover, disruption of Btg2 and Yy1 markedly attenuated GHmediated induction of hepatic gluconeogenesis. Our findings suggest that BTG2 and YY1 are key regulators of GH-induced hepatic gluconeogenesis. Therefore, BTG2 and YY1 may be novel potential therapeutic targets to combat metabolic dysfunction in response to the GH-dependent signaling pathway.

Results
Growth hormone increases hepatic BTG2 and YY1 gene expression. Our previous report demonstrated that GH increased hepatic gluconeogenesis, but this phenomenon was abolished by metformin-ataxia telangiectasia mutated (ATM)-AMP-activated protein kinase (AMPK)-small heterodimer partner (SHP) signaling pathway 26 . Here, we examined more deeply the role of GH on hepatic gluconeogenesis in mouse livers and primary mouse hepatocytes. GH treatment significantly increased the levels of Pck1 and G6pc along with the increased expression of Btg2 and Yy1 (Fig. 1a) in primary mouse hepatocytes. As expected, glucose production was efficiently elevated by GH treatment (Fig. 1b). Consistent with primary mouse hepatocytes data, GH challenge increased the mRNA levels of Btg2, Yy1, Pck1, and G6pc in mouse livers (Fig. 1c). Similarly, GH exposure significantly increased blood glucose levels relative to that of the control groups (Fig. 1d). Overall, these findings (a) Mouse primary hepatocytes (MPH) were treated with growth hormone (GH, 500 ng/ml) for 3 h. Gene expressions were analyzed by qPCR using the indicated primers. (b) Glucose output assay in MPH exposed to GH for 3 h. (c) Male C57BL/6 wild-type (WT) mice were injected intraperitoneally with GH (2 μg/g) for 7 days. (d) Blood glucose concentrations were measured from the indicated groups. n = 5 mice per group. *P < 0.05 vs. untreated control cells and mice. www.nature.com/scientificreports/ strongly suggest that GH plays an important role in hepatic gluconeogenesis and it can also induce Btg2 and Yy1 gene expression.
Growth hormone-mediated induction of hepatic gluconeogenesis is BTG2 dependent. Next, we investigated the critical role of BTG2 as a key modulator of gluconeogenesis using an adenoviral delivery system for the overexpression of Btg2 (Ad-Btg2) or the control green fluorescent protein (Ad-GFP) in primary mouse hepatocytes. As shown in Fig. 2a, the Btg2 mRNA was robustly increased by Ad-Btg2. Overexpression of Btg2 significantly increased the expression of Yy1, Pck1, and G6pc compared to Ad-GFP control groups, but not the expression of specificity protein 1 (Sp1), a zinc finger transcription factor known to regulate many cellular processes. Interestingly, glucose production was also increased by Ad-Btg2 compared to Ad-GFP control groups (Fig. 2b). Next, we further verified whether BTG2 modulates GH-mediated induction of gluconeogenic gene expression and glucose production in primary mouse hepatocytes. The GH-induced protein and mRNA levels of BTG2, YY1, PCK1, and G6PC were dramatically downregulated by endogenous knockdown of Btg2 (Fig. 2c,d).
Consistently, the increase of hepatic glucose production induced by GH treatment was markedly impaired when Btg2 was silenced (Fig. 2e). Taken together, these findings imply the role of BTG2 in mediating GH-induced hepatic gluconeogenesis.
Growth hormone-and BTG2-induced hepatic gluconeogenesis depends on YY1. To investigate the vital role of YY1 on hepatic gluconeogenesis by GH in primary mouse hepatocytes and the liver of mice. Yy1 expression was successfully overexpressed by adenoviral delivery system. Overexpression of Ad-Yy1 significantly augmented Pck1 and G6pc mRNA expression compared to Ad-GFP control groups in primary mouse hepatocytes and mouse livers, but not Btg2 expression ( Fig. 3a,b). Notably, glucose production was efficiently elevated by GH treatment (Fig. 3c). We further explored whether YY1 is involved in hepatic gluconeogenic gene regulation and glucose production. The protein and mRNA levels of YY1 were successfully silenced in GH-treated primary hepatocytes using lentiviral delivery system for shRNA Yy1 (shYy1). The increase of gluconeogenic genes by GH treatment was markedly reduced in Yy1 silenced group (Fig. 3d,e). Interestingly, GH-mediated induction of hepatic glucose production was also prominently decreased in Yy1 knockdown group (Fig. 3f).
To determine whether BTG2-mediated induction of gluconeogenic key enzymes and glucose production can be modulated by YY1, we evaluated the effect of Yy1 on the regulation of hepatic gluconeogenesis using Ad-Btg2 and shYy1 in primary mouse hepatocytes. Ad-Btg2 significantly increased the expression of YY1, PCK1, and G6PC genes, while this phenomenon was markedly diminished by silencing of Yy1 in primary hepatocytes, but not the expression of BTG2 (Fig. 3g,h). Similarly, Ad-Btg2-mediated increase in glucose production was markedly negated by silencing of Yy1 (Fig. 3i). Overall, these results suggest that GH-and BTG2-stimulated hepatic gluconeogenesis is mediated by YY1.

YY1 is a novel regulator of hepatic gluconeogenic gene transcription.
To determine whether the transcriptional activity of Yy1 in response to GH treatment regulates hepatic gluconeogenic gene expression at the transcriptional level, we examined transient transfection assays using luciferase reporter constructs containing the Pck1 and G6pc gene promoters in AML-12 cells. As shown in Fig. 4a, GH treatment significantly increased the activity of Pck1 and G6pc gene promoters in hepatocytes. In addition, transiently expressed Btg2 and Yy1 significantly elevated the promoter activities of Pck1 and G6pc as compared to the control groups. To identify the putative transcription activation site by GH on hepatic gluconeogenic gene promoter, serial deletion constructs of the Pck1 and G6pc gene promoters were used for transient transfection and luciferase reporter gene assay. The activity of Pck1 promoter induced by GH treatment was retained with deletion up to − 800 bp and this activation was completely lost around the − 490 bp construct indicating the presence of GH activation site between − 800 and − 490 bp of Pck1 promoter (Fig. 4b). Moreover, G6pc promoter activity in response to GH exposure was retained with a deletion up to − 500 bp, and it was lost when used the minimal promoter (− 200 bp) construct (Fig. 4c). These observations indicate that the YY1-binding element required for the GH response is located within the region between − 800 and − 490 bp on the Pck1 gene promoter and − 500 and − 200 bp on the G6pc gene promoter.
Our in-silico analysis predicted that there is YY1-binding site on Pck1 and G6pc promoters. To further evaluate the functional significance of the YY1-binding region on the Pck1 and G6pc gene promoter, site-directed mutagenesis was carried out on the Pck1 and G6pc gene promoters. Wild-type (Pck1 wt) and the mutant reporter plasmid (Pck1 mt) were transiently transfected with Btg2 and Yy1 in hepatocytes. GH treatment as well as overexpression of Btg2 and Yy1 significantly increased Yy1-dependent activity of Pck1 gene promoter, and this phenomenon was prominently abolished in Yy1-mutant Pck1 promoter (Fig. 4d). Similarly, GH treatment or transiently expressed Btg2 and Yy1 efficiently enhanced G6pc gene promoter, and this increase was significantly hampered in the YY1-binding mutated (mt) G6pc promoter (Fig. 4e). Overall, these results suggest that the YY1-binding site can be sufficient to mediate the activation of Pck1 and G6pc gene promoters in response to GH treatment. Finally, we conducted chromatin immunoprecipitation (ChIP) assays in primary mouse hepatocytes to identify the YY1-binding site on the Pck1 and G6pc gene promoter. GH exposure strongly enhanced YY1 occupancy on the proximal region (Pro, − 800/− 600) of the Pck1 promoter but not in the non-specific distal region (Dis, − 2000/− 1800) of the Pck1 promoter (Fig. 4f). Moreover, we identified GH-induced YY1 recruitment on the proximal region (Pro, − 300/− 100) of the G6pc promoter but not in the distal region (Dis, − 2000/− 1800) of the G6pc promoter (Fig. 4g). Overall, these findings strongly suggest that YY1 is recruited to both Pck1 and G6pc gene promoters to mediate GH-induced Pck1 and G6pc gene transcription. www.nature.com/scientificreports/ these findings, we suggest that the BTG2-YY1 signaling network exerts as a critical factor for the regulation of hepatic gluconeogenesis during the GH-dependent pathway.
Cui et al. have demonstrated that upregulation of multiple target genes (BTG2, c-FOS, and SOCS3) by GH treatment are mediated by specific mechanisms involving C/EBPβ and basic leucine zipper (bZIP) family transcription factors in adipocytes 27 . However, the potential link between GH and BTG2 in the regulation of hepatic gluconeogenesis has not been studied yet. Consistent with previous report, we found that GH exposure significantly increased the expression of BTG2 and YY1 genes, along with the increased key hepatic gluconeogenic gene (Pck1 and G6pc) expression as well as glucose levels in mouse livers and primary hepatocytes (Fig. 1). Therefore, we speculate that BTG2 might be an important factor to regulate hepatic gluconeogenic gene expression and glucose production in response to GH signaling.
Our previous study has revealed that the induction of BTG2 by gluconeogenic signals (fasting state, forskolin, and glucagon treatment) positively regulates hepcidin gene expression and hepatic hepcidin production involved in iron metabolism by stimulating YY1 expression 23 . BTG2 also elevated hepatic gluconeogenesis via the induction of Nur77 in the livers of diabetic mice 20 . However, several other studies have demonstrated that hepatic gluconeogenesis is regulated by hepatocyte nuclear factor (HNF)-4α, HNF-6, and STAT5 during GH exposure 26,28,29 . Particularly, we investigated whether BTG2-YY1 signaling pathway may affect hepatic gluconeogenic gene expression and glucose production in response to GH treatment in primary mouse hepatocytes and mouse livers. As anticipated, GH significantly induced hepatic gluconeogenic gene expression, glucose production, and blood glucose levels ( Fig. 1), whereas this stimulatory effect of GH was strikingly reduced in Btg2 or Yy1 silenced group (Figs. 2, 3). Our current study suggests that GH is a crucial regulator of hepatic gluconeogenesis by upregulating the BTG2-YY1 signaling network. Therefore, the identified BTG2-YY1 signaling pathway seems to be critical for hepatic GH-dependent gluconeogenic signaling.
YY1 is well-characterized to regulate hepcidin gene expression through BTG2-YY1 signaling network in mouse livers and primary hepatocytes 23 . In addition, BTG2 participates in the regulation of hepatic glucose metabolism through Nur77 and CREB induction both in vivo and in vitro 19,20 . Based on these findings, we proposed a novel molecular mechanism that BTG2-YY1 axis mediated hepatic gluconeogenic gene expression. As shown in Fig. 4, recruitment of BTG2 and YY1 on key gluconeogenic enzyme promoters were confirmed in hepatocytes. In response to GH treatment, endogenous YY1 was directly recruited to the YY1-binding site (proximal) region of both Pck1 and G6pc gene promoters. Overall, our findings suggest a novel link between hepatic gluconeogenic gene transcription and BTG2-YY1 signaling pathway. However, we cannot rule out the possibility that one or the other unexplored mechanisms by unknown transcription factors or coregulators to regulates target gene transcription.
In conclusion, this present study demonstrates that GH augments hepatic gluconeogenesis by upregulating the BTG2-YY1 signaling pathway. Moreover, it suggests that BTG2-YY1 signaling pathway mediates GH-induced hepatic gluconeogenesis. Therefore, as described in Fig. 5, we propose a novel molecular mechanism involved in hepatic glucose metabolism by the BTG2-YY1 signaling network that may provide a better understanding to develop a novel therapeutic agent for metabolic dysfunction like diabetes.

Materials and methods
Animal experiments. 8-week-old male C57BL/6 mice (Samtako, Osan, Republic of Korea) were used for the following experiments. For the growth hormone (GH) stimulation experiments, wild-type (WT) mice were injected intraperitoneally with GH (ProSpec, Tany Technogen, Ltd., Rehovot, Israel) (2 μg/g of body weight) for 7 days, as previously described 26 . At the end of the specified experiments, 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 of the Kyungpook National University according to the