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Phosphoenolpyruvate carboxykinase (PEPCK) is considered to be the rate-limiting enzyme for gluconeogenesis. Alterations in levels of transcription regulated PEPCK have been shown to accurately reflect changes in PEPCK activity as well as rates of gluconeogenesis.［1］In hepatocytes, insulin resistance can result from PEPCK over-expression.［2］ This also appears to involve disturbed insulin signaling towards the regulation of PEPCK expression.［2,3］Therefore, the activity of PEPCK is increased in diabetes. This observation emphasizes the role that PEPCK could play in the metabolic alterations associated with overt type 2 diabetes and perhaps in the pathogenesis of diabetes as well. Antihyperglycemic effect of metformin is mainly a consequence of reduced hepatic glucose output. This can be accounted for mainly by inhibition of gluconeogenesis. However, the molecular basis for metformin's effect on hepatocytes is largely unknown. This also includes possible effects of metformin on insulin signaling and regulation of PEPCK in hepatocytes. With these facts as background, the aim of the present study was therefore to investigate the effect and mechanism of metformin on the expression of PEPCK gene in hepatocytes, and to determine whether the effects of metformin in hepatocytes are transmitted throughout the known insulin signaling pathways.
METHODS
Materials
Dulbecco's Modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL, USA. RNAzol was purchased from WAK Chemie, Germany. Random prime kit, PCR DIG (digoxin) probe synthesis kit and Dig Easy Hyb solution were obtained from Roche, USA. First strand cDNA synthesis kit was obtained from MBI Fermentas, Germany. The dig luminescent detection kit was purchased from Boehringer. Human insulin (Actrapid) was obtained from Novo Nordisk (Germany). Metformin, 8-Br-cAMP, dexamethasone, wortmannin, UO126 (1,4-diamino-2,3-dicyano-1,4-bisc 2-aminopherylthio, butadiene) and other reagents were bought from Sigma, USA.
Cell culture
H4IIE rat hepatoma cells were grown to confluence in 10 cm plastic culture dishes containing 10 ml DMEM supplemented with 10% FBS, 25 mmol/L HEPES (pH 7.4), 100 U/ml penicillin, 100 mg/ml streptomycin and 25 mmol/L glutamine. Twelve hours before each experiment, the medium was replaced with serum-free DMEM. The cells were incubated at 37℃ in a humidified atmosphere of 5% CO［2］ and 95% air. Confluent H4IIE cells were preincubated with or without the concentrations of metformin for 16 h. During the last 4 h of incubation, cells were treated with or without 100 μmol/L cAMP and 500 nmol/L dexamethasone either in the absence or in the presence of the concentrations of insulin (as specified in the figure legends). In some experiments, cells were preincubated with 500 nmol/L wortmannin, 100 μmol/L UO126 for 30 min prior to the addition of metformin to the medium. Cells treated without these compounds were used as basal controls. Incubations were stopped by quickly removing the medium and cells were washed three times with ice-cold PBS.
Isolation of total RNA and Northern blot
RNA isolation was performed according to the RNAzol method. For Northern blot, 20 μg aliquots of RNA were separated by 1% agarose gel electrophoresis and transferred onto nylon membranes (Amersham Pharmacia Biotech, USA) by Northern blot with 10×SSC (0.15 mol/L sodium citrate, 1.5 mol/L NaCl, pH 7.0) overnight. RNA was bound to membranes by a 2 min exposure to UV light of 254 nm. The probes were produced using a PCR Dig probe synthesis kit. Consistent volumes of cDNA-probes (2 μl labeled probe/10 ml hybridization solution) were denatured at 95℃ for 10 min. The membranes were hybridized with Dig Easy Hyb solution in a forced air rotary hybridization oven at 48℃ for 16 h. After hybridization, membranes were washed twice with 2×SSC/0.1% SDS for 5 min at 25℃ and then washed twice with 0.1×SSC/0.1% SDS at 68℃ for 15 min. Detection of hybridized RNA species was carried out using a Dig luminescent detection kit at 20℃. Then, the membrane was exposed to X-ray and resulting bands were visualized. During the experiments, β-actin served as a control for assessment of equivalent RNA loading
Statistical analysis
Blot signal intensities were quantified by Image Quant scanning densitometry (Molecular Dynamics, USA). The average densitometric value for the levels in control was arbitrarily set at 1.0. Results were expressed as mean±s.Results between experimental groups were compared by Student's t test. Statistical significance was considered at P<0.05.
RESULTS
Effects of metformin on the expression of the PEPCK gene in H4IIE cells
Given the low basal level of PEPCK transcription in the absence of other agents, transcriptional regulation of PEPCK has been best studied using the ability of insulin to counteract stimulation of gene transcription by glucagon (or cAMP) and glucocorticoids.［4,5］Thus, for the purpose of comparison, the regulation of insulin and cAMP/dexamethasone on the transcription of PEPCK gene was first investigated. Compared to the basal level, cAMP and dexamethasone stimulated the expression of PEPCK mRNA was increased by 5.7 fold (P<0.001). At a concentration of 10 nmol/L, insulin almost completely inhibited the induction of PEPCK mRNA by cAMP and dexamethasone (P<0.001). Compared to cells incubated with cAMP and dexamethasone alone, the addition of insulin led to a reduction of PEPCK mRNA by 91% (P<0.001) at 1 nmol/L insulin, by 82% (P<0.01) at 0.1 nmol/L insulin and by 6 % (P=0.8132) at 0.01 nmol/L insulin (Fig. 1)
The following experiments were carried out using a concentration of 0.1 nmol/L insulin, as this concentration had the lowest detectable but significant inhibitory effect on PEPCK gene expression. Treatment with 0.1 mmol/L metformin alone for 16 h significantly decreased basal PEPCK mRNA levels by 75% (P<0.001). When cells were incubated with cAMP and dexamethasone, simultaneous treatment with metformin had no significant effect on the increase of PEPCK mRNA. In contrast, when 0.1 nmol/L insulin was present together with cAMP/dexamethasone, the addition of metformin was associated with a 94% decrease in PEPCK gene expression compared to cells stimulated with cAMP and dexamethasone alone (P<0.001) (Fig. 2). The concentration of metformin was selected based upon the dose-response experiment shown in Fig. 3, in which 0.1 mmol/L metformin consistently produced the highest inhibitory effect at a therapeutic concentration.
Effects of inhibitors on the insulin signaling pathway
To further clarify the mechanism of metformin's action and to determine whether the effects of metformin are transmitted throughout the known insulin signaling pathways, we examined the inhibitory effect of metformin in the presence of inhibitors of the insulin signaling cascade. The inhibitors were wortmannin, which inhibits PI 3K, and UD126, a MAPK inhibitor,［6］which binds to MEK1 and MEK2 and inhibits both molecules. As shown in Fig. 4, in the presence of both 0.1 mmol/L metformin alone and metformin with 0.1 nmol/L insulin together, neither wortmannin nor UD126 had any effect on metformin-inhibited PEPCK mRNA level. In the absence of metformin, either wortmannin alone or wortmannin with UD126 together almost completely blocked the inhibitory effect of insulin on cAMP/dexamethasone-induced PEPCK gene expression. With wortmannin/insulin/cAMP/dexamethasone, PEPCK mRNA was 82% of that treated with cAMP/dexamethasone (P<0.01). With wortmannin/UD126/insulin/cAMP/dexamethasone, PEPCK mRNA was 95% of that treated with cAMP/dexamethasone (P<0.01). Treatment of UD126 alone had no significant effect on the inhibitory action of insulin and/or metformin on PEPCK mRNA (Fig. 4) .
DISCUSSION
PEPCK is the rate-limiting enzyme in gluconeogenesis. Its expression is controlled at the transcriptional level.［1,5］ Using stable transfectants with PEPCK promoter sequence ligated to the CAT reporter gene, the H4IIE cell line was widely used to study the regulation of PEPCK gene transcription by numerous hormones and other agents, such as glucagon, glucocorticoids and insulin.［5］Based on these data, H4IIE cells were chosen for the present study as a cell model to examine the effect of metformin on the expression of the PEPCK gene, which is regulated by glucagon, glucocorticoids and insulin.Liver, a key target of insulin action, controls fasting blood glucose primarily by regulating the rate of gluconeogenesis. Increased gluconeogenesis is believed to make a major contribution to fasting hyperglycemia in type 2 diabetes. The inhibition of endogenous glucose production through suppression of gluconeogenesis is one of the main antihyperglycemic effects of metformin.［7-9］Although the drug has been extensively studied, little is known regarding the exact molecular mechanism of metformin action in liver.
A large amount of data demonstrated that metformin inhibits total hepatic glucoseoutput by inhibiting gluconeogenesis in a dose-dependent manner. In isolated rat hepatocytes, metformin has been shown to inhibit glucose production and, in particular, gluconeogenesis in doses ranging from high physiological to pharmacological concentrations (100 to 5000 μmol/L).［9］This effect appears to be dose-dependent and is obviously observed in the presence of insulin. However, in the absence of insulin, metformin may directly suppress gluconeogenesis at pharmacological concentrations.［9］ Metformin has also been reported to enhance pyruvate kinase activity, decrease hepatic lactate uptake in the perfused liver［9］and to inhibit hepatic glucose-6 phosphatase activity in insulin-resistant rats fed a high-fat diet.［10］In addition, metformin has also been reported to decrease glucose production by reducing the flux through pyruvate carboxylase-phosphoenolpyruvate carboxykinase (PC-PEPCK).［11］
The present study demonstrated that an additional molecular mechanism of metformin's action in H4IIE rat hepatoma cells might exist to inhibit PEPCK gene expression. Treatment of metformin alone decreased basal PEPCK mRNA level. Furthermore, metformin inhibited cAMP and dexamethasone-induced PEPCK gene expression through interaction with insulin. However, in the absence of insulin in the culture medium, metformin had no effect on cAMP- and dexamethasone-induced levels of PEPCK mRNA. In contrast, insulin had a clear inhibitory effect on cAMP- and dexamethasone-induced expression of PEPCK gene. The results suggested that insulin and metformin could inhibit PEPCK gene expression via different mechanisms. On the other hand, in the presence of low insulin concentration with only mild suppression of PEPCK gene expression, addition of metformin significantly decreased cAMP- and dexamethasone-stimulated PEPCK mRNA expression. This suggested a potentiation of insulin action by metformin. Thus, metformin inhibited PEPCK mRNA expression via two distinct mechanisms, with one being insulin-independent and the other in conjunction with insulin.
The present data have given further evidence to this point by using insulin signal pathway inhibitors.The regulation of PEPCK gene expression by insulin has been studied in most detail and has served as a useful model.［5］Insulin has been shown to inhibit basal and cAMP/dexamethasone-stimulated PEPCK gene expression in H4IIE cells.［5］Although the regulation of PEPCK is a critical physiological site of insulin action, the signal transduction pathways involved in these actions of insulin are only now beginning to be elucidated.［4,12］Recently, conflicting reports have suggested that insulin inhibits cAMP-induced PEPCK gene expression via activating the Ras/MAPK pathway or via the PI 3K pathway.［13］The PI 3K inhibitor wortmannin and LY 294002 have been shown to block the effects of insulin on PEPCK gene transcription.［4］Recently, Favata et al［6］recommended a highly selective inhibitor of MEK1 and MEK2 in the MAPK pathway, UO126. It has shown that this compound is able to inhibit both MEK1 and MEK2. In contrast, the frequently used MAPK inhibitor PD098059 inhibits only MEK1. In the present stydy, cells in subsequent experiments were treated with metformin in the presence or absence of wortmannin and/or UO126, respectively. The data revealed that neither wortmannin nor UO126 had any effect on the observed inhibition of basal PEPCK mRNA levels by metformin. In the presence of insulin and metformin together, wortmannin caused a partial inhibition of cAMP and dexamethasone-induced PEPCK gene expression. In the presence of insulin alone, wortmannin completely reverse cAMP and dexamethasone-stimulated PEPCK mRNA levels. In contrast, UO126 was shown to have no effect on both metformin's and insulin's ability to inhibit expression of PEPCK gene. These results suggest that the inhibitory effects of insulin on PEPCK gene expression are mediated presumably through the PI 3K pathway, not MAPK pathway.
On the other hand, our data suggests that inhibition of the PEPCK gene expression is induced through at least two pathways, of which one is a PI 3K-mediated insulin-dependent pathway and another is an insulin-independent pathway. Metformin inhibits PEPCK mRNA level mainly through the insulin-independent pathway. The exact molecular mechanism of this inhibitory effect of metformin on PEPCK gene expression remains to be clarified. There are two possible mechanisms, one is to directly inhibit the PEPCK gene expression; another is to indirectly reduce PEPCK mRNA levels. For example, metformin could inhibit the expression of a transcription factor, which may be necessary for PEPCK gene activity in hepatocytes. The dose-response experiment in the present study revealed that metformin in doses ranging from physiological to pharmacological concentrations (0.01 to 1 mmol/L) inhibited either basal PEPCK mRNA levels in the absence of insulin or cAMP/dexamethasone-induced PEPCK gene expression in the presence of insulin. These effects were dose-dependent. This dose of drug is consistent with the current recommended therapeutic dose of metformin at between 500 to 2250 mg/day in vivo. Recently, Fulgencio et al［14］also reported that 50 to 500 μmol/L metformin decreased PEPCK gene expression in isolated rat hepatocytes. Since these effects of metformin on PEPCK gene expression were observed at concentrations reached in the serum of metformin-treated patients, these data could be somewhat suggestive of the actual situation of metformin action in vivo.
In the present study, when the dose of metformin was further increased to 10 mmol/L, the effects of metformin were partially blocked. It is likely that at this high pharmacological concentration, metformin may inhibit some proteins or kinase activity that are necessary in regulation of metformin or insulin on PEPCK gene expression. A possible example could be the expression or phosphorylation of some insulin signaling proteins. We have further confirmed this hypothesis in HepG2 cells, in which, 10 mmol/L metformin inhibited tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and the association of IRS-1 with PI 3K.［15］These results suggest that a possible mechanism by which metformin reduces gluconeogenesis is to inhibit PEPCK gene expression. So far, the observations are mainly in vitro, in vivo studies remain to be further investigated
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