Regulation of hepatic gluconeogenesis by nuclear factor Y transcription factor in mice

Hepatic gluconeogenesis is essential to maintain blood glucose levels, and its abnormal activation leads to hyperglycemia and type 2 diabetes. However, the molecular mechanisms in the regulation of hepatic gluconeogenesis remain to be fully defined. In this study, using murine hepatocytes and a liver-specific knockout mouse model, we explored the physiological role of nuclear factor Y (NF-Y) in regulating hepatic glucose metabolism and the underlying mechanism. We found that NF-Y targets the gluconeogenesis pathway in the liver. Hepatic NF-Y expression was effectively induced by cAMP, glucagon, and fasting in vivo. Lentivirus-mediated NF-Y overexpression in Hepa1-6 hepatocytes markedly raised the gluconeogenic gene expression and cellular glucose production compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF-Y subunit A (NF-YA) attenuated gluconeogenic gene expression and glucose production. We also provide evidence indicating that CRE-loxP-mediated, liver-specific NF-YA knockout compromises hepatic glucose production. Mechanistically, luciferase reporter gene assays and ChIP analysis indicated that NF-Y activates transcription of the gluconeogenic genes Pck1 and G6pc, by encoding phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase), respectively, via directly binding to the CCAAT regulatory sequence motif in their promoters. Of note, NF-Y enhanced gluconeogenesis by interacting with cAMP-responsive element–binding protein (CREB). Overall, our results reveal a previously unrecognized physiological function of NF-Y in controlling glucose metabolism by up-regulating the gluconeogenic genes Pck1 and G6pc. Modulation of hepatic NF-Y expression may therefore offer an attractive therapeutic approach to manage type 2 diabetes.

Glucose is a primary fuel for mammals. Blood glucose levels are mainly maintained by the hormone-regulated processes of hepatic glucose output and peripheral glucose uptake (1). Under short-term fasting conditions, the continued demand for glucose is supplied by the process of glycogenolysis, which is the breakdown of glycogen into free glucose. Prolonged fasting or starvation will cause the depletion of the finite hepatic glycogen storage. To avoid hypoglycemia, new glucose is synthesized via the process of gluconeogenesis (2). Gluconeogenesis is stimulated by glucagon and glucocorticoids, and massive elevation of serum glucose concentrations is observed upon simultaneous treatment with glucocorticoid and glucagon in animals, whereas fasting significantly elevates the serum concentration of these hormone (3,4). It is generally accepted that excessive hepatic gluconeogenesis is the major contributor to the elevated blood glucose levels observed in patients with type 2 diabetes (5).
Gluconeogenesis in the liver is largely regulated at the transcriptional level by control of PEPCK 3 and G6Pase, which are the major rate-limiting enzymes for this glucose-generating pathway (6). These enzymes catalyze the conversion of oxaloacetate and glucose-6-phosphate to phosphoenolpyruvate and glucose, respectively. Expression of these enzymes is controlled via hormonal modulation of transcription factors and coactivators (7). Abundant evidence indicates that the cAMP signaling pathway is central to the hormonal control of gluconeogenesis (8), and cAMP-responsive element (CRE)-binding protein (CREB) is a particularly important effector of the cAMP pathway in liver (9,10). Both Pck1 and G6pc genes encoding PEPCK and G6Pase, respectively, possess CRE in their promoter regions, and binding of CREB to CRE increases the transcriptional activity of these genes. In addition, CREB could induce expression of peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣), which in turn stimulates the transcriptional activity of factors such as glucocorticoid receptor and FOXO1 on the promoters of gluconeogenic genes (11). However, hepatocytes lacking PGC-1␣ retain some ability to respond to cAMP (12), indicating that other pathways also contribute to control of gluconeogenic gene expression by the cAMP axis.
Nuclear factor-Y (NF-Y), a ubiquitous transcription factor conserved in evolution, is composed of three subunits including NF-YA, NF-YB, and NF-YC. NF-Y induces gene expression by binding to the CCAAT box in the proximal promoter region, so it is also named as the CCAAT-binding factor (13). It has been known that many genes are regulated by NF-Y, so NF-Y plays multiple roles in various contexts, e.g. cell cycle (14), embryonic development (15), endoplasmic reticulum stress (16), fatty acid metabolism, muscle cell differentiation (17), neurodevelopment (18), and so on. In addition, several extracellular signals, such as hormones, inflammatory signals, and growth factors, could promote NF-Y expression in a cell type-specific manner (19). Given the diverse pathways inducing NF-Y expression, it is speculated that the NF-Y function also depends highly on cell type and context. However, the NF-Y function in the regulation of hepatic gene expression has not previously been explored.
We hypothesized that gluconeogenesis could a NF-Y target in the liver for the following reasons. 1) It is known that the NF-Y expression is responsive to the cAMP signaling pathways in pancreatic ␤ cell line (20); 2) NF-Y is a ubiquitously evolutionally conserved CCAAT box motif-binding transcription factor; 3) the CCAAT box located at the promoter region has been shown to mediate the cAMP stimulatory effects (21)(22)(23); and 4) cAMP as the secondary messenger plays a central role in the hepatic gluconeogenesis controlled by hormones. This raised the possibility that NF-Y could be one of the cAMP downstream mediators regulating glucose metabolism. Our results showed that NF-Y expression is effectively induced by cAMP, glucagon, and fasting in vivo. Furthermore, we provided evidence indicating that NF-Y regulates gluconeogenic gene expression and hepatic glucose production. Our study further supports evidence that NF-Y enhances gluconeogenesis by physically interacting with CREB and activating transcription of Pck1 or G6pc via directly binding to its promoter region.

cAMP axis induces hepatic NF-YA expression
To determine the NF-Y function in hepatic glucose metabolism, we stimulated Hepa1-6 mouse hepatocytes with different concentrations (0, 1, 10, and 20 mol/liter) of 8-Br-cAMP, which is a stable cAMP analog and is known to activate gluconeogenesis. As shown in Fig. 1a, 8-Br-cAMP treatment significantly up-regulated mRNA expressions of the gluconeogenesis genes G6pc and Pck1 in a dose-dependent manner. Treatment of 8-Br-cAMP also induced mRNA expressions of all three subunits of NF-Y including NF-A, NF-YB, and NF-YC in Hepa1-6 hepatocytes (Fig. 1a). Simultaneously, the NF-YA protein was also significantly stimulated by 8-Br-cAMP treatment (Fig. 1b).
To further confirm that NF-Y is involved into the cAMP axis in vivo, we treated mice with 300 g/kg glucagon, a hormone produced by ␣ cells activates the hepatic cAMP signaling pathway (24). In line with the effects observed in cultured Hepa1-6 hepatocytes, glucagon treatment significantly stimulated mRNA Figure 1. Induction of NF-Y expression in liver in response to the glucagon-cAMP axis and fasting. a and b, Hepa1-6 mouse hepatocytes were stimulated with different concentration (0, 1, 10, and 20 M) of 8-Br-cAMP. a, mRNA expression levels of NF-Y subunits and gluconeogenic genes were measured by qRT-PCR. b, NF-YA protein levels were determined by Western blotting. c, C57BL/6 mice (n ϭ 5/group) were challenged for 1 h with 300 g/kg body weight glucagon; hepatic mRNA expressions of NF-Y subunit genes were analyzed by RT-qPCR. d, C57BL/6 mice (n ϭ 5/group) fed ad libitum, fasted for 24 h, or fasted for 24 h and then refed for 24 h. Hepatic mRNA expression levels of NF-Y subunits and gluconeogenic genes were measured by RT-qPCR. The data represent means Ϯ S.D. of three independent experiments. *, p Ͻ 0.05 versus vehicle treatment.

NF-Y regulates gluconeogenesis
expressions of NF-YA, NF-YB, and NF-YC in mouse liver (Fig.  1c). Furthermore, mRNA expressions of all three subunits were induced in livers of 24-h fasted mice and were reduced upon refeeding (Fig. 1d), a typical regulatory manner characterized by the gluconeogenic gene. These findings provided evidence of NF-Y being induced in response to cAMP.

NF-Y regulates gluconeogenesis gene expression and glucose metabolism
PEPCK and G6Pase are the primary enzymes in the gluconeogenic pathway in the liver. It is well known that the cAMP signaling pathway plays a central role in controlling the expression of G6pc and Pck1 (25,26). The response of NF-Y to cAMP challenge in the mouse liver suggested that this transcription factor might mediate gluoconeogenic gene expression. To prove this hypothesis, we used lentivirus vectors to express all three subunits of NF-Y simultaneously or empty vector control (pLVX) in Hepa1-6 hepatocytes. Transduction of recombinated lentivirus encoding NF-Y significantly induced both mRNA expression (Fig. 2a) and protein levels ( Fig. 2b) of G6Pase and PEPCK. The functional consequence of forced expression of NF-Y is confirmed by the stimulation of glucose production from pyruvate and lactate in Hepa1-6 hepatocytes (Fig. 2c).
To further confirm the effect of NF-Y expression on the gluconeogenic pathway, we employed the CRISPR-Cas9 system to knock out NF-YA in Hepa1-6 hepatocytes. The small guide RNA (sgRNA) effectively abolished NF-YA expression. Simul-taneously, NF-YA knockout attenuated both mRNA levels ( Fig.  2d) and protein expressions (Fig. 2e) of G6Pase and PEPCK. Correspondingly, the diminished expression of NF-YA significantly blunted glucose production (Fig. 2f). Taken together, these findings suggest that NF-Y might be involved in the transcriptional process of hepatic gluconeogenesis.

Basal phenotyping of liver-specific Nf-ya knockout mice
Subsequently, we examined the effects of NF-Y on hepatic glucose metabolism in vivo. A total body knockout of Nf-ya is lethal (15). Thus, to study the function of NF-Y in liver in animals, we generated a liver-specific NF-Y knockout by crossing an albumin-CRE (Alb-Cre) mouse to an Nf-ya flox/flox mouse, in which exons 3-8 of 9 total coding exons are flanked by loxP sequences (Fig. 3a). Through two rounds of crossing, we obtained liver-specific Nf-ya knockout mice Nf-ya flox/flox/ Alb-Cre, which was referred as Nf-ya LKO and was confirmed by genotyping as well as by performing Western blotting to assay Nf-ya protein expression. The littermates Nf-ya flox/flox were used as controls.
Nf-ya LKO mice showed significant reductions in body weight (Fig. 3b) and the total body fat percentage (Fig. 3c), although liver weight/body weight ratio remained similar to that in control mice (Fig. 3d). Under fasting conditions, the Nf-ya LKO mice showed significantly reduced levels of blood glucose (Fig. 3e) and serum glucagon ( Fig. 3f) with similar serum insulin levels (Fig. 3g) compared with littermate controls. Periodic acid-Schiff (PAS) staining indicated that the a-c, Hepa1-6 cells were transfected with lentivirus vectors to express all three subunits of NF-Y simultaneously or empty vector control (pLVX) and then selected stable cells with 1.5 g/ml puromycin. Expression of gluconeogenic gene mRNAs (a) and proteins (b) were induced, and glucose production (c) was stimulated in NF-Y over expression stable cells. d-f, NF-YA knockout by the CRISPR-Cas9 system in Hepa1-6 cells. Stable cells were selected with 1.5 g/ml puromycin. Expression of gluconeogenic genes mRNAs (d) and proteins (e) were down-regulated, and glucose production (f) was reduced in NF-YA knockout stable cells. The protein and mRNA levels of the indicated genes were assayed by Western blotting and real-time PCR, respectively. Glucose production from pyruvate and lactate in Hepa1-6 cells were assayed as described under "Materials and Methods." The values are means Ϯ S.D. of three independent experiments. *, p Ͻ 0.05 versus control (unpaired Student's t test).

NF-Y regulates gluconeogenesis
production of hepatic glycogens in the Nf-ya LKO mice was significantly lower than that of littermate control mice (Fig. 3h), which was further confirmed by quantitative enzymatic assay of hepatic glycogen content (Fig. 3i).

Deletion of NF-Y suppresses the hepatic gluconeogenesis in vivo
In line with the difference in fasting glucose levels, mRNA expression of the gluconeogenic genes G6pc and Pck1 was inhibited in Nf-ya LKO mice after 24 h of fasting compared with littermate controls (Fig. 4a). Consistent with mRNA expression, the protein levels of G6PC and PEPCK were significantly lowered in Nf-ya LKO mice (Fig. 4b). To further investigate whether knockout of Nf-ya in the liver give rise to a decreased glucose production, we conducted a pyruvate-tolerance test, which consisted of analyzing the rise of glycemia in response to a pyruvate injection in a fasted subject. An obviously elevated glucose level was observed in WT control mice, and the maximal increase of 64% is observed after 30 -60 min. In contrast, the blood glucose level in overnight fasted Nf-ya LKO mice was already reduced by 30% compared with control animals and then stayed significantly lower after the pyruvate injection (Fig. 4c). The corresponding glycemia area under the curve value in Nf-ya LKO mice was 60% of that observed in control

NF-Y regulates gluconeogenesis
mice (Fig. 4d). These results clearly indicated an attenuated gluconeogenesis ability in the liver of Nf-ya LKO mice. We further directly measured glucose production in isolated hepatocytes from Nf-ya LKO or control mice. Indeed, glucose production was dramatically reduced in primary hepatocytes from Nf-ya LKO mice compared with controls (Fig. 4e). Together, these observations clearly indicate that NF-Y drives glucose production in mice livers.
Additionally, we also explored the effect of hepatic Nf-ya knockout on lipogenesis. We found that Nf-ya LKO mice had increased expression of lipolytic gene Pnpla2 (also known as Atgl) and decreased expression of lipogenic gene Acaca and Fasn (also known as Acc1 and Fas, respectively) (Fig. 5a). Correspondingly, Nf-ya LKO mice displayed reduced serum levels of triglyceride (Fig. 5b) and cholesterol (Fig. 5c), implying that NF-Y has a marked regulatory impact on lipid metabolism. c, pyruvate-tolerance test showing glucose production in response to pyruvate challenge. The mice were fasted for 16 h and intraperitoneally injected with sodium pyruvate (2 g/kg body weight). After injection, blood glucose was monitored at the designated time points. d, the areas under the curves (AUC) were calculated. e, glucose production (glucose output) was measured in the culture medium of primary hepatocytes isolated from Nf-ya LKO and littermate control mice. The values are expressed as means Ϯ S.D. *, p Ͻ 0.05 versus control (unpaired Student's t test).

NF-Y regulates gluconeogenesis NF-Y is not necessary for glucagon-mediated gluconeogenesis in vivo
In the fasted state, glucagons secreted from pancreatic ␣ cells to increase hepatic glucose production. We explored whether NF-Y is necessary for glucagon induction of gluconeogenic genes. Nf-ya in Hepa1-6 hepatocytes was knocked out via the CRISPR-Cas9 system, and then the cells were treated with cAMP. Nf-ya knockout was found to significantly reduce the gluconeogenic gene expression and the cAMP stimulatory effects on gluconeogenic genes Pck1 and G6pc (Fig. 6a).
To further explore the physiological effects of NF-Y deficiency on hepatic glucose metabolism in response to the glucagon stimulation, we performed a glucagon challenge assay to determine hepatic glucagon sensitivity in Nf-ya LKO mice. Prior to the glucagon administration, mice were injected with somatostatin intraperitoneally to inhibit endogenous glucagon production. G6pc and Pck1 were significantly down-regulated at the transcript levels at both baseline and in response to glucagon stimulation in Nf-ya LKO mice compared with control mice (Fig. 6b). However, the Nf-ya LKO mice respond robustly to glucagon administration with respect to the expression of G6pc and Pck1. Moreover, the relative fold increase in G6pc and Pck1 expression with glucagon treatment appears similar in Nf-ya LKO mice and littermate controls. (Fig. 6b). Consistent with the stimulatory effects on the gluconeogenic gene expression, glucagon produces a robust increase in the glucose excursion in Nf-ya LKO mice, although this appears modestly enhanced in littermate controls (Fig. 6c). These data indicated that NF-Y is not required for glucagon-mediated induction of the expressions of gluconeogenic genes in vivo.
The induction of gluconeogenesis by glucagon-dependent fasting depends on CREB and PGC-1␣ transcriptional activation, CREB is one of the PGC-1␣ transcription factors (11,27). We examined the phosphorylation status of CREB. Although the fasting-induced hepatic CREB phosphorylation was markedly lower in Nf-ya LKO mice compared with control mice, the protein levels of PGC-1␣ were similar in Nf-ya LKO and control livers (Fig. 6d). Taken together, these observations suggested that NF-Y largely regulates gluconeogenic gene expression, which may be cAMP/CREB axis-independent.

NF-Y is a direct regulator of gluconeogenesis genes
We next wanted to specify the mechanism for NF-Ymediated gluconeogenic gene expression. Because NF-Y is a transcription factor, we determined whether NF-Y can modulate gluconeogenic gene promoter transcriptional activity. Hepa1-6 hepatocytes were transiently cotransfected with a luciferase reporter vector driven by the mouse G6pc (Ϫ1441 to ϩ38 bp) or Pck1 (Ϫ1265 to ϩ308 bp) promoter fragment and all three lentiviral NF-Y subunits. The results in Fig. 7a showed that inclusion of NF-Y resulted in markedly higher activities of both G6pc and Pck1 promoters in transient transfection assays.
We further examine whether NF-Y exerts its effect on gene expression through direct promoter interactions. NF-Y often binds to CCAAT motif in regulated genes promoters. Sequence analysis showed the presence of a potential CCAAT motif within 1.5 kb upstream from mRNA start site of G6pc and Pck1 promoters, respectively (Fig. 7b, left panel). The occupancy of NF-Y over each putative CCAAT motif on G6pc and Pck1 promoters was confirmed by ChIP assay (Fig. 7b, right panel).
To further determine whether NF-Y activates G6pc and Pck1 promoters through binding to the CCAAT motif, constructs with site-directed mutagenesis (deletions) of CCAAT motif in the G6pc and Pck1 promoters were generated. When the CCAAT motif in the G6pc or Pck1 promoter was deleted, the stimulating effect was ablated (Fig. 7c).
CREB is one of important effectors of the hepatic cAMP signal pathways, the cAMP-CREB axis is generally accepted as a critical signal for the gluconeogenesis stimulation, prompting us to determine whether CREB would interact with NF-Y. Indeed, CREB interacted with NF-YA in a coimmunoprecipitation study (Fig. 7d), suggesting that NF-Y could function as a coactivator for CREB. Taken together, these data demonstrated that gluconeogenic genes G6pc and Pck1 were direct targets of NF-Y in hepatocyte cell lines.

Discussion
Gluconeogenesis is essential for the glucose production during fasting to maintain circulating levels of glucose. Glucagon

NF-Y regulates gluconeogenesis
and its downstream cAMP/CREB-signaling pathway play a crucial role in the activation of glucose production especially in the early phase of fasting (8). CREB promotes the transcription of gluconeogenic genes by binding to the promoter. In addition, cAMP-CREB axis also stimulates the expression of PGC-1␣, which is a transcriptional coactivator facilitating the biological activities of other transcription factors, such as the glucocorticoid receptor, FOXO1, and HNF4␣ (11).
In this study, we identified NF-Y as a previously unrecognized modulator mediating hepatic glucose production. Hepatic NF-Y expression is potently induced in response to treatments of cAMP, glucagon, and fasting. Gluconeogenesis involves multiple stepwise chemical reactions in which G6Pase and PEPCK are the rate-limiting enzymes, NF-Y activates the transcription of Pck1 or G6pc via a unique regulatory sequence in their promoters. Our results also indicate that NF-Y stimulates CREB expression both in vitro and in vivo, and it works together with CREB to promote transcription of gluconeogenic genes. The exact underlying mechanism is not known, but it appears that NF-Y has a role in the assembly of transcription complexes at the proximal promoter region. NF-Y interacts with other transcriptional protein, such as TATA box-binding protein, to form promoter complexes and to facilitate the initiation of the gene transcription (13).
NF-Y is an evolutionarily conserved CCAAT motif-binding transcription factor, and the NF-Y-binding CCAAT box is usually located between Ϫ60 and Ϫ100 bp upstream of the transcription starting site (28). In the present study, an inverted NF-Y-binding CCAAT motif was identified in the promoters of G6pc and Pck1, which is speculated to be responsible for the cAMP-stimulated gene expression. Indeed, NF-Y-binding CCAAT box has been involved into the cAMP stimulation of some genes (21)(22)(23)29). In human pinealocytes, an inverted NF-Y-binding CCAAT box in the promoter of the Tph (tryptophan hydroxlase) gene is necessary for mediating cAMP stimulatory effects (30). Additionally, a NF-Y-binding CCAAT motif in the rat hexokinase II (Hk2) gene promoter can confer cAMP inducibility sufficiently in L6 myotubes (31). Our study clearly demonstrates that NF-Y confers cAMP responsiveness to gluconeogenic genes in mice hepatocytes.
Glucagon and its downstream cAMP/CREB-signaling pathway play a fundamental role in the hepatic glucose production in the fasted state. In the current study, Nf-ya LKO mice showed reduced glycemia and gluconeogenic gene expression compared with littermate controls. Surprisingly, the serum glucagon level is also significantly lower in the Nf-ya LKO mice than that of controls. There is no expected counter-regulation to compensate the reduction in gluconeogenic enzymes in the Nf-ya LKO mice. This may be attributed to the glucagon role in regulating hepatic glucose metabolism. Glucagon produced by ␣ cells of the pancreas generally elevates the blood glucose concentration by promoting glycogenolysis in the liver; it has hardly any effect on gluconeogenesis (32). However, the production of hepatic glycogens in the Nf-ya LKO mice is significantly lower than that of control mice. Thus, Nf-ya LKO mice might not require much more glucagon to promote glycogenolysis than control mice.

NF-Y regulates gluconeogenesis
In the present study, we also explored whether NF-Y is necessary for glucagon-mediated gluconeogenesis in mice livers. We found that the stimulatory effects of glucagon on the expression of the gluconeogenic genes are similar between control and Nf-ya LKO mice. Furthermore, glucagon also induce a robust increase in glycemia in Nf-ya LKO mice. Our data suggest that NF-Y is not necessary for glucagon-mediated induction of the expression of gluconeogenic genes in vivo. Thus, we speculated that NF-Y largely regulates gluconeogenic gene expression independently of the cAMP/CREB axis.
The cAMP pathway is also used by other hormones to signal the glucose metabolism (33). Thus, it is very likely that NF-Y also takes part in mediating their responsiveness. Postprandially, blood insulin levels elevate. Insulin pathway has long been recognized to be a dominant over gluconeogenic stimuli and suppress hepatic glucose production (6). Therefore, it is required to explore where insulin fits in the NF-YA pathway in the future study. In addition, the biology of the three subunits of NF-Y is complex. NF-YA and NF-YC have multiple splicing isoforms with different function and expression levels in different tissues, and the subunits are subject to post-translational modifications, such as phosphorylations, acetylations, and ubiquitinations (34). It is possible that insulin-signaling pathways modify NF-Y and alter the protein degradation of NF-Y.
Additionally, because insulin signaling is essential to the activities of some gluconeogenic regulator, such as PGC-1␣ and FOXO1, NF-YA may be involve in the insulin pathway by altering the activities of such factors.
As a whole, this work reveals a previously unrecognized physiological function of NF-Y in controlling glucose metabolism. We identified that NF-Y binding to the promoter of Pck1 or G6pc to transcriptionally modulate hepatic gluconeogenesis. Elevated hepatic gluconeogenesis is primarily responsible for the increase in fasting hepatic glucose production in patients with diabetes. Effective management of dysregulated gluconeogenesis is emerging as a promising therapeutic strategy to alleviate hyperglycemia in diabetes. Several antidiabetic drugs, such as metformin, regulate blood glucose by transcriptional inhibition of the gluconeogenic program (35). Modulation of hepatic NF-Y expression may therefore offer an attractive therapeutic approach to manage type 2 diabetes.

NF-Y regulates gluconeogenesis
ondary antibodies were obtained from Sigma-Aldrich. Fetal bovine serum was from Hyclone (Logan, UT). Sperm DNA and BSA were from New England BioLabs. ELISA kits for glucagon and insulin were from Crystal Chem (Elk Grove Village, IL). Glycogen assay kit was from BioAssay Systems (Hayward, CA). The ChIP assay kit and PCR purification kit were from Beyotime Biotechnology (Shanghai, China). pGL3-basic reporter vector and Dual-Glo TM luciferase assay kit were from Promega (Shanghai, China). Site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Lipofectamine TM 2000 and TRIzol reagent were from Invitrogen. Antibodies against PEPCK and ␣-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against G6PC, PEPCK, NF-YA, NF-YB, and NF-YC were from Cell Signal Technology (Danvers, MA). Antibody against CREB and pCREB were from HuaAn Biotechnology (Hangzhou, China). SYBR Green Master Mix was from Applied Biosystems (Foster City, CA). All of the chemicals were dissolved in the appropriate media solution or DMSO and then used at the indicated concentrations.

Mice
The mice were housed under a 12-h light/12-h dark cycle and followed free access to normal chow diet. All animal experiments were carried according to the National Institute of Health ethical guidelines. Animal care and experimental procedures were approved by the ethics committee of animal experimentation of Sichuan University. Nf-ya flox mice were a gift from N. Nobuyuki's laboratory at Juntendo University Graduate School of Medicine (Tokyo, Japan). Nf-ya flox mice were maintained on a C57BL6J background and genotyped according to the protocol described previously (15). Alb-Cre mice harboring a transgene containing Cre recombinase driven by the albumin promoter (36) were purchased from The Jackson Laboratory (Bar Harbor, ME). The Nf-ya flox/flox mice bred with the Alb-Cre mice (homozygous for WT Nf-ya) generated Nf-ya flox/wt;Cre mice. These mice were then mated via brother and sister generate Nf-ya flox/flox;Cre mice, which were used as liver-specific NF-YA knockout (LKO) mice. The littermates Nf-ya wt/flox;Cre and Nf-ya flox/flox were used as controls for each subsequent experiment. 10-week-old male mice were overnight fasted and then killed. Their livers and plasma were collected for further analysis.

Metabolic analysis
Serum concentrations of insulin and glucagon were quantified by ELISA according to the manufacturer's instructions. Serum concentrations of triglyceride and total cholesterol were measured by an automated Monarch device (Instrumentation Laboratory, Lexington, MA). Body fat mass was measured using peripheral dual-energy X-ray absorptiometry in a Lunar PIXImus II densitometer (PIXImus; GE-Lunar, Madison, WI) as described previously (37), and anesthetized mice were placed on a positioning tray ventral side down with the legs extended away from the body. A pyruvate-tolerance test was performed after 16 h of fasting (5:00 p.m. to 9:00 a.m.). 10-week-old male mice were injected intraperitoneally with sodium pyruvate (2 g/kg). The glucagon challenge was performed by injecting glucagon (300 g/kg) subcutaneously 15 min after the intraperito-neal injection of somatostatin (10 mg/kg). Blood glucose levels were determined from the tail vein at the indicated time points using an automatic glucose monitor (OneTouch; LifeScan).

Hepatic glycogen staining and measurement
Hepatic glycogen was stained by PAS following the standard protocol. Briefly, formalin-fixed, paraffin-embedded mice liver tissue was sectioned, deparaffinized, rehydrated, and oxidized in 0.5% periodic acid solution. Then the oxidized tissue sections were incubated in Schiff's reagent. To quantify hepatic glycogens, mice liver tissue were homogenized in ice-cold citrate buffer (0.1 M, pH 4.2). Homogenates were immediately subjected to glycogen measurement using the commercial glycogen assay kit following the manufacturer's instructions. Hepatic glycogen content was presented after normalization to liver mass.

Plasmid and virus
The promoters of G6pc (Ϫ1441 to ϩ38 bp) and Pck1 (Ϫ1265 to ϩ308 bp) were amplified from C57BL6J mouse genomic DNA and cloned into pGL3-basic reporter vector. The putative NF-Y binding site in the G6pc or Pck1 promoter was mutated or deleted using a QuikChange lightning site-directed mutagenesis kit, and the mutations were further confirmed by DNA sequencing. The cDNAs encoding full-length mouse Nf-ya, Nf-yb, and Nf-yc were amplified by RT-PCR, respectively, which were then subcloned into the pLVX-puro vector via EcoRI and XbaI restriction sites to produce the recombinant lentivirus plasmid. In CRISPR-Cas9 system, two pairs of sgRNAs (pair 1, 5Ј-CACCGATTGTGCCATCTGCATT-AAC-3Ј and 5Ј-AAACGTTAATGCAGATGGCACAATC-3Ј; and pair 2, 5Ј-CACCGCAGATACAGTTGGTGCCTCC-3Ј and 5Ј-AAACGGAGGCACCAACTGTATCTGC-3Ј) targeting the coding region in exon 4 or 5 of Nf-ya were synthesized according to CRISPR Design (Massachusetts Institute of Technology) and cloned into the pSpCas9(BB)-2A vector (Addgene plasmid entry 48138) to produce the recombinant lentivirus plasmid. Each recombinant lentivirus plasmid was then transfected into HEK293 cells with viral packaging vectors (pMD2G and psPAX2) by Lipofectamine TM 2000 to allow packaging and amplification of the recombinant lentivirus.

Cell culture
Hepa1-6 cells and 293T cells were purchased from American Type Culture Collection (Manassas, VA). Mouse primary hepatocytes were isolated from Nf-ya flox/flox;Cre mice and their littermate control mice by liver perfusion with type II collagenase and grown on collagen-coated plates, and cell number and viability were assessed using trypan blue. The cells were cultured in DMEM containing 4.5 g/liter glucose and 2 mM L-glutamine at 37°C with a humidified 5% CO 2 atmosphere. The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 mg/ml streptomycin.

NF-Y overexpression and knockdown
Hepa1-6 cells plated on 6-well plates were coinfected with recombinant Nf-ya, Nf-yb, and Nf-yc lentiviruses in overexpression assay, and infected with recombinant Nf-ya Crispr-Cas9

NF-Y regulates gluconeogenesis
lentivirus in knockdown assay. 1.5 g/ml puromycin was used for stable selection.

Dual-luciferase activity measurement
Luciferase activity assay was carried out as described previously (38). Briefly, HEK293 cells plated on 96-well plates were transfected transiently with 20 ng/well of G6pc or Pck1 promoter reporter plasmid or control vector using Lipofectamine TM 2000. After 12 h of treatment, the cells were infected with vehicle or Nf-ya, Nf-yb, and Nf-yc lentiviruses for an additional 24 h. The luciferase activity was assayed by using the Dual-Glo TM luciferase system according to the manufacturer's protocols. The firefly luciferase activity was normalized to the Renilla luciferase activity (firefly luciferase/Renilla luciferase) and presented as relative luciferase activity.

Glucose output assay
Stable Hepa1-6 cells with Nf-y overexpression or Nf-ya knockdown and primary mouse hepatocytes seeded on 6-well plates were subjected to glucose production. The cells were washed three times with PBS and then incubated in glucose production medium (phenol-red-free, glucose-free DMEM containing 10 M 8-Br-cAMP, 20 mM sodium lactate, and 2 mM pyruvate) for 6 h. Glucose concentrations in the media were quantified in triplicate using a colorimetric glucose oxidase assay (Applygen Technologies, Beijing, China) and normalized to the total protein content of cells.

ChIP assays
ChIP followed by PCR was conducted using the inputs according to the ChIP assay kit protocol. Briefly, the cells were fixed, lysed, and sonicated. The protein-DNA complexes were immunoprecipitated with the indicated antibodies. Purified DNA was subsequently subjected to PCR using the following primers. The primers amplifying G6pc promoter region are 5Ј-GCAGCCTCTAGCACTGTCAAGCA-3Ј and 5Ј-GCCCTGG-ATTCAGTCTGTAGGTCAA-3Ј; the primers amplifying Pck1 promoter region are 5Ј-AGTTGCCTGTCACCATCCTCC-3Ј and 5Ј-GTAAAGACTTCTCTGCGAGCCAC-3Ј. Meanwhile, the region flanking the putative binding site in the promoter was amplified by following primer pairs to be as negative controls: for the G6pc promoter, 5Ј-TGTAGTTCAATCCTCAATACC-3Ј and 5Ј-GAGGTCAGCGATCACAGACAC-3Ј; and for the Pck1 promoter, 5Ј-TTAGAGCAGGGGTCAGTA TG-3Ј and 5Ј-GAGACGCCTCTTGGACTTCA-3Ј.

Coimmunoprecipitation
Proteins were extracted from NF-Y stable cells by using radioimmune precipitation assay lysis buffer, and protein extracts were quantified using BCA assay. 2 g of antibody against NF-Y or CREB and 20 l of protein A/G resin were suspended in 500 l of cold PBS binding for 9 h and were washed with cold PBS before being blocked overnight with 1% BSA in PBS. The antibody-resin complex was then incubated with 2 mg of total protein in 1 ml of lysis buffer overnight at 4°C on a rotating wheel. The immunoprecipitated complexes were collected by centrifugation at 4°C and then eluted with the lysis buffer. The eluted protein samples were separated on SDS-PAGE and followed by immunoblotting using the anti-NF-Y or CREB body.

Real-time quantitative PCR analysis
Total RNA was extracted from Hepa1-6 cells, primary hepatocytes, and mouse liver using TRIzol reagent and quantified on a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA synthesis and real-time quantitative PCR (qPCR) analysis were performed as described (39). In brief, qPCRs were performed using ABI PRISM 7500 sequence detection system (Applied Biosystems) using the mixture composed of cDNA, SYBR Green PCR Master Mix, and gene-specific primers (Table S1). All measurements were normalized to Gapdh expression, and the relative gene expression between groups was calculated using a comparative Ct (⌬⌬Ct) method. qPCRs were conducted in triplicate.

Protein extraction and Western blotting
As previously described (40), liver tissue or Hepa1-6 were lysed in radioimmune precipitation assay buffer. The proteins were quantified using a BCA protein assay kit. For Western blotting, proteins (20 g) from each sample was separated by SDS-PAGE (10%) and then transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat milk repaired in Tween-TBS buffer and immunoblotted with the antibodies against target proteins overnight. The blots were subsequently washed and then incubated with the horseradish peroxidase-conjugated secondary antibodies. The immunoreactive bands were visualized by using an enhanced chemiluminescence kit and quantified by volume densitometry using the UN-SCAN-IT Gel 5.1 software (Silk Scientific Inc., Orem, UT).

Statistical analyses
The data are presented as means Ϯ S.D. of at least three independent experiments, unless otherwise indicated in the figure legends. Statistical analyses were performed with GraphPad Prism. Differences between two groups were analyzed by using unpaired two-tailed Student's t test. A p value of Ͻ0.05 was considered statistically significant.