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Hepatic gluconeogenesis is the major contributor to the hyperglycemia observed in both patients and animals with type 2 diabetes. The transcription factor FOXO1 plays a dominant role in stimulating hepatic gluconeogenesis. FOXO1 is mainly regulated by insulin under physiological conditions, but liver-specific disruption of Foxo1 transcription restores normal gluconeogenesis in mice in which insulin signaling has been blocked, suggesting that additional regulatory mechanisms exist. Understanding the transcriptional regulation of Foxo1 may be conducive to the development of insulin-independent strategies for the control of hepatic gluconeogenesis. Here, we found that elevated plasma levels of adenine nucleotide in type 2 diabetes are the major regulators of Foxo1 transcription. We treated lean mice with 5′-AMP and examined their transcriptional profiles using RNA-seq. KEGG analysis revealed that the 5′-AMP treatment led to shifted profiles that were similar to db/db mice. Many of the upregulated genes were in pathways associated with the pathology of type 2 diabetes including Foxo1 signaling. As observed in diabetic db/db mice, lean mice treated with 5′-AMP displayed enhanced Foxo1 transcription, involving an increase in cellular adenosine levels and a decrease in the S-adenosylmethionine to S-adenosylhomocysteine ratio. This reduced methylation potential resulted in declining histone H3K9 methylation in the promoters of Foxo1, G6Pc, and Pepck. In mouse livers and cultured cells, 5′-AMP induced expression of more FOXO1 protein, which was found to be localized in the nucleus, where it could promote gluconeogenesis. Our results revealed that adenine nucleotide-driven Foxo1 transcription is crucial for excessive glucose production in type 2 diabetic mice.
The liver plays a central role in whole-body homeostasis and metabolic health. Many metabolic functions, including lipid processing and distribution, amino acid synthesis, and gluconeogenesis, are performed or controlled by the liver (
Regulation of glucose-6-phosphatase gene expression by protein kinase Balpha and the forkhead transcription factor FKHR. Evidence for insulin response unit-dependent and -independent effects of insulin on promoter activity.
). The phosphorylation state of FOXO1 determines its cellular localization and transcription activity of FOXO1, which is mainly regulated by the insulin signaling pathway under physiological conditions (
), insulin has no effects on Foxo1 transcription. The mechanism of hepatic Foxo1 transcriptional regulation in type 2 diabetes mellitus is unknown. The factors that stimulate hepatic Foxo1 transcription are generally considered to be less important than FOXO1 protein phosphorylation and are almost ignored in type 2 diabetes. Our previous observations demonstrate that the elevation of plasma adenine nucleotides is an upstream regulator in type 2 diabetic db/db mice (
). In the present study, we demonstrated that adenine nucleotides stimulate hepatic Foxo1 transcription via suppressing histones methylation in type 2 diabetic db/db mice, indicating that adenine nucleotides-driven hepatic Foxo1 transcription is crucial for excessive glucose production in type 2 diabetic mice.
Adenine nucleotide activates Foxo signaling
To explore the unbiased biological association of the role of adenine nucleotides in diabetes, we used RNA sequencing to measure all poly A-containing transcripts in the livers of control and model mice. The significantly enriched pathways were identified by using the KEGG database. Mapping of annotated DEGs to KEGG pathways resulted in 249 DEGs of 42 pathways and 656 DEGs of 44 pathways in the livers of 5′-AMP-treated (Fig. 1A, upper) and db/db mice (lower), respectively (Supporting information S1–S4). Among these, a total of 42 KEGG pathways were disrupted simultaneously in both mouse models, indicating a high degree of similarity in gene transcription changes between the AMP group and the DB group. The 175 genes that are significantly changed in the AMP group overlapped with the same genes in the DB group (Fig. 1B, left). A heat map of the 175 genes with significant regulatory functions was constructed (Fig. 1B, right). Pearson r analysis showed that there was a strong correlation between biological repeats in each group. Next, we used ingenuity pathway analysis to identify the top 20 classic pathways implicated by gene expression changes, revealing many metabolic pathways, including glucagon signaling, insulin signaling, and insulin resistance, which showed consistent changes in the AMP and the DB group (Fig. 1C). Moreover, the Foxo signaling pathway was found to be one of the largest spots and was shown in the KEGG enrichment diagram of the AMP group (Fig. 1D), and the gluconeogenesis-related genes in the Foxo signaling pathway were activated (Fig. 1E). Similarly, the Foxo signaling pathway in the DB group has changed (Supporting Information S5). Although KEGG pathway analysis showed some changes between the AMP group and the DB group, there was no significant difference in the pathways related to type 2 diabetes (Fig. S1). Therefore, we speculate that 5′-AMP is likely to regulate hepatic gluconeogenesis through Foxo signaling.
To verify the reliability of RNA-Sequencing data, eight responsive genes of 175 genes changed both in the AMP and the DB group were analyzed by qRT-PCR analysis. As shown in Figure 2A, the mRNA levels of Mfsd2a, Slc25a25, Etnppl, Actg1, Klf3, Foxo1, G6Pc, and Pepck were elevated in both groups. 5′-AMP significantly increased the transcription of gluconeogenesis-related genes including Foxo1, G6Pc, and Pepck, which were closely related to Foxo signaling and insulin resistance. Then, we investigated the function of 5′-AMP in regulating hepatic gluconeogenesis in vivo. 5′-AMP increased the glucose area under the curve during the pyruvate tolerance test (PTT) (Fig. 2B). PTT experiments confirmed that gluconeogenesis also increased in the db/db mice (Fig. 2B). Using different gluconeogenic substrates, we also found that 5′-AMP significantly increased glucose appearance with intraperitoneal (i.p.) administration of glycerol (Fig. 2C), lactate (Fig. 2D), fructose (Fig. 2E), and glutamine (Fig. 2F), respectively. The same results were obtained with db/db mice (Fig. 2, C–F). PTT experiments confirmed that 5′-AMP accelerated the consumption of gluconeogenic substrate pyruvate (Fig. 2G). Moreover, inhibition of FOXO1 function by selective FOXO1 inhibitor AS1842856 (
To investigate the underlying mechanism of 5′-AMP-dependent hepatic gluconeogenesis, we first examined the effects of 5′-AMP on the expression levels of Foxo1 mRNA and protein. 5′-AMP caused a dose-dependent increase in the mRNA levels of Foxo1, Pepck, and G6Pc in mouse livers (Fig. S2). Western blotting analysis showed that the FOXO1 protein level was also increased in 5′-AMP-treated livers (Fig. 3A). Moreover, 5′-AMP decreased the phosphorylation level of FOXO1 (Fig. 3B). Immunofluorescence analysis showed that the fluorescence intensity of FOXO1 was significantly increased after 5′-AMP, and the immunofluorescence of FOXO1 was mainly localized in the nucleus (Fig. 3C). These observations indicate that 5′-AMP enhances Foxo1 transcription as well as FOXO1 nuclear translocation.
5′-AMP-stimulated Foxo1 transcription is related to cellular methylation potential
To clarify how 5′-AMP stimulates hepatic Foxo1 transcription, a transcription inhibitor actinomycin D (AD) was used to explore the underlying mechanism. The results revealed that the AD significantly blocked the transcription level of Foxo1 in the livers of 5′-AMP-treated mice and db/db mice, excluding the possibility of posttranscriptional regulation of Foxo1 mRNA by 5′-AMP (Fig. 4, A and B), suggesting that 5′-AMP increased Foxo1 mRNA level through a process involving de novo gene transcription. Because 5′-AMP can be dephosphorylated into adenosine by 5′-nucleotidase anchored on the cell membranes, the function of 5′-AMP may play a role via increasing intracellular adenosine levels or acting on specific cell surface receptors (
). Administration of adenosine also increased hepatic Foxo1 transcription (Fig. 4C) and promoted gluconeogenesis in mice (Fig. 4D). To determine whether the effects of adenosine were due to the engagement of adenosine receptors, mice were treated with the broad-spectrum adenosine receptor antagonist theophylline. Theophylline treatment did not lower Foxo1 mRNA levels seen in 5′-AMP mice (Fig. 4E). Next, we found that a methyl donor betaine reduced Foxo1 mRNA in the livers of 5′-AMP-treated mice (Fig. 4F) and db/db mice (Fig. 4G). Cycloleucine acts as a competitive inhibitor of methionine S-adenosyl transferase involved in regulating the ratio of S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH), the cellular methylation potential (
). The methylation inhibitor cycloleucine significantly increased Foxo1 mRNA levels in WT livers (Fig. 4F). HPLC analysis showed that betaine, but not theophylline, increased the ratio of SAM/SAH in the livers of 5′-AMP mice (Fig. 4, H and I) and db/db mice (Fig. 4J). As expected, cycloleucine significantly reduced the ratio of SAM to SAH in mouse livers (Fig. 4I). These results demonstrate that 5′-AMP-stimulated Foxo1 transcription is closely related to cellular methylation potential. Then we examined whether 5′-AMP influences DNA methylation. As shown in Figure 4K, the DNA methylation levels of the Foxo1 promoter remained unchanged in the livers of 5′-AMP-treated mice. Similar results were observed in that of db/db mice (Fig. 4K).
). Increased histones methylation of some specific amino acid residues is generally associated with transcriptional silencing and heterochromatin formation, which ensures stable repression and genomic integrity (
). Then we detected a significant reduction in total H3K9me2 methylation in the livers of 5′-AMP-treated mice (Fig. 5A). The observation of a reduction in methylation epitopes associated with transcriptional repression (H3K9) chromatin suggests that hypomethylation may occur at the genome-wide level. Then, chromatin immunoprecipitation (ChIP) analysis was performed to compare histone methylation levels in Foxo1, G6Pc, and Pepck promoter regions. Compared with the control group, 5′-AMP resulted in a significant decrease of H3K9me2 levels in the promoter regions of Foxo1, G6Pc, and Pepck (Fig. 5B). The results of the ChIP analysis were confirmed by ChIP-qPCR (Fig. 5C). We also detected a significant reduction in H3K9 methylation in db/db livers by western blotting analysis (Fig. 5D). ChIP assay and qPCR analysis demonstrated that the H3K9me2 levels, as observed in 5′-AMP-treated mice, were markedly decreased in the promoter regions of Foxo1, G6Pc, and Pepck in db/db mice (Fig. 5, E and F). Together, our results suggest that the overall decrease of H3K9me2, specifically the reduction of Foxo1, G6Pc, and Pepck promoter locus, results in transcriptional stimulation of these genes in 5′-AMP-treated and db/db mice.
Adenine nucleotide promotes Foxo1 transcription in cultured cells
To confirm the results we obtained from the mouse livers, we used HepG2 cells to investigate the effects of 5′-AMP on the regulation of Foxo1 in vitro. The results showed that 5′-AMP resulted in a significant increase of glucose production in HepG2 cells (Fig. S3). HPLC analysis revealed that 5′-AMP caused a significant decrease in the SAM/SAH ratio (Fig. 6A). Then, we investigated whether nonspecific adenosine receptor antagonist (theophylline) and adenosine transporter inhibitor (dipyridamole) affect cell methylation potential and are related to Foxo1 transcription. As shown in Figure 6A, theophylline did not affect the 5′-AMP-decreased cellular methylation potential, and the adenosine transport inhibitor dipyridamole significantly inhibited the effect of 5′-AMP on methylation potential (Fig. 6A). To further explore the possible involvement of cellular methylation potential, we added SAM and the methylation inhibitor cycloleucine to observe the changes in the SAM/SAH ratio (Fig. 6B). The addition of SAM restored the 5′-AMP-decreased ratio of SAM/SAH. Twenty-four hours after the incubation of cycloleucine, the SAM/SAH ratio decreased significantly (Fig. 6B). The qRT-PCR analysis showed that 5′-AMP significantly increased Foxo1 mRNA levels in HepG2 cells, and the addition of theophylline did not affect the regulation of Foxo1 transcription by 5′-AMP (Fig. 6C). Notably, dipyridamole significantly decreased 5′-AMP-stimulated Foxo1 transcription (Fig. 6C). In HepG2 cells, cycloleucine increased Foxo1 transcription like 5′-AMP (Fig. 6D), and SAM eliminated the effects of 5′-AMP on Foxo1 transcription regulation (Fig. 6D), further indicating that 5′-AMP regulates Foxo1 transcription by affecting the methylation potential of cells. Immunofluorescence analysis was performed to show the subcellular location of FOXO1. While FOXO1 was mainly located in the cytoplasm in control cells, 5′-AMP significantly increased FOXO1accumulation in the nucleus (Fig. 6E). The theophylline did not affect 5′-AMP-regulated FOXO1 nuclear accumulation, but dipyridamole inhibited the appearances of FOXO1 in the nucleus. Moreover, SAM increased the intracellular cytoplasmic FOXO1 protein level and reduced the nuclear FOXO1 accumulation. Contrarily, cycloleucine markedly increased the appearances of FOXO1 in the nucleus (Fig. 6E).
Hepatic Foxo1 transcription is activated in the livers of patients with insulin resistance and type 2 diabetic mice (
). The molecular mechanism that stimulates hepatic Foxo1 transcription in type 2 diabetes is unknown and almost ignored. In the present study, we showed that 5′-AMP increased FOXO1 accumulation in the nucleus both in vivo and in vitro, thus promoting gluconeogenesis. This characterization was initially based on the induction of Foxo1 at the transcription level, for the first time revealing that adenine nucleotides-driven Foxo1 transcription is essential for excess glucose production in type 2 diabetes.
Free fatty acid (FFA) is an important link between obesity, insulin resistance, and type 2 diabetes (
). In our observations, adenosine transport inhibitor, but not adenosine receptor antagonist, significantly reduced 5′-AMP-stimulated Foxo1 transcription. Adenosine also has its direct biochemical function, which is not directly related to the adenosine receptor pathway (
). Under the pathological state of type 2 diabetes, the sustained high concentration of adenosine may passivate the regulatory effect of adenosine receptors. Our results suggest that 5′-AMP-stimulated Foxo1 transcription is related to the transports rather than cell surface receptors.
Exogenous 5′-AMP results in a dose-dependent elevation in adenosine levels in the liver, thereby increasing SAH and decreasing SAM/SAH ratio (
). Our data showed that 5′-AMP promoted hepatic gluconeogenesis and increased glucose output, which was mediated by the methylation ability associated with the SAM/SAH ratio. SAH is a powerful inhibitor of all methylation reactions. The concentrations of SAM and SAH are associated with diabetes (
). In our observation, the adenosine-driven SAM/SAH ratio did not change the DNA methylation level of the Foxo1 gene, but it resulted in insufficient histone methylation associated with the Foxo1 promoter region. Methylation of H3K9 is very common in transcriptional silencing (
Therefore, the insufficient methylation of histone H3K9 in the Foxo1 promoter region led to the activation of Foxo1 gene transcription. In addition, the change of SAM/SAH ratio implied that hypomethylation may occur at the whole genome level. Thus, other genes related to diabetes pathways are probably also involved. In fact, we found that Mfsd2a, Slc25a25, Etnppl, Actg1, and Klf3 were indeed elevated in both the AMP and the DB groups. Our analysis suggests a far more substantial role for adenine nucleotides in diabetes development.
Our previous observation showed that plasma 5′-AMP was elevated in type 2 diabetic db/db mice, and exogenous 5′-AMP caused type 2 diabetes-like hyperglycemia in lean mice (
). Since adenosine was a potential inhibitor of insulin receptor (IR) tyrosine kinase, insulin-stimulated IR autophosphorylation was significantly attenuated by 5′-AMP treatment, resulting in a reduction of insulin sensitivity (
). Together, it strongly suggests that adenine nucleotides and insulins may antagonize each other on blood glucose homeostasis.
In conclusion, we revealed a novel molecular mechanism underlying that adenine nucleotides regulate Foxo1 transcription for excessive glucose production in type 2 diabetes (Fig. 7). The mechanism of stimulating Foxo1 transcription in type 2 diabetes is almost ignored. Our findings extend the current understanding of epigenetic regulation of hepatic gluconeogenesis through Foxo1, highlight the crucial role of adenine nucleotides in the development of type 2 diabetes, and reveal a novel strategy for diabetes treatment.
Eight-week-old male C57BL/6, C57BL/Ks db/db mice, and their lean littermates (+/+) were used in this study. Mice were housed in a standard animal facility under a 12-h/12-h light/dark cycle with free access to food and water. All procedures were approved by the Animal Care and Use Committee at Nanjing University of Science and Technology (ACUC-NUST-20170223).
Treatment of 5′-AMP, betaine, cycloleucine, and actinomycin D
5′-AMP (adenosine 5′-monophosphate disodium salt) was solvated in saline and administered to mice by intraperitoneal injection in doses of 0.5 or 1 μmol/g body weight at zeitgeber time (ZT)1. Adenosine was solvated in saline and administered to mice by intraperitoneal injection in doses of 0.2 μmol/g body weight at ZT1. Saline was injected as a control. To change cellular methylation potential, betaine was supplemented in the drinking water at a concentration of 2% (wt/vol) for 2 weeks before the test (
). To inhibit mRNA transcription, actinomycin D was intraperitoneally (3 nmol/g body weight) injected into C57BL/6, C57BL/Ks db/db mice, and their lean littermates at ZT0. The transcriptional inhibitor actinomycin D was administered 60 min before the administration of 5′-AMP. To explore the role of adenosine receptors, adenosine receptor antagonist theophylline (10 μg/g, i.p.) was used 30 min before the administration of 5′-AMP. To inhibit FOXO1, mice were treated with AS1842856 (30 μg/g, i.p.) or the carrier solution 60 min before the administration of 5′-AMP. One hour after the injection of 5′-AMP, adenosine, or cycloleucine, all mice were sacrificed by cervical dislocation, and the livers were removed and freeze-clamped in liquid nitrogen for further analysis. Blood samples were immediately centrifuged at 5000 g for 5 min at 20 °C. The obtained plasma was immediately used. 5′-AMP, betaine, cycloleucine, theophylline, adenosine, and actinomycin D were from Sigma. AS1842856 was from MCE (HY-100596, MCE, China).
Gluconeogenesis tests were performed as described previously (
). Briefly, mice were fasted overnight (16 h) prior to injected i.p. with pyruvate (2 g/kg BW), glycerol (2 g/kg BW), lactate (1.5 g/kg BW), fructose (2 g/kg BW), or glutamine (1.5 g/kg BW), respectively. 5′-AMP (0.5 μmol/g i.p.) or adenosine (0.2 μmol/g i.p.) was mixed with gluconeogenic substrates and coinjected into mice. Blood glucose levels were determined from the tail vein at 0, 15, 30, 60, and 120 min after injection, with a One Touch Ultra Blood Glucose Meter (Lifescan).
Measurement of pyruvate concentration
Hepatic pyruvate concentration was assayed with commercial kits according to the manufacturers' instructions (Jiancheng). Livers were homogenized in ice-cold PBS. Then, the homogenates were collected for pyruvic acid determination. Pyruvate concentration was normalized to the protein concentration of the samples.
RNA sequencing and analysis
Purified total RNA from the livers of 5′-AMP-treated, db/db, and control mice was used for RNA-sequencing preparation. cDNA library construction and sequencing were performed by Beijing Genomics Institute using a BGISEQ-500 sequencer. High-quality reads were aligned to the mouse genome (mm10) by using Bowtie2. The expression levels for each of the genes were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM).
Promoter methylation analysis
Quantitative DNA promoter methylation of Foxo1 was performed by bisulfite sequencing PCR (BSP) (
). The primer sequences were shown in Table S1. Amplified DNA was ligated into the pCR2.1 vector (Invitrogen) and transformed into competent E. coli DH5α. Ten clones per sample were selected and sequenced. The results were analyzed by Biq Analyzer software.
HPLC analysis of SAM and SAH
SAM and SAH were extracted from frozen liver samples or PBS washed cell monolayers using 0.4 N perchloric acid (
). The mobile phase contained 0.1 M sodium acetate, 5 mM heptanesulfonic acid adjusted to pH 4.5 with acetic acid, and 5.5% acetonitrile. The samples were eluted on a reversed-phase C18 column at room temperature with an invariable gradient at a flow rate of 0.8 ml/min. Characteristic peak spectra and retention times compared with those of the standards were used to identify SAM and SAH. Quantitation was based on peak areas. SAM was from Solarbio. SAH was from Sigma.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from livers and cells with Karrol reagent (Karroten Scientific) according to the manufacturer's instructions. Reverse transcript reaction was carried out by reverse transcript kit (Invitrogen) according to the manufacturer's protocol. Real-time PCR was performed with the SYBR Green PCR Kit (Applied Biosystems) following the manufacturer's instructions on an ABI 7300 real-time PCR system (Applied Biosystems) in a 20-μl volume. The relative mRNA levels of Foxo1, Pepck, and G6Pc were quantified, with β-Actin used for normalization. The primer sequences used for quantitative RT-PCRs were shown in Table S2.
Western blot analysis
Fresh livers and cells were homogenized, and proteins were extracted with an Extraction Reagent (KeyGEN) according to the manufacturer's instructions. The extraction was separated by SDS-PAGE 10%–15% polyacrylamide gel. The membranes were incubated with primary antibodies anti-FOXO1, phospho-FOXO1 (Ser-256), anti-Histone H3, and anti-Di-Methyl-Histone H3 Lys9 (Cell Signaling), respectively, following by incubation with HRP-conjugated secondary antibody (Boster) and detection by enhanced chemical luminescence kit (Thermo scientific). β-actin was used as a control.
Chromatin immunoprecipitation assays
ChIP assays were performed as described previously (
). Cross-linked chromatin was immunoprecipitated with 5 μg of anti-Histone H3, anti-Di-Methyl-Histone H3 Lys9, respectively, or negative control rabbit IgG. Immunoprecipitated DNA was then used as a template for PCR. The primer sequences used for PCR were listed in Table S3.
Immunofluorescence and confocal microscopy were performed as described previously (
). Briefly, liver tissues were immersed in cryo-embedding medium and then sectioned into 10-μm-thick slices using a cryotome (Leica Microsystems). The cultured cells were washed three times with fresh PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 (Sigma) for 10 min. Slices and fixed cells were incubated with primary antibodies (anti-FOXO1, Cell Signaling) and secondary antibodies step by step. Nuclei were stained with DAPI. Fluorescence images of liver slices were observed with fluorescence microscopy (Eclipse 800; Nikon). The fluorescence images of cultured cells were acquired on a superresolution DeltaVision OMX imaging system (GE Healthcare).
HepG2 cells were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin (10 μl/ml of medium, Gibco) at 37 °C in 5% (v/v) CO2. To investigate the mechanisms responsible for the adenosine-mediated effects, the medium was removed and replaced with fresh medium, containing various concentrations of the compounds to be tested, for another 24 h: nontreated, 5′-AMP (0.5 mM) alone, 5′-AMP (0.5 mM) plus theophylline (100 μM), or dipyridamole (0.1 μM). To further confirm the regulatory effect of cell methylation potential, cells were treated with 5′-AMP for 24 h with or without 2 mM SAM. To inhibit methylation, cells were treated with 20 mM cycloleucine for 24 h. Theophylline, dipyridamole, SAM, and cycloleucine were purchased from Sigma.
Glucose production in cells
A previously established protocol was followed to estimate glucose production (
). HepG2 was plated in 6-well plates in 5% CO2 incubator at 37 °C for 24–48 h. After 70–80% confluency cells were incubated overnight in DMEM media containing 2% charcoal-treated FBS and 1% antibiotics. Cells were washed three times with PBS to remove all traces of glucose and incubated with media containing 2% charcoal-treated FBS, phenol red, and glucose-free media for 2 h. Cells were treated with 0.5 mM 5′-AMP in glucose production assay medium (phenol red and glucose-free DMEM) containing 2 mM sodium pyruvate and 20 mM sodium lactate, pH 7.4, and incubated up to 24 h. A quantity of 300 μl of the medium was sampled for measurement of glucose concentration using a glucose assay kit (GAGO20, Sigma). Glucose values were normalized with cellular total protein concentrations.
The results were presented as means ± SEM. Statistical difference between groups was determined by Student's t test, and comparisons among groups were performed using ANOVA. A p-value of less than 0.05 indicated statistical significance.
All data of this study are available in this article and in the supporting information. All source data generated for this study and relevant information are available from the corresponding author.
The authors declare that they have no conflicts of interest with the contents of this article.
This work was supported by the grant from the National Natural Science Foundation of China (numbers 31871178, 31861163004).
W. G., R. C., and J. Z. conceptualization; J. Z. funding acquisition; W. G., Y. Z., and R. C. investigation; Y. Z., Y. Y, Z. D., and X. X. methodology; J. Z. project administration; Y. Y., Z. D., X. X., and R. C. resources; J. Z. supervision; Y. Z., Y. Y., Z. D., X. X., D. W., S. W., and R. C. validation; W. G., R. C., and J. Z. writing-original draft; W. G., D. W., S. W., R. C., and J. Z. writing-review and editing.
Regulation of glucose-6-phosphatase gene expression by protein kinase Balpha and the forkhead transcription factor FKHR. Evidence for insulin response unit-dependent and -independent effects of insulin on promoter activity.