Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of both GKRP and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.     

Glucagon induces translocation of glucokinase from the cytoplasm to the nucleus of hepatocytes by transfer between 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase-2 and the glucokinase regulatory protein

Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25 mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes.     

Contributions of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 to the elevated glycolysis in hepatocytes from Zucker fa/fa rats

The insulin-resistant Zucker fa/fa rat has elevated hepatic glycolysis and activities of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 (PFK2). The latter catalyzes the formation and degradation of fructose-2,6-bisphosphate (fructose-2,6-P(2)) and is a glucokinase-binding protein. The contributions of glucokinase and PFK2 to the elevated glycolysis in fa/fa hepatocytes were determined by overexpressing these enzymes individually or in combination. Metabolic control analysis was used to determine enzyme coefficients on glycolysis and metabolite concentrations. Glucokinase had a high control coefficient on glycolysis in all hormonal conditions tested, whereas PFK2 had significant control only in the presence of glucagon, which phosphorylates PFK2 and suppresses glycolysis. Despite the high control strength of glucokinase, the elevated glycolysis in fa/fa hepatocytes could not be explained by the elevated glucokinase activity alone. In hepatocytes from fa/fa rats, glucokinase translocation between the nucleus and the cytoplasm was refractory to glucose but responsive to glucagon. Expression of a kinase-active PFK2 variant reversed the glucagon effect on glucokinase translocation and glucose phosphorylation, confirming the role for PFK2 in sequestering glucokinase in the cytoplasm. Glucokinase had a high control on glucose-6-phosphate content; however, like PFK2, it had a relative modest effect on the fructose-2,6-P(2) content. However, combined overexpression of glucokinase and PFK2 had a synergistic effect on fructose-2,6-P(2) levels, suggesting that interaction of these enzymes may be a prerequisite for formation of fructose-2,6-P(2). Cumulatively, this study provides support for coordinate roles for glucokinase and PFK2 in the elevated hepatic glycolysis in fa/fa rats.     

Glucose-induced dissociation of glucokinase from its regulatory protein in the nucleus of hepatocytes prior to nuclear export

The glucose phosphorylating enzyme glucokinase regulates glucose metabolism in the liver. Glucokinase activity is modulated by a liver-specific competitive inhibitor, the glucokinase regulatory protein (GRP), which mediates sequestration of glucokinase to the nucleus at low glucose concentrations. However, the mechanism of glucokinase nuclear export is not fully understood. In this study we investigated the dynamics of glucose-dependent interaction and translocation of glucokinase and GRP in primary hepatocytes using fluorescence resonance energy transfer, selective photoconversion and fluorescence recovery after photobleaching. The formation of the glucokinase:GRP complex in the nucleus of primary hepatocytes at 5 mmol/l glucose was significantly reduced after a 2 h incubation at 20 mmol/l glucose. The GRP was predominantly localized in the nucleus, but a mobile fraction moved between the nucleus and the cytoplasm. The glucose concentration only marginally affected GRP shuttling. In contrast, the nuclear export rate of glucokinase was significantly higher at 20 than at 5 mmol/l glucose. Thus, glucose was proven to be the driving-force for nuclear export of glucokinase in hepatocytes. Using the FLII12Pglu-700μ-δ6 glucose nanosensor it could be shown that in hepatocytes the kinetics of nuclear glucose influx, metabolism or efflux were significantly faster compared to insulin-secreting cells. The rapid equilibration kinetics of glucose flux into the nucleus facilitates dissociation of the glucokinase:GRP complex and also nuclear glucose metabolism by free glucokinase enzyme. In conclusion, we could show that a rise of glucose in the nucleus of hepatocytes releases active glucokinase from the glucokinase:GRP complex and promotes the subsequent nuclear export of glucokinase.     

Activation and translocation of glucokinase in rat primary hepatocytes monitored by high content image analysis

In the liver, glucokinase (GK) regulatory protein (GKRP) negatively modulates the metabolic enzyme GK by locking it in an inactive state in the nucleus. Here, the authors established a high content screening assay in the 384-well microplate format to measure the nucleus-to-cytoplasm translocation of GK by reagents that destabilize the interaction between GK and GKRP. As a cellular model system, primary rat hepatocytes endogenously expressing both GK and GKRP at physiological levels were used. The GK translocation assay was robust, displayed limited day-to-day variability, and delivered good Z' statistics. The increase of the glucose concentration in the extracellular medium from a low glucose situation (2.8 mM) to beyond its physiological set point value of 5 mM was found to drive GK from the nucleus into the cytoplasm. Likewise, both fructose (converted intracellularly into fructose-1-phosphate) and a known allosteric GK activator were found to induce the export of GK from the nucleus and to synergistically enhance the effects of medium or high glucose concentrations with respect to GK translocation. Transfer of the high content screening format to a semiautomated medium throughput screening platform enabled the profiling of large compound numbers with respect to allosteric activation of GK.     

Subcellular localization of liver FBPase is modulated by metabolic conditions

In primary cultured hepatocytes, fructose-1,6-bisphosphatase (FBPase) localization is modulated by glucose, dihydroxyacetone (DHA) and insulin. In the absence of these substrates, FBPase was present in the cytoplasm, but the addition of glucose or DHA induced its translocation to the nucleus. As expected, we observed the opposite effect of glucose on glucokinase localization. The addition of insulin in the absence of glucose largely increased the amount of nuclear FBPase. Moreover, at high concentrations of glucose or DHA, FBPase shifted from the cytosol to the cell periphery and co-localized with GS. Interestingly, the synthesis of Glu-6-P and glycogen induced by DHA was not inhibited by insulin. These results indicate that FBPase is involved in glycogen synthesis from gluconeogenic precursors. Overall, these findings show that translocation may be a new integrative mechanism for gluconeogenesis and glyconeogenesis.     

Mechanism of the stimulatory effect of a potassium-rich medium on the phosphorylation of glucose in isolated rat hepatocytes.

We have investigated the mechanism by which the replacement of a Na(+)-rich medium by a K(+)-rich medium causes an increase in the apparent affinity of glucokinase (hexokinase IV or D) for glucose in isolated hepatocytes [Bontemps, F., Hue, L. & Hers, H. G. (1978) Biochem. J. 174, 603-611]. The stimulatory effect of a K(+)-rich medium on the rate of glucose phosphorylation, as assessed by the release of tritium from [2-3H]glucose, was only partially additive with the effect of fructose, suggesting that it was also due to a decrease in the inhibition exerted on glucokinase by its regulatory protein. Measurements of metabolites indicated that the effect of the K(+)-rich medium was neither due to the formation of fructose 1-phosphate, nor to changes in the concentrations of fructose 6-phosphate or Pi, two other effectors of the regulatory protein. Replacement of Na+ by K+ in the medium resulted in a time-dependent and dose-dependent increase in cell volume that paralleled the changes in the rate of detritiation observed at 5 mM glucose. The water and chloride contents, estimated using radiolabelled compounds, were threefold and tenfold higher, respectively, in K+ cells than in Na+ cells, and the intracellular Cl- concentration about threefold higher (94 versus 29 meq/l). The effects of the K(+)-rich medium on cell volume, Cl- concentration and rate of detritiation were greatly reduced by including 80 mM trehalose or sucrose in the medium at the start of the incubation. Addition of trehalose to cells incubated for 45-50 min in the K(+)-rich medium caused an immediate decrease in cell volume whereas the rate of detritiation and the Cl- concentration underwent a transient increase followed by a decrease. Replacement of KCl by KBr, potassium acetate or potassium trichloroacetate in the K(+)-rich medium resulted in different relationships between cell volume and the rate of detritiation, in agreement with the differential effect of these salts on the activity of purified glucokinase assayed in the presence of regulatory protein. From these results we conclude that the increase in the activity of glucokinase induced by a KCl-rich medium is at least partly due to an increase in the concentration of Cl-, which relieves the inhibition exerted by the regulatory protein on purified glucokinase.     

Evidence for glucose and sorbitol-induced nuclear export of glucokinase regulatory protein in hepatocytes

Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1-P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the regulatory protein of glucokinase (GKRP) also translocates from the nucleus during substrate-induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin-1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.     

Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone

The conversion of glucose into glucose 6-phosphate in an extract of isolated rat hepatocytes incubated in the presence of MgATP was studied spectrophotometrically at 340nm and also by a radiochemical procedure based on the release of (3)H from [2-(3)H]glucose. Both methods gave similar results. The glucose-saturation curve was sigmoidal and the shape of this curve was not influenced by the ionic composition of the incubation medium. The activity at 0.5mm-glucose was only 1-2% of V(max.), indicating a virtual absence of low-K(m) hexokinase in the preparation. The radiochemical method was also used for the determination of glucose phosphorylation by intact hepatocytes. The glucose-saturation curve was also markedly sigmoidal, but the s(0.5) (substrate concentration at half-maximal velocity) and the Hill coefficient were larger than in extracts of hepatocytes. These two parameters became smaller when cells were incubated in a medium in which Na(+) ions were replaced by K(+) ions. The increased rate of phosphorylation at low glucose concentration in a K(+) medium was accompanied by an increased rate of metabolite recycling between glucose and glucose 6-phosphate and also by an increased uptake of glucose. In both media phosphorylation of glucose was inhibited co-operatively by N-acetylglucosamine. Calculations indicate that this inhibition would reach 100% at saturation of the inhibitor, although at lower concentrations of N-acetylglucosamine it was smaller than expected from the known K(i) of N-acetylglucosamine for glucokinase. The rate of phosphorylation of glucose was proportional to the amount of glucokinase in hepatocytes from newborn rats and in conditions such as starvation and diabetes in which the total amount of glucokinase in the liver is decreased. In the same conditions, glucose 6-phosphatase activity was either normal or increased. It is concluded that the phosphorylation of glucose in isolated hepatocytes follows sigmoidal kinetics, which can be explained by the activity of glucokinase alone with no participation of low-K(m) hexokinase or of glucose 6-phosphatase.     

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.     

Analysis of the cooperativity of human beta-cell glucokinase through the stimulatory effect of glucose on fructose phosphorylation

Using overexpressed Escherichia coli sorbitol-6-phosphate dehydrogenase to monitor fructose 6-phosphate formation, we found that the stimulation of fructose phosphorylation by glucose was reduced in two human beta-cell glucokinase mutants with a low Hill coefficient or when the activity of wild type glucokinase was decreased by replacing ATP with poorer nucleotide substrates. Mutation of two other residues, neighboring glucose-binding residues in the catalytic site, also reduced the affinity for glucose as a stimulator of fructose phosphorylation. Among a series of glucose analogs, only 3, all substrates of glucokinase, stimulated fructose phosphorylation; other analogs were either inactive or inhibited glucokinase. Glucose increased the apparent affinity for inhibitors that are glucose analogs but not for the glucokinase regulatory protein or palmitoyl-CoA. These data indicate that the stimulatory effect of glucose on fructose phosphorylation reflects the positive cooperativity for glucose and is mediated by binding of glucose to the catalytic site. They support models involving the existence of two slowly interconverting conformations of glucokinase that differ through their affinity for glucose and for glucose analogs. We show by computer simulation that such a model can account for the kinetic properties of glucokinase, including the differential ability of mannoheptulose and N-acetylglucosamine to suppress cooperativity (Agius, L., and Stubbs, M. (2000) Biochem. J. 346, 413-421).     

A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate

At a concentration of 1 mM, fructose 1-phosphate stimulated about twofold, and glucose 6-phosphate inhibited by about 30%, the phosphorylation of 5 mM glucose in high-speed supernatants prepared from rat liver or from isolated hepatocytes, but did not affect, or barely so, the activity of a partially purified preparation of glucokinase. Anion-exchange chromatography of liver extracts separated glucokinase from a fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive inhibitor of that enzyme. This inhibitor could be further purified by chromatography on phospho-Ultrogel. It was destroyed by trypsin and was heat-labile. It inhibited glucokinase competitively with respect to glucose and its inhibitory effect was greatly reinforced by fructose 6-phosphate although not by glucose 6-phosphate. Fructose 1-phosphate relieved the enzyme of the inhibitory effect of the regulator and antagonised the effect of fructose 6-phosphate in a competitive manner. It is concluded that the regulator plays a role in the physiological control of the activity of glucokinase, particularly with respect to the stimulatory effect of fructose in isolated hepatocytes (see preceding paper in this journal).     

Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding

The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase in hepatic glucose metabolism.     

Alpha- and beta-anomeric preference of glucose-induced insulin secretion at physiological and higher glucose concentrations, respectively

We determined the anomeric preference of glucose phosphorylation by islet glucokinase, glucose utilization by pancreatic islets, and insulin secretion induced by glucose over a wide range of glucose concentrations. alpha-D-Glucose was phosphorylated faster than beta-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM). At 20 mM, there was no significant difference in phosphorylation rate between the two anomers. Similar patterns of anomeric preference were observed both in islet glucose utilization and in glucose-induced insulin secretion. The present study affords strong evidence that glucokinase is responsible for the anomeric preference of glucose-stimulated insulin secretion through anomeric discrimination in islet glucose utilization.     

Intracellular distribution of hepatic glucokinase and glucokinase regulatory protein during the fasted to refed transition in rats.

We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1- and 2-h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.     

The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte

Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte.     

Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes

Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose. The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05-1 mM) caused a concentration-dependent and transient increase in the fructose 1-phosphate content. Glucagon (1 microM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1-phosphate in cells incubated with fructose, whereas the addition of 0.1 microM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1-phosphate concentration. Sorbitol had a similar effect to fructose in causing the formation of fructose 1-phosphate. D-Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier. The effect of D-glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon. Both fructose and D-glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3H2O from [2-3H]glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier. Glucagon and ethanol antagonised the effect of low concentrations of fructose or D-glyceraldehyde on the detritiation of glucose. These results support the proposal that fructose 1-phosphate mediates the effects of fructose, D-glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein.     

Inhibition of DNA synthesis by TGF-beta 1 coincides with inhibition of phosphorylation and cytoplasmic translocation of p53 protein.

Immunostaining demonstrated that p53 protein was localized in the cytoplasm of growing MCF-7 cells and in the nuclei of cells that were growth arrested by serum starvation. Serum stimulation of the arrested cells induced marked increases in DNA synthesis and p53 phosphorylation, and translocation of the protein from the nucleus to the cytoplasm at 20 h after the stimulation. This increase in the DNA synthesis that was significantly inhibited by TGF-beta 1 was coincident with the inhibition of phosphorylation and cytoplasmic translocation of the p53 protein.     

Regulation of the gene expression of glucokinase and L-type pyruvate kinase in primary cultures of rat hepatocytes by hormones and carbohydrates

The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3 h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)