Analytical Methods and Bioassays for Cytotoxicity and Antidiabetic Properties of Aquilaria crassna Leaf Extracts in HepG2 Cells.

Aquilaria crassna is a herbal plant that has recently been reported to possess several biological activities. A. crassna leaf extracts have been demonstrated to have a glucose-lowering effect in animal models. However, it is unclear what phytochemical compounds mediate this antidiabetic property. Here, we describe analytical methods for identifying such compounds from dried leaves by differential extractions with ethanol, butanol, ethyl acetate, and water, respectively. The phytochemical compounds in each fraction were further identified by gas chromatography-mass spectrometry. The cytotoxicity of these fractions was tested against a HepG2 cell line, while the rate of glucose utilization was determined using glucose oxidase assay. Lastly, the inhibitory effect on suppression of hepatic glucose production in HepG2 cells was determined by quantitative real-time PCR of genes encoding pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, and liver glycogen synthase.

c-Myc directly targets an over-expression of pyruvate carboxylase in highly invasive breast cancer.

Here we showed that the c-Myc oncogene is responsible for overexpression of pyruvate carboxylase (PC) in highly invasive MDA-MB-231 cells. Pharmacological inhibition of c-Myc activity with 10074-G5 compound, resulted in a marked reduction of PC mRNA and protein, concomitant with reduced cell growth, migration and invasion. This growth inhibition but not migration and invasion can be partly restored by overexpression of PC, indicating that PC is a c-Myc-regulated pro-proliferating enzyme. Analysis of chromatin immunoprecipitation sequencing of c-Myc bound promoters revealed that c-Myc binds to two canonical c-Myc binding sites, locating at nucleotides -417 to -407 and -301 to -291 in the P2 promoter of human PC gene. Mutation of either c-Myc binding site in the P2 promoter-luciferase construct resulted in 50-60% decrease in luciferase activity while double mutation of c-Myc binding sites further decreased the luciferase activity in MDA-MB-231 cells. Overexpression of c-Myc in HEK293T cells that have no endogenous c-Myc resulted in 250-fold increase in luciferase activity. Mutation of either E-boxes lowered luciferase activity by 50% and 25%, respectively while double mutation of both sites abolished the c-Myc transactivation response. An electrophoretic mobility shift assay using nuclear proteins from MDA-MB-231 confirmed binding of c-Myc to both c-Myc binding sites in the P2 promoter. Bioinformatic analysis of publicly available transcriptomes from the cancer genome atlas (TCGA) dataset revealed an association between expression of c-Myc and PC in primary breast, as well as in lung and colon cancer tissues, suggesting that overexpression of PC is deregulated by c-Myc in these cancers.

CCAAT-enhancer binding protein-α (C/EBPα) and hepatocyte nuclear factor 4α (HNF4α) regulate expression of the human fructose-1,6-bisphosphatase 1 (FBP1) gene in human hepatocellular carcinoma HepG2 cells.

Fructose-1,6-bisphosphatase (FBP1) plays an essential role in gluconeogenesis. Here we report that the human FBP1 gene is regulated by two liver-enriched transcription factors, CCAAT-enhancer binding protein-α (C/EBPα) and hepatocyte nuclear factor 4α (HNF4α) in human hepatoma HepG2 cells. C/EBPα regulates transcription of FBP1 gene via binding to the two overlapping C/EBPα sites located at nucleotide -228/-208 while HNF4α regulates FBP1 gene through binding to the classical H4-SBM site and direct repeat 3 (DR3) located at nucleotides -566/-554 and -212/-198, respectively. Mutations of these transcription factor binding sites result in marked decrease of C/EBPα- or HNF4α-mediated transcription activation of FBP1 promoter-luciferase reporter expression. Electrophoretic mobility shift assays of -228/-208 C/EBPα or -566/-554 and -212/-198 HNF4α sites with nuclear extract of HepG2 cells overexpressing C/EBPα or HNF4α confirms binding of these two transcription factors to these sites. Finally, we showed that siRNA-mediated suppression of C/EBPα or HNF4α expression in HepG2 cells lowers expression of FBP1 in parallel with down-regulation of expression of other gluconeogenic enzymes. Our results suggest that an overall gluconeogenic program is regulated by these two transcription factors, enabling transcription to occur in a liver-specific manner.

Insights into Transcriptional Regulation of Hepatic Glucose Production.

Maintenance of systemic glucose homeostasis is pivotal in animals because most tissues, especially brain and red blood cells, rely on glucose as the sole energy source. The liver protects the body from hypoglycemia because it possesses two biochemical pathways, namely gluconeogenesis and glycogenolysis which provide glucose during starvation period. Posttranslational regulation by allosteric effectors and/or reversible phosphorylation of the key enzymes involved in these two pathways provide the rapid response for the immediate increase in the enzyme activities to accelerate rates of gluconeogenesis and glycogenolysis, but these mechanisms are insufficient for long-term control. Glucoregulatory hormones can alter the rate of enzyme synthesis at the transcriptional step by modulating the key transcription factors and coactivators, such as CREB/CRTC2, FoxO1, nuclear receptors, C/EBPα, hepatocyte nuclear factors, PGC1α, and CLOCK genes. Precise and well-coordinated regulation of activities of these transcription factors at the right time enables liver to synthesize or suppress glucose production, thus maintaining the proper function of tissues and organs during starvation and feeding cycles. Loss of function mutation or deregulation of these key transcription factors and coactivators can result in the pathophysiological condition, such as type 2 diabetes.

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter.

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (-386/-374), non-perfect DR1 (-118/-106) and HNF4α-specific binding motif (H4-SBM) (-26/-14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.

Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

Pyruvate carboxylase (PC) is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney) cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an) activator element(s) for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y), as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription.

Functional characterization of the proximal promoter of the murine pyruvate carboxylase gene in hepatocytes: role of multiple gc boxes.

Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis in the liver. The murine PC gene possesses two promoters, the proximal (P1) and the distal (P2) which mediate production of distinct tissue-specific mRNA isoforms. By comparing the luciferase activities of 5'-nested deletions of the P1-promoter in the AML12 mouse hepatocyte cell line, the critical cis-acting elements required for maintaining basal transcription were located within the 166 nucleotides proximal to the transcription start site. Three GC boxes were identified within this region and shown by gel shift and ChIP assays to bind Sp1/Sp3. Over-expression of Sp1/Sp3 in AML12 and NIH3T3 cells increased P1-promoter activity, with Sp1 being a stronger activator than Sp3. Mutation of any one of the three GC boxes dramatically reduced basal promoter activity by 60-80% suggesting that all three boxes are equally strong regulatory elements. In AML12 cells, over-expression of Sp1/Sp3 restored the transcriptional activity of GC1 and GC2 but not GC3 mutants to levels similar to that of the WT construct, suggesting that GC3 is particularly critical for Sp1/Sp3-mediated induction. In NIH3T3 cells, however, the three boxes were equally important, indicating that the GC boxes differentially contribute to transcriptional regulation of the P1-promoter in the two cell lines. Mutants harboring two disrupted GC boxes showed a further decrease in promoter activity similar to the triple GC box mutant. Neither Sp1 nor Sp3 was able to fully restore the promoter activities of these mutants to that the WT level.

Identification of the cyclic AMP responsive element (CRE) that mediates transcriptional regulation of the pyruvate carboxylase gene in HepG2 cells.

Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis. Here we investigated the effect of various hormones including cAMP, dexamethasone and insulin on the abundance of PC mRNA in the human hepatocyte cell line, HepG2. Treatment of HepG2 cells with 1 microM of glucagon increased the expression of PC mRNA threefold within 72 h. Treatment with 1mM 8-Br-cAMP caused the abundance of PC mRNA to increase by 2-3-fold by 48 h, peak at fourfold at 72 h, and remain unchanged to 96 h. This is in contrast to phosphoenolpyruvate carboxykinase (PEPCK) for which expression was decreased after 72 h, suggesting a distinct difference in the control of these two enzymes in the long term. Dexamethasone or insulin alone did not affect the abundance of PC mRNA whereas treatment of HepG2 cells with the combination of 1mM 8-Br-cAMP and 0.5 microM dexamethasone further increased the abundance of PC mRNA, suggesting the predominant role of 8-Br-cAMP over dexamethasone. Transient transfection of the luciferase reporter construct driven by a 1.95 kbp 5'-flanking sequence of the mouse PC gene and a plasmid encoding the human cAMP-responsive element binding protein increased luciferase reporter activity to 7-fold similar to that observed with a PEPCK promoter-luciferase reporter construct. Deletion of the 5'-flanking region of the PC gene to 781 bp resulted in the complete loss of CREB-mediated induction of reporter gene, suggesting the presence of the cAMP-responsive unit is located between 1.95 kbp and 781 bp upstream of the mouse PC gene. Electrophoretic mobility shifted and chromatin immunoprecipitation assays demonstrated that CREB bind to -1639/-1631 CRE of mouse PC gene in vitro and in vivo, respectively.

Transcriptional regulation of the distal promoter of the rat pyruvate carboxylase gene by hepatocyte nuclear factor 3beta/Foxa2 and upstream stimulatory factors in insulinoma cells.

PC (pyruvate carboxylase) plays a crucial role in intermediary metabolism including glucose-induced insulin secretion in pancreatic islets. In the present study, we identified two regions of the 1.2 kb distal promoter, the -803/-795 site and the -408/-403 E-box upstream of the transcription start site, as the important cis-acting elements for transcriptional activation of the luciferase reporter gene. Site-directed mutagenesis of either one of these sites in the context of this 1.2 kb promoter fragment, followed by transient transfections in the insulinoma cell line, INS-1, abolished reporter activity by approx. 50%. However, disruption of either the -803/-795 or the -408/-403 site did not affect reporter gene activity in NIH 3T3 cells, suggesting that this promoter fragment is subjected to cell-specific regulation. The nuclear proteins that bound to these -803/-795 and -408/-403 sites were identified by gel retardation assays as HNF3beta (hepatocyte nuclear factor 3beta)/Foxa2 (forkhead/winged helix transcription factor box2) and USFs (upstream stimulatory factors), USF1 and USF2, respectively. Chromatin immunoprecipitation assays using antisera against HNF3beta/Foxa2, USF1 and USF2 demonstrated that endogenous HNF3beta/Foxa2 binds to the -803/-795 Foxa2 site, and USF1 and USF2 bind to the -408/-403 E-box respectively in vivo, consistent with the gel retardation assay results. Although there are weak binding sites located at regions -904 and -572 for PDX1 (pancreatic duodenal homeobox-1), a transcription factor that controls expression of beta-cell-specific genes, it did not appear to regulate PC expression in INS-1 cells in the context of the 1.2 kb promoter fragment. The results presented here show that Foxa2 and USFs regulate the distal promoter of the rat PC gene in a cell-specific manner.