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Purβ promotes hepatic glucose production by increasing Adcy6 transcription

Objective Enhanced glucagon signaling and hepatic glucose production (HGP) can account for hyperglycemia in patients with obesity and type 2 diabetes. However, the detailed molecular mechanisms underlying the enhanced HGP in these patients are not fully understood. Here, we identify Purβ as a positive regulator of HGP and study its molecular mechanisms in the regulation of HGP both in vivo and in vitro. Methods Adenovirus-mediated knockdown or overexpression of Purβ was performed in either primary hepatocytes or the livers of db/db mice. Glucose metabolism, insulin sensitivity, and HGP were determined by glucose, insulin, and lactate tolerance tests, respectively. Purβ/ADCY6 protein levels, glucagon signaling (p-CREB/CREB), and insulin signaling (p-Akt/Akt) were measured by immunoblotting. Gene expression was measured by RNA-seq and real-time quantitative polymerase chain reaction. Luciferase reporter and chromatin immunoprecipitation assays were used to study the interaction between Purβ and the Adcy6 promoter. Results Purβ was abnormally elevated in obese mice and was also increased under fasting conditions or via the glucagon signaling pathway, which promoted HGP by increasing Adcy6 expression. Liver-specific knockdown of Purβ in db/db mice significantly ameliorated hyperglycemia and glucose intolerance by suppressing the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway. Consistent with this observation, the knockdown of Purβ also inhibited glucose production in isolated primary hepatocytes by inhibiting the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway, whereas the overexpression of Purβ promoted glucose production by activating this signaling pathway. Mechanistically, Purβ directly binds to the promoter of the Adcy6 gene and thereby promotes its transcription. Conclusions Taken together, these results illustrate a new model in which Purβ functions to regulate the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway to help maintain glucose homeostasis.


INTRODUCTION
The liver is the main organ producing glucose via glycogenolysis and gluconeogenesis in response to fasting. Under fasting conditions, hepatic glucose production (HGP) is increased in response to elevated blood glucagon levels [1,2]. Conversely, under fed conditions, HGP is suppressed by increased plasma insulin levels [1]. Thus, the HGP rate is determined by a balance between glucagon and insulin. Glucagon, via its G proteinecoupled receptors, stimulates cAMP-mediated activation of protein kinase A (PKA), and PKA phosphorylates cAMP response element-binding protein (CREB) on Ser 133 [3,4], which in turn activates the transcription of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), both key gluconeogenic genes [3,4]. Insulin inhibits HGP by suppressing the expression of PEPCK and G6Pase [1,5]. In patients with obesity and type 2 diabetes, HGP is abnormally elevated, thus contributing to hyperglycemia and glucose intolerance [2,5]. Enhancement of the glucagon signaling pathway and hepatic insulin resistance are two major causes of enhanced HGP [2]. However, the detailed molecular mechanisms underlying this enhanced HGP in patients with obesity are not fully understood.
Purine-rich element binding proteins, such as Pura, Purb, and Purg, which form the evolutionarily conserved Pur family, are able to bind to purine-rich single-or double-stranded DNA or RNA [6]. Pur proteins play important roles in development, especially in the development of brain [7,8] and myeloid cells [8], and they are also involved in many human diseases, including cancer [9e11] and fragile-X mental retardation syndrome [7,8,12,13]. Recently, Purb has been shown to bind to lncRNA (lnc-HOXA1) and mediate repression of Hoxa1 transcription [14]. In addition, Purb binds to DNA and negatively or positively regulates gene expression in a cell typeespecific manner. For example, Purb serves as a repressor that inhibits the expression of smooth muscle a-actin in fibroblasts and vascular smooth muscles [15,16], whereas it also acts as a transcription factor that increases mTOR and SREBP1 expression in mammary epithelial cells [17]. However, whether Purb regulates HGP is largely unknown. Here we identify Purb as a positive regulator of HGP. Purb, induced by fasting or glucagon, promotes HGP by increasing Adcy6 transcription, leading to cAMP accumulation, increased PKA activity, CREB activation, and increased transcription of PEPCK and G6Pase, both key gluconeogenic genes. Purb is abnormally elevated in mice with obesity, and liver-specific knockdown of Purb in db/db mice significantly ameliorates hyperglycemia and glucose intolerance by suppressing the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway. Consistent with this observation, in isolated primary hepatocytes, knockdown of Purb also inhibits glucose production by inhibiting this signaling pathway. Moreover, Purb binds to the promoter of the Adcy6 gene, promoting its expression and activating the cAMP/PKA/CREB signaling pathway. These results support a new model in which Purb regulates the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway to help maintain glucose homeostasis, indicating that Purb/ADCY6 may serve as a promising drug target for the treatment of hyperglycemia in patients with obesity.

MATERIALS AND METHODS
2.1. Animals C57BL/6 and db/db mice were purchased from GemPharmatech (Nanjing, China). Mice were housed on a 12-h light/12-h dark cycle and fed either a normal chow or a high-fat diet with free access to water. All animal procedures described in this study were performed in adherence with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and with the approval by the Institutional Animal Care and Use Committee of Harbin Institute of Technology. Liver-specific Purb knockdown db/db mice were generated via tail vain injection of a purified adenovirus expressing shPURB1 for 8e12 days, and the experiments were conducted in conscious mice. In glucose tolerance tests, insulin tolerance tests, glucagon tolerance tests, and lactate tolerance tests, mice were fasted 6 h (8:00 AMe2:00 PM) and intraperitoneally injected with glucose (0.5 g/kg body weight), insulin (4 U/kg body weight), glucagon (6 mg/kg body weight), and sodium lactate (0.5 g/kg body weight), respectively. Blood glucose was monitored after injection. Blood samples were collected from tail veins, and blood glucose was measured as described previously [18,19]. Plasma insulin was measured using insulin enzyme-linked immunosorbent assay (ELISA) kits (MS100, EZassay). Liver triacylglycerol (TAG) levels were measured using Free Glycerol Reagent (Sigma).
2.2. Primary hepatocyte cell cultures, adenoviral infection, and HGP assays Primary hepatocytes were isolated from C57BL/6 mice by liver perfusion with type II collagenase (Worthington Biochem, Lakewood, NJ) and cultured at 37 C and 5% CO 2 in DMEM medium supplemented with 5% fetal bovine serum. For Purb overexpression experiments, primary hepatocytes were infected with bGal or Purb adenoviruses overnight. For knockdown of Purb, two target sequences (shPURB1: GTCGGTATGCAGATGAAATGA and shPURB2: GATGAAAT-GAAAGAGATCCAG) were selected. For Purb knockdown experiments, primary hepatocytes were infected with Scramble, shPURB1, or shPURB2 adenoviruses for 30 h. These hepatocytes were used for immunoblotting, quantitative polymerase chain reaction, RNA sequencing, cAMP production, PKA activity, and HGP assays. For HGP assays, these hepatocytes were incubated in Hank's Balanced Salt Solution supplemented with 10 mM lactate and 1 mM pyruvate in the presence or absence of glucagon (100 nM) for 4 h. Glucose in the medium was measured and normalized to total protein levels.
2.4. Quantitative real-time PCR (RT-qPCR) analysis qPCR analysis was performed as previously described [18,19,22]. Briefly, total RNAs were extracted from indicated hepatocytes or livers using TriPure Isolation Reagent (Roche, Mannheim, Germany). The first-strand cDNAs were synthesized using random primers and M-MLV reverse transcriptase (Promega, Madison, WI). RNA abundance was measured using the Roche LightCycler 480 real-time PCR system (Roche, Mannheim, Germany). The expression of individual genes was normalized to the expression of 36B4, a housekeeping gene. Primers 2.5. Nuclear run-on RT-qPCR Nascent mRNA levels were measured by nuclear run-on RT-qPCR following a published protocol [23]. Briefly, primary hepatocytes were infected with bGal or Purb adenoviruses overnight. Nuclei were extracted in NP-40 lysis buffer (10 mM Tris HCl pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , and 0.5% NP-40) and then incubated with BrUTP and unlabeled ribonucleotides in a transcription reaction buffer supplemented with 100U RNase OUT, 0.5 mM BrUTP, 1 mM ATP, 1 mM GTP, 1 mM CTP, and 0.5 mM UTP at 30 C for 30 min. Nuclear RNA was extracted using TriPure Isolation Reagent. Labeled and unlabeled nuclear RNA samples were immunoprecipitated with anti-BrdU antibodies. Nascent Adcy6 mRNA levels were then measured by RT-qPCR and normalized by 36B4.
2.6. cAMP and PKA activity assays Mice were fasted for 20e24 h, and livers were harvested for cAMP and PKA activity assays. Primary hepatocytes were infected with indicated adenoviruses and then treated with 100 nM glucagon for 10 min. cAMP was measured using an ELISA kit (H164-1-2, Nanjing Jiancheng). For PKA activity assays, livers and hepatocytes were lysed in buffer containing 20 mM MOPS, 50 mM b-glycerolphosphate, 50 mM sodium fluoride, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 10 mg/ml leupeptin and aprotinin. PKA activity assays were performed following the manufacturer's protocol (ab9435, Abcam).

RNA sequencing
Total RNAs were extracted from hepatocytes using TriPure Isolation Reagent (Roche, Mannheim, Germany). RNA-seq was performed by using the Illumina NovaSeq 6000 platform. Paired-end clean reads were aligned to the mouse reference genome (Ensemble_GRCm38.96) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1) as described previously [19]. RNA-seq data that support the findings of this study have been deposited in GEO under accession code GSE136728.

Luciferase reporter assays
Mouse Adcy6 promoter (À2001 to À1 or À1001 to À1) luciferase reporter plasmids and b-galactosidase reporter plasmids were transiently cotransfected with Purb expression plasmids into HEK293T cells using polyethylenimine reagents. Cells were collected 24 h after transfection, and luciferase activity was measured using a luciferase assay system (Promega Corporation). Luciferase activity was normalized to b-galactosidase levels.

Statistical analysis
Data were presented as means AE SEM. Differences between groups were analyzed by two-tailed Student's t tests. P < 0.05 was considered statistically significant.

RESULTS
3.1. Identification of Purb as a positive regulator of HGP HGP is increased during fasting via enhanced glucagon/cAMP/CREB signaling. Thus, fasting-induced genes may regulate HGP. We first measured the levels of the Purb mRNA and protein in the livers of fasted mice via qPCR and immunoblotting analyses, respectively. As shown in Figure 1A,B, fasting increased the levels of both Purb mRNA and protein. As fasting increases glucagon secretion and its signaling pathway in the liver, these observations raise the possibility that glucagon may increase the levels of hepatic Purb in primary hepatocytes. To test this hypothesis, primary hepatocytes were isolated from C57BL/6 mice and treated with glucagon. As shown in Figure 1C,D, glucagon treatment increased both the Purb mRNA and protein levels.
To determine whether Purb was essential for HGP, primary hepatocytes from C57BL/6 mice were infected with an Ad-shPURB1 or Ad-shPURB2 adenovirus to knockdown Purb expression, and HGP assays were then performed. Adenovirus-mediated expression of shPURB1 or shPURB2 in primary hepatocytes led to a significant reduction in Purb ( Figure 1E), which resulted in a decrease in HGP under both basal and glucagon-treated conditions ( Figure 1F). To determine whether Purb promotes HGP, primary hepatocyte cultures were prepared from C57BL/6 mice and infected with Purb or bGal adenoviruses. The results indicated that recombinant Purb was dramatically increased in Purb adenovirus-infected hepatocytes ( Figure 1G). These infected hepatocytes were then treated with or without glucagon and subjected to HGP assays. The results demonstrated that Purb overexpression could significantly enhance HGP under both basal and glucagon-treated conditions ( Figure 1H). These results demonstrated that Purb could function to promote HGP.
3.2. Liver-specific knockdown of Purb protects against hyperglycemia and glucose intolerance in obesity Enhanced HGP contributes to hyperglycemia in both patients and rodents with obesity. To determine whether Purb expression is abnormally increased under such conditions, both Purb mRNA and protein levels were measured using qPCR and western blot analysis, respectively, in two obese mouse models (high-fat-dieteinduced obesity and leptin receptor deficiency [db/db] mice). Both Purb mRNA and protein levels were significantly increased in the livers of these models (Figure 2A,B), and these data indicate that Purb might function to regulate hepatic glucose metabolism in both patients and rodents with obesity. To determine whether Purb could regulate hepatic glucose metabolism in obese mice, we generated liver-specific Purb knockdown db/db mice via tail-vain injection of a purified adenovirus expressing shPURB1 and measured the blood glucose and glucose tolerance in these mice. The expression of Purb was significantly decreased in the liver of Purb-KD db/db mice compared with control db/db mice ( Figure 2C), but body weight was similar between Purb-KD and control db/db mice ( Figure 2D). However, fasting blood glucose was 42.8% lower in Purb-KD than in the control db/db mice ( Figure 2E), and blood glucose levels were also reduced by 36.8% in the former mice group under fed conditions ( Figure 2F). Plasma insulin levels were also significantly decreased in Purb-KD db/db mice ( Figure 2G). Glucose tolerance in Purb-KD db/db mice was significantly improved ( Figure 2H). The area under the curve (AUC) was decreased by 62.9% in Purb-KD db/db mice ( Figure 2I). To further assess insulin sensitivity in these mice, insulin tolerance tests were performed. Surprisingly, exogenous insulin reduced blood glucose in these two groups to a similar extent ( Figure 2J). These data suggest that the knockdown of Purb can ameliorate hyperglycemia and glucose intolerance in db/db mice independent of insulin sensitivity.
3.3. Purb does not regulate hepatic insulin signaling, steatosis, or inflammation To further determine whether Purb regulates glucose metabolism independent of insulin signaling in obesity, we measured insulin-induced phosphorylation of Akt in Purb-KD db/db mice. The phosphorylation (Ser 473 and Thr 308) of Akt was similar in Purb-KD and control db/db mice ( Figure 3A). Insulin sensitivity was also associated with hepatic steatosis and inflammation. As shown in Figure 3B,C, liver-specific knockdown of Purb did not alter liver TAG levels or the expression of inflammatory genes such as IL1b, IL6, and TNFa. Furthermore, insulin-induced phosphorylation (Ser 473 and Thr 308) of Akt was not altered by either knockdown or overexpression of Purb in primary hepatocytes ( Figure 3D,E). These data demonstrated that Purb could regulate hepatic glucose metabolism independent of insulin signaling, hepatic steatosis, and inflammation.

Liver-specific knockdown of Purb decreases glucagon sensitivity and gluconeogenesis in obesity
In both patients and rodents with obesity and type 2 diabetes, plasma glucagon levels, glucagon sensitivity, and glucagon/CREB signaling are abnormally increased, contributing to higher HGP and hyperglycemia. To determine whether Purb regulates glucagon-induced gluconeogenesis, glucagon tolerance tests and lactate tolerance tests were measured in Purb-KD and control db/db mice. Exogenous glucagon markedly increased blood glucose levels in the control db/db mice; however, its ability to increase blood glucose was severely impaired in Purb-KD db/db mice ( Figure 4A), with the AUC decreased by 46% in Purb-KD db/db mice ( Figure 4B). Hepatic gluconeogenesis, as estimated by lactate tolerance tests, was also significantly lower in Purb-KD db/db mice, with the AUC reduced by 64.7% ( Figure 4C,D). To determine whether hepatic Purb modulates glucagon-stimulated CREB phosphorylation, Purb-KD and control db/db mice were fasted for w20 h, and liver tissue extracts were immunoblotted using a phospho-CREB (pSer133) antibody. This analysis indicated that CREB phosphorylation was decreased by 62% in Purb-KD db/db mice ( Figure 4E). In addition, total CREB levels were slightly increased in Purb-KD db/db mice compared with the control db/db mice ( Figure 4E), possibly compensating for the decreased p-CREB levels.
To determine whether Purb regulates the hepatic gluconeogenic program, liver mRNA was extracted and used to measure the mRNA abundance of key genes via qPCR. The expression of G6Pase and PEPCK was dramatically decreased by 56.7% and 56.2% in Purb-KD db/db mice, respectively ( Figure 4F,G). These data suggest that hepatic knockdown of Purb can ameliorate hyperglycemia and glucose intolerance, and this most likely occurs via a decrease in HGP in obese mice.

Purb promotes the hepatic gluconeogenic program in primary hepatocytes
Knockdown of Purb decreased HGP, whereas overexpression of Purb increased HGP in primary hepatocytes ( Figure 1E-H), indicating that Purb could cell-autonomously regulate HGP. To further confirm that Purb cell-autonomously regulates the hepatic gluconeogenic program, the expression of G6Pase and PEPCK and its upstream regulator p-CREB levels were measured in Purb knockdown or overexpressing hepatocytes. The expression of G6Pase and PEPCK was significantly reduced in Purb knockdown hepatocytes ( Figure 5A,B), which was most likely due to decreased p-CREB levels ( Figure 5C). Conversely, the expression of G6Pase and PEPCK was significantly increased in Figure 4: Knockdown of Purb decreases glucagon sensitivity and gluconeogenesis in obesity. (AeB) Purb-KD and control db/db mice were fasted for 6 h and intraperitoneally injected with glucagon (6 mg/kg body weight). Blood glucose was monitored after injection, and AUC was calculated. (CeD) Hepatic gluconeogenesis was measured by injection of sodium lactate (0.5 g/kg body weight) in Purb-KD and control db/db mice fasted for 6 h, and AUC was calculated. (E) p-CREB, CREB, Purb, and Tubulin protein levels were measured by western blot. (FeG) G6Pase and PEPCK mRNA levels were measured by qPCR. n ¼ 7e8. *p < 0.05. **p < 0.01. hepatocytes overexpressing Purb ( Figure 5D,E), likely because of increased p-CREB levels ( Figure 5F). However, CREB protein levels did not show any change in Purb-overexpressing and Purb KD hepatocytes ( Figure 5C, F, and Supplemental Fig. 1), suggesting that Purb does not regulate CREB expression.
3.6. Purb promotes hepatic cAMP production and PKA activity Both cAMP and PKA function upstream of the CREB signaling pathway. To determine whether Purb can regulate hepatic cAMP production and PKA activity, intracellular cAMP levels and PKA activity were measured in Purb-overexpressing hepatocytes, PurbKD hepatocytes, and livers of PurbKD db/db mice. Overexpression of Purb in primary hepatocytes increased cAMP levels and PKA activity ( Figure 6A,B), whereas knockdown of Purb decreased cAMP levels and PKA activity both in vitro (Figure 6C,D) and in vivo ( Figure 6E,F). These data suggest that Purb could promote the CREB signaling pathway and hepatic gluconeogenic program by increasing hepatic cAMP production and PKA activity. Figure 5: Purb promotes the hepatic gluconeogenic program in primary hepatocytes. (AeB) Primary hepatocytes were prepared from C57BL/6 males (8e10 weeks) and infected with Scramble, shPURB1, or shPURB2 adenoviruses for 30 h and then treated with or without glucagon for 2 h. The abundance of G6Pase and PEPCK mRNA levels was measured by qPCR (n ¼ 3e4). (C) Primary hepatocytes were infected with Scramble, shPURB1, or shPURB2 adenoviruses for 30 h and then treated with or without glucagon for 30 min. p-CREB, CREB, Purb, and Tubulin protein levels were measured by immunoblotting. (DeE) Primary hepatocytes were infected with bGal or Purb adenovirus overnight and then treated with or without glucagon for 2 h. The abundance of G6Pase and PEPCK mRNA levels was measured by qPCR (n ¼ 4). (F) Primary hepatocytes were infected with bGal or Purb adenovirus overnight and then treated with or without glucagon for 30 min. p-CREB, CREB, Flag, and Tubulin protein levels were measured by immunoblotting. *p < 0.05. **p < 0.01. Original Article 92 3.7. Purb promotes Adcy6 expression Hepatic cAMP homeostasis is controlled by adenylate cyclases (ADCYs) and phosphodiesterases (PDEs), and Purb promotes hepatic cAMP production either by increasing expression of ADCYs or by decreasing expression of PDEs. To address this possibility, RNA sequencing (RNA-seq) analysis was performed in Purb-overexpressing and PurbKD hepatocytes. As shown in Figure 7A, the expression of liverenriched PDEs, such as Pde4b, Pde3b, Pde8a, Pde7a, Pde6d, and Pde5a, did not show any correlated change in Purb-overexpressing and Purb KD hepatocytes. However, the expression of liver-enriched Adcy6 was upregulated by overexpression of Purb and downregulated by knockdown of Purb ( Figure 7B). RT-qPCR and Figure 6: Purb promotes hepatic cAMP production and PKA activity. (AeB) Primary hepatocytes were infected with bGal or Purb adenovirus overnight and then treated with glucagon (100 nM) for 10 min. cAMP levels and PKA activity were measured using commercial kits (n ¼ 5e6). (CeD) Primary hepatocytes were prepared from C57BL/6 males (8e10 weeks) and infected with Scramble or shPURB1 adenoviruses for 30 h and then treated with glucagon (100 nM) for 10 min. cAMP levels and PKA activity were measured using commercial kits (n ¼ 5e6). (EeF) cAMP levels and PKA activity in Purb-KD and control db/db mice were measured using commercial kits (n ¼ 7e8). *p < 0.05. **p < 0.01. immunoblotting analysis showed that overexpression of Purb increased both the ADCY6 mRNA and protein levels ( Figure 7C), whereas knockdown of Purb decreased ADCY6 expression ( Figure 7D). Furthermore, liver-specific knockdown of Purb in db/db mice also resulted in a significant decrease in both the ADCY6 mRNA and protein levels ( Figure 7E). Interestingly, similar to Purb, hepatic ADCY6 expression could also be induced by fasting ( Figure 7F) and glucagon (Supplemental Fig. 2). Hepatic ADCY6 expression was also abnormally elevated in the two obese mouse models ( Figure 7G,H). These data demonstrate that Purb could promote HGP and lead to hyperglycemia by increasing Adcy6 expression.
3.8. Purb directly binds to the Adcy6 promoter and increases its transcription To further test whether Purb promotes Adcy6 expression at the transcriptional level, nascent Adcy6 mRNA levels were measured by performing nuclear run-on RT-qPCR. As shown in Figure 8A, the nascent Adcy6 mRNA level was significantly increased in Purb-overexpressing hepatocytes, indicating that Purb promoted Adcy6 transcription. Furthermore, ChIP assays showed that Purb directly binds to the Adcy6 promoter at À1558 to À1850 (Figure 8B), which is a purine-enriched region. In addition, Purb increased Adcy6 promotercontrolled luciferase activity in a dose-dependent manner, and deletion of the purine-enriched region abolished this enhancement ( Figure 8C). These results demonstrated that Purb directly binds to the Adcy6 promoter and increases its transcription.

DISCUSSION
HGP is controlled by glucagon and insulin, and enhanced glucagon signaling in the liver leads to increased HGP, contributing to hyperglycemia in both patients and rodents with obesity and type 2 diabetes luciferase reporter plasmids and b-galactosidase reporter plasmids were transiently cotransfected with Purb expression plasmids into HEK293T cells. Luciferase activity was measured 24 h after transfection and normalized to bGal levels (n ¼ 6). *p < 0.05. **p < 0.01. [3,4]. The detailed molecular mechanisms underlying this enhanced HGP are not yet fully understood. In this study, Purb was identified as a positive regulator of HGP and was shown to promote HGP by increasing Adcy6 expression and subsequent activation of the cAMP/PKA/CREB signaling pathway, which contributes to the occurrence of hyperglycemia in obesity. This study provides several lines of evidence indicating that Purb acts as a positive regulator of HGP. First, hepatic Purb expression was increased by fasting and glucagon. Glucagon could activate CREB via the cAMP/PKA signaling pathway. We noted that there are four binding sites of CREB at the promoter (À22 to À29; À98 to À105; À1337 to À1344; and À1349 to À1356) of PURB gene by searching the JASPAR database [24], indicating that glucagon induces Purb expression through the activation of CREB. Knockdown of Purb decreased the ability of glucagon to stimulate CREB phosphorylation, the expression of G6Pase and PEPCK, and glucose production in primary hepatocytes, whereas Purb overexpression had the opposite effects. Purb did not regulate CREB expression, indicating that Purb enhances glucagon-induced CREB phosphorylation mainly through the activation of the upstreams of CREB. Mechanistically, Purb likely increases Adcy6 transcription by directly binding to its promoter, which then leads to increased cAMP production, enhanced PKA activity, and increased HGP. Both Purb and ADCY6 were induced at a later stage by fasting and then promoted HGP for survival during prolonged fasting. Second, hepatic Purb and ADCY6 are abnormally elevated in obesity and type 2 diabetes mouse models. Hepatocyte-specific knockdown of Purb in db/db mice severely impaired the ability of glucagon to increase blood glucose levels and inhibited CREB phosphorylation and PKA activity (due to decreased cAMP production) by suppressing Adcy6 expression in the liver. These observations indicate that hepatic Purb/ ADCY6 could promote the glucagon signaling pathway, contributing to the occurrence of hyperglycemia in type 2 diabetes. Surprisingly, Purb does not regulate insulin signaling, although Purb-KD db/db mice displayed reduced plasma insulin levels, probably secondary to a decrease in blood glucose in Purb-KD db/db mice given that low blood glucose stimulates less insulin secretion from b cells.
Hepatic deletion of TRAF2 also shows similar phenotypes [20]. Liverspecific knockdown of Purb in db/db mice did not alter hepatic insulin signaling, steatosis, or inflammation. Exogenous insulin reduced blood glucose to a similar degree in both Purb-KD and control db/db mice, suggesting that hepatic Purb does not alter hepatic or systemic insulin sensitivity. Consistently, in isolated primary hepatocytes, neither knockdown nor overexpression of Purb altered the insulin-induced phosphorylation of Akt, further supporting the conclusion that hepatic Purb does not regulate insulin sensitivity. In summary, Purb was shown to positively regulate HGP by directly binding to the promoter of the Adcy6 gene, increasing its expression and thereby enhancing the glucagon/cAMP/PKA/CREB signaling pathway. Purb/ADCY6 is abnormally elevated in obese mice and is also increased by fasting or via the glucagon signaling pathway. Hepatic knockdown of Purb in db/db mice significantly ameliorated hyperglycemia and glucose intolerance by suppressing the ADCY6/cAMP/ PKA/CREB signaling pathway. These data suggest that Purb/ADCY6 might serve as an important drug target for the treatment of hyperglycemia in patients with type 2 diabetes.

DISCLOSURE STATEMENT
All authors have nothing to declare.

AUTHOR CONTRIBUTIONS
L.J. performed most of the experiments. Y.J. and X.L. researched data. Z.C. designed the project and wrote the manuscript.