Up-regulation of liver glucose-6-phosphatase in x-linked hypophosphatemic mice.

We previously showed that a phosphate-deficient diet resulting in hypophosphatemia upregulated the catalytic subunit p36 of rat liver glucose-6-phosphatase, which is responsible for hepatic glucose production. A possible association between phosphate and glucose homeostasis was now further evaluated in the Hyp mouse, a murine homologue of human X-linked hypophosphatemia. We found that in the Hyp mouse as in the dietary Pi deficiency model, serum insulin was reduced while glycemia was increased, and that liver glucose-6-phosphatase activity was enhanced as a consequence of increased mRNA and protein levels of p36. In contrast, the Hyp model had decreased mRNA and protein levels of the putative glucose-6-phosphate translocase p46 and liver cyclic AMP was not increased as in the phosphate-deficient diet rats. It is concluded that in genetic as in dietary hypophosphatemia, elevated glucose-6-phosphatase activity could be partially responsible for the impaired glucose metabolism albeit through distinct mechanisms.

Probing into the function of the gene product responsible for glycogen storage disease type Ib.

This study aimed at directly assessing glucose 6-phosphate (G6P) transport by intact rat liver microsomes. Tracer uptake from labeled G6P occurred with T(1/2) values that proved insensitive to unlabeled G6P or 100 microM vanadate, and could not be activated over background levels by intravesicular phosphate in the complete absence of G6P hydrolysis. [(32)P]Phosphate efflux was similarly unaffected by G6P or phosphate in the incubation medium. We conclude that the gene product responsible for glycogen storage disease type Ib is functionally distinct from the bacterial hexose phosphate transporter, which operates as an obligatory phosphate:phosphate or G6P:phosphate exchanger.

Influence of long-term diabetes on renal glycogen metabolism in the rat.

BACKGROUND/AIMS: The effects of acute insulin deficiency on the kidney have been investigated in animal models of experimental diabetes; however, the impact of long-term diabetes has not been determined. METHODS: We measured renal glycogen contents in streptozotocin (STZ)-diabetic rats 3 weeks (n = 12) or 9 months (n = 12) after the induction of diabetes, and in 2 groups of control rats of similar age (n = 16 and n = 12, respectively), in the fed state and after a 24-hour fast. RESULTS: Diabetic rats had high glucose levels, low insulin but normal glucagon concentrations in portal blood. In the fasting state, kidney glycogen content was very low in both young control and young diabetic rats (54 +/- 15 and 189 +/- 26 microg/g, respectively, mean +/- SD); in contrast, glycogen levels were markedly elevated in rats with long-standing diabetes as compared to old nondiabetic animals (2,628 +/- 1,023 +/- and 1,968 +/- 989 microg/g of diabetic rat, fasting and fed, respectively, p < 0.001 vs. 0 +/- 0 and 4 +/- 6 microg/g of control rats). On electron microscopy, large glycogen clusters were localized to the renal tubules. Kidney phosphorylase activity was higher, and synthase activity lower in diabetic than control rats (p < 0.05 for both), whereas kidney glycogen was strongly related to plasma glucose levels, suggesting that the enzyme changes were secondary to glycogen accumulation itself. Renal hexosephosphates and fructose-2,6-bisphosphate contents were both increased in long-term diabetic rats (p < 0.05), implying enhanced fluxes through both glycolysis and gluconeogenesis. CONCLUSION: In chronic, untreated diabetes glycogen accumulates in the renal tubules; prolonged hyperglycemia is the sole driving force for this phenomenon.

An integrated view of the kinetics of glucose and phosphate transport, and of glucose 6-phosphate transport and hydrolysis in intact rat liver microsomes.

The dynamics of the glucose 6-phosphatase system were investigated in intact rat liver microsomes using a fast-sampling, rapid-filtration apparatus. Glucose and phosphate transport followed single exponential kinetics, appeared to be homogeneous, was unaffected by unlabeled substrate concentrations up to 100 mM, proved insensitive to various potential inhibitors, and demonstrated similarly low energies of activation. The extent of tracer accumulation from glucose 6-phosphate depended on which of the glucose or phosphate moieties was the labeled species in the parent molecule. The rates of tracer equilibration reflected those of glucose or phosphate transport but similar initial rates of uptake were observed for the glucose and phosphate products of hydrolysis. However, the latter accounted for only 12-13% of the steady-state rate of total glucose production. It is concluded that tracer uptake cannot represent substrate transport, that labeled glucose 6-phosphate at best represents a tiny fraction of the intramicrosomal glucose or phosphate pools, and that glucose 6-phosphate transport is not an obligatory prerequisite to its hydrolysis. The latter conclusion invalidates a major postulate of the substrate transport-catalytic unit concept but proves compatible with a conformational model whereby glucose 6-phosphate transport and hydrolysis are tightly coupled processes while glucose and phosphate share, along with water and a variety of other organic and inorganic solutes, a common porelike structure for their transport through the microsomal membrane.

Overexpression of the P46 (T1) translocase component of the glucose-6-phosphatase complex in hepatocytes impairs glycogen accumulation via hydrolysis of glucose 1-phosphate.

The final step of gluconeogenesis and glycogenolysis is catalyzed by the glucose-6-phosphatase (Glc-6-Pase) enzyme complex, located in the endoplasmic reticulum. The complex consists of a 36-kDa catalytic subunit (P36), a 46-kDa glucose 6-phosphate translocase (P46), and putative glucose and inorganic phosphate transporters. Mutations in the genes encoding P36 or P46 have been linked to glycogen storage diseases type Ia and type Ib, respectively. However, the relative roles of these two proteins in control of the rate of glucose 6-phosphate hydrolysis have not been defined. To gain insight into this area, we have constructed a recombinant adenovirus containing the cDNA encoding human P46 (AdCMV-P46) and treated rat hepatocytes with this virus, or a virus encoding P36 (AdCMV-P36), or the combination of both viruses, resulting in large and equivalent increases in expression of the transgenes within 8-24 h of viral treatment. The overexpressed P46 protein was appropriately targeted to hepatocyte microsomes and caused a 58% increase in glucose 6-phosphate hydrolysis in nondetergent-treated (intact) microsomal preparations relative to controls, whereas overexpression of P36 caused a 3.6-fold increase. Overexpression of P46 caused a 50% inhibition of glycogen accumulation in hepatocytes from fasted rats incubated at 25 mm glucose relative to cells treated with a control virus (AdCMV-betaGAL). Furthermore, in hepatocytes from fed rats cultured at 25 mm glucose and then exposed to 15 mm glucose, AdCMV-P46 treatment activated glycogenolysis, as indicated by a 50% reduction in glycogen content relative to AdCMV-betaGAL-treated controls. In contrast, overexpression of P46 had only small effects on glycolysis, whereas overexpression of P36 had large effects on both glycogen metabolism and glycolysis, even in the presence of co-overexpressed glucokinase. Finally, P46 overexpression enhanced glucose 1-phosphate but not fructose 6-phosphate hydrolysis in intact microsomes, providing a mechanism by which P46 overexpression may exert its preferential effects on glycogen metabolism.

Chronic intraperitoneal insulin delivery, as compared with subcutaneous delivery, improves hepatic glucose metabolism in streptozotocin diabetic rats.

We have previously shown that chronic insulin treatment by the intraperitoneal route normalizes the elevated glucose production (GP) in streptozotocin (STZ) diabetic rats, while insulin delivered by the subcutaneous route only partially normalizes GP. To investigate the biochemical mechanism of the effect of chronic insulin delivery by either route on hepatic glucose metabolism, we measured the hepatic activity of glucose 6-phosphatase (G6Pase) and glucokinase (GK). Four groups of rats were used: (1) nondiabetic rats (N, n = 7), (2) untreated STZ diabetic rats (D, n = 8), (3) diabetic rats treated intraperitoneally (IP, n = 6), or (4) subcutaneously (SC, n = 8) (both 3 U of insulin/d). Glucose levels, higher in D, were normalized by insulin treatment regardless of route. Peripheral insulin levels were lowest in D and highest in SC as expected (N, 162 +/- 18 pmol/L; D, 66 +/- 12; IP, 360 +/- 96; SC, 798 +/- 198). STZ diabetes resulted in a 10-fold decrease in GK (P < .001), and a 2-fold increase in G6Pase activity (P < .01). Both intraperitoneal and subcutaneous treatments normalized G6Pase activity. In contrast, with subcutaneous but not intraperitoneal treatment, GK activity was still 35% less than normal (SC v N, P < .05). Glucose 6-phosphate (G6P) levels did not differ among the groups. In summary: (1) the increase in GP in D reflected increased activity of G6Pase and reduced activity of GK, (2) the partial suppression of GP with subcutaneous insulin treatment reflected correction of increased G6Pase activity, but only partial correction of low GK activity, and (3) the normalization of GP with intraperitoneal insulin treatment reflected correction of both increased G6Pase activity and low GK activity. Our current studies indicate that chronic intraperitoneal insulin treatment is superior to subcutaneous treatment with regard to hepatic glucose metabolism.

Dietary P(i) deprivation in rats affects liver cAMP, glycogen, key steps of gluconeogenesis and glucose production.

We previously reported [Xie, Li, Méchin and van de Werve (1999) Biochem. J. 343, 393-396] that dietary phosphate deprivation for 2 days up-regulated both the catalytic subunit and the putative glucose-6-phosphate translocase of the rat liver microsomal glucose-6-phosphatase system, suggesting that increased hepatic glucose production might be responsible for the frequent clinical association of hypophosphataemia and glucose intolerance. We now show that liver cAMP was increased in rats fed with a diet deficient in P(i) compared with rats fed with a control diet. Accordingly, in the P(i)-deficient group pyruvate kinase was inactivated, the concentration of phosphoenolpyruvate was increased and fructose 2, 6-bisphosphate concentration was decreased. Phosphoenolpyruvate carboxykinase activity was marginally increased and glucokinase activity was unchanged by P(i) deprivation. The liver glycogen concentration decreased in the P(i)-deficient group. In the fed state, plasma glucose concentration was increased and plasma P(i) and insulin concentrations were substantially decreased in the P(i)-deficient group. All of these changes, except decreased plasma P(i), were cancelled in the overnight fasted P(i)-deficient group. In the fasted P(i)-deficient group, immediately after a glucose bolus, the plasma glucose level was elevated and the inhibition of endogenous glucose production was decreased. However, this mild glucose intolerance was not sufficient to affect the rate of fall of the glucose level after the glucose bolus. Taken together, these changes are compatible with a stimulation of liver gluconeogenesis and glycogenolysis by the P(i)-deficient diet and further indicate that the liver might contribute to impaired glucose homeostasis in P(i)-deficient states.

Ontogeny of the catalytic subunit and putative glucose-6-phosphate transporter proteins of the rat microsomal liver glucose-6-phosphatase system.

The catalytic subunit (p36) and putative glucose-6-phosphate (G6P) transporter (p46) protein levels of the rat glucose-6-phosphatase (G6Pase) system were studied in relation to G6Pase hydrolytic activity and G6P uptake in liver microsomes during the fetal to neonatal period. The mean G6P hydrolytic activity in liver microsomes increased significantly from the 20th to 21st day of gestation (from 6 to 22 mU/mg protein) and was further enhanced by 3-fold 6 hours after birth, with a maximal activity at 1 day of age (112 mU/mg protein). In contrast, G6P uptake into the vesicles was undetectable before birth, appeared after day 1 (656 pmol/mg protein), and decreased after day 2 (about 330 pmol/mg protein). Immunoblot analysis showed that the mean p36 protein level was low (< 1.6 arbitrary units [AU]) during gestation, increased sharply (to about 4.0 AU) during the first day, and remained stable afterward. Unlike p36, p46 protein was present before birth at values comparable to those postpartum. P46 increased from 3.2 AU at 20 days to 4.6 AU at 21 days of gestation, and decreased transiently after birth. These results show that (1) G6Pase hydrolytic activity before birth can occur without detectable G6P uptake function; (2) the presence of the putative G6P transporter protein is not sufficient to elicit G6P uptake; and (3) full G6Pase activity requires optimal expression of both p36 and p46 proteins. These data are discussed in relation to the function of G6Pase.

Glucose-6-phosphate transporter and receptor functions of the glucose 6-phosphatase system analyzed from a consensus defined by multiple alignments.

The cDNA encoding the protein (P46) that is mutated in glycogen storage disease type-1b (GSD-1b) has been previously cloned by homology with bacterial sequences of the uhp (upper hexose phosphate) system. Hydropathic profiles, transmembrane-prediction analysis, and a multiple alignment of 14 sequences related to P46 (with percentage of identity around 30%) allowed to identify two large domains in the proteins linked by a large variable loop. Highly conserved transmembrane (TM) segments, TM1 and TM4 in the first domain and TM5 in the second one, were identified almost in all the integral proteins related to P46. The multiple alignment allowed definition of a consensus involving the 14 sequences related to P46. The detailed comparison of the consensus with the UhpT (the bacterial G6P transporter) and with UhpC (the bacterial G6P receptor) sequences reveals that the P46 protein could carry both G6P receptor and transporter functions.

Distinct hormone stimulation and counteraction by insulin of the expression of the two components of glucose 6-phosphatase in HepG2 cells.

We found recently (J. Biol. Chem. 274, 33866-33869, 1999) that the expression of the catalytic subunit (p36) and putative glucose 6-phosphate translocase (p46) of the liver glucose 6-phosphatase system was stimulated by cyclic AMP and glucose and repressed by insulin. We now further show in HepG2 cells that whereas insulin (0.01-10 nM) suppressed p36 mRNA, it only reduced p46 mRNA by half at 1 microM. Cyclic AMP (0.01-100 microM) caused a 2.7-fold increase in p36 mRNA but barely increased p46 mRNA. In contrast, dexamethasone (0.1-100 nM) increased both p36 and p46 mRNA by more than 3-fold. The effects of cyclic AMP and dexamethasone were counteracted by 1 microM insulin. The endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (1-100 nM) increased p36 mRNA by 2-fold but not p46 mRNA. It thus appears that the hormonal changes which affect p36 alone concur with known modifications in glucose production; those that affect both p36 and p46 are rather consistent with glucose storage.

New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system.

The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.

Insulin-induced Ca(2+) entry in hepatocytes is important for PI 3-kinase activation, but not for insulin receptor and IRS-1 tyrosine phosphorylation.

Insulin produces an influx of Ca(2+) into isolated rat hepatocyte couplets that is important to couple its tyrosine kinase receptor to MAPK activity (Benzeroual et al., Am. J. Physiol. 272, (1997) G1425-G1432. In the present study, we have examined the implication of Ca(2+) in the phosphorylation state of the insulin receptor (IR) beta-subunit and of insulin receptor substrate-1 (IRS-1), as well as in the stimulation of PI 3-kinase activity in cultured hepatocytes. External Ca(2+) chelation (EGTA 4 mM) or administration of Ca(2+) channel inhibitors gadolinium 50 microM or nickel 500 microM inhibited insulin-induced PI 3-kinase activation by 85, 50 and 50%, respectively, whereas 200 microM verapamil was without effect. In contrast, the insulin-induced tyrosine phosphorylation of IR beta-subunit and of IRS-1 was not affected by any of the experimental conditions. Our data demonstrate that the stimulation of PI 3-kinase activity by the activated insulin receptor, but not the phosphorylation of IR beta-subunit and IRS-1, requires an influx of Ca(2+). Ca(2+) thus appears to play an important role as a second messenger in insulin signaling in liver cells.

Diabetes affects similarly the catalytic subunit and putative glucose-6-phosphate translocase of glucose-6-phosphatase.

The effect of streptozocin diabetes on the expression of the catalytic subunit (p36) and the putative glucose-6-phosphate translocase (p46) of the glucose-6-phosphatase system (G6Pase) was investigated in rats. In addition to the documented effect of diabetes to increase p36 mRNA and protein in the liver and kidney, a approximately 2-fold increase in the mRNA abundance of p46 was found in liver, kidney, and intestine, and a similar increase was found in the p46 protein level in liver. In HepG2 cells, glucose caused a dose-dependent (1-25 mM) increase (up to 5-fold) in p36 and p46 mRNA and a lesser increase in p46 protein, whereas insulin (1 microM) suppressed p36 mRNA, reduced p46 mRNA level by half, and decreased p46 protein by about 33%. Cyclic AMP (100 microM) increased p36 and p46 mRNA by >2- and 1.5-fold, respectively, but not p46 protein. These data suggest that insulin deficiency and hyperglycemia might each be responsible for up-regulation of G6Pase in diabetes. It is concluded that enhanced hepatic glucose output in insulin-dependent diabetes probably involves dysregulation of both the catalytic subunit and the putative glucose-6-phosphate translocase of the liver G6Pase system.

Up-regulation of liver glucose-6-phosphatase in rats fed with a P(i)-deficient diet.

Because P(i) deprivation markedly affects the Na/P(i) co-transporter in kidney and has been related to insulin resistance and glucose intolerance, the effect of a P(i)-deficient diet on the liver microsomal glucose-6-phosphatase (G6Pase) system was investigated. Rats were fed with a control diet (+P(i)) or a diet deficient in phosphate (-P(i)) for 2 days and killed on the morning of the third day, after an overnight fast (fasted) or not (fed). Kinetic parameters of P(i) transport (t((1/2)) and equilibration) into liver microsomes were not changed by the different nutritional conditions. In contrast, it was found that G6Pase activity was significantly increased in the (-P(i)) groups. This was due to an increase in the V(max) of the enzyme, without change in the K(m) for G6P. There was no correlation between liver microsomal glycogen content and G6Pase activity, but both protein abundance and mRNA of liver 36 kDa catalytic subunit of G6Pase (p36) were increased. The mRNA of the putative G6P translocase protein (p46) was changed in parallel with that of the catalytic subunit, but the p46 immunoreactive protein was unchanged. These findings indicate that dietary P(i) deficiency causes increased G6Pase activity by up-regulation of the expression of the 36 kDa-catalytic-subunit gene.

Evidence that the transit of glucose into liver microsomes is not required for functional glucose-6-phosphatase.

We show that the production of glucose from glucose-6-phosphate hydrolysis outside microsomes is a function of glucose-6-phosphatase independent of its property to form glucose inside microsomes. Indeed, during development (before 1 day of age), mouse liver microsomes had glucose-6-phosphatase producing glucose solely outside microsomes. Furthermore, in vivo treatment of rats with the glucocorticoid analogue triamcinolone resulted in increased glucose-6-phosphatase activity outside but not inside microsomes and without change in the catalytic subunit 40 kDa glucose-6-phosphatase mRNA abundance or protein level, indicating that other factors induced by triamcinolone (e.g., altered membrane lipid environment and/or a regulatory protein) were responsible for the activity change. Triamcinolone treatment also lessened the inhibition of glucose-6-phosphatase by pyridoxal 5'-phosphate (PLP), but this effect was not due to an interaction of PLP with the active site. Accordingly, reversal of the inhibition was observed after permeabilization of the microsomes. The two distinct orientations of liver microsomal glucose-6-phosphate phosphohydrolase suggest different physiological roles played by this enzyme in the endoplasmic reticulum membrane.

Dexfenfluramine modulates hepatic glycogen metabolism by a calcium-dependent pathway.

In this study, the mechanism of action of dexfenfluramine (DEXF) at the hepatic level was investigated. The drug is shown to bind to the alpha 1-adrenergic receptor and to increase intracellular calcium in isolated rat hepatocytes, thereby activating phosphorylase via a calcium-dependent mechanism. Moreover, phosphorylase activation by DEXF was inhibited by different agents that interfere with the alpha 1-adrenergic signalling system: prazosin, phorbol 12 alpha-myristate 13 beta-acetate (PMA), and DEXF itself. We also show that phosphorylase activation induced by catecholamines and analogues (epinephrine, phenylephrine), whose actions are mediated by a calcium-dependent mechanism, was counteracted by the drug in the submillimolar range (0.1-1 mM). The activation of glycogenolysis by the drug is accompanied by a stimulation of the glycolytic flux (54% increase in lactate plus pyruvate accumulation), consistent with an increase in fructose-2,6-bisphosphate (F-2,6-BP) levels (36%). These results indicate that the interaction of DEXF with the alpha 1-adrenergic receptor channels glucose 6-phosphate derived from glycogen away from glucose production into the glycolytic pathway.

Insulin induces Ca2+ influx into isolated rat hepatocyte couplets.

Isolated rat hepatocyte couplets were used to study the direct effect of insulin on intracellular Ca2+ homeostasis. Insulin induced a dose-dependent increase in hepatocellular Ca2+ that was gradual, generally monophasic, and reversible. Chelation of extracellular Ca2+ abolished the insulin-induced Ca2+ response, and this suppression was not related to an effect on insulin binding, as indicated by displacement studies. We thus tested the effect of several Ca2+ channel inhibitors on insulin-induced Ca2+ influx. Verapamil at 20 or 200 microM was without effect, whereas 500 microM nickel and 50 microM gadolinium strongly inhibited insulin-induced Ca2+ entry. Finally, we tested whether insulin-induced Ca2+ movements were implicated in the stimulation of mitogen-activated protein kinase (MAPK) activity, which we measured with the use of an immune-complex assay. Verapamil was without effect on the insulin-dependent stimulation of p44mapk activity, whereas addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, nickel, or gadolinium strongly inhibited the effect of the peptide hormone. Our results indicate that insulin triggers Ca2+ influx into hepatocytes, possibly through the opening of channels on the plasma membrane, and that this effect is important for insulin activation of MAPK.

Activation of mitogen-activated protein kinase in freshly isolated rat hepatocytes by both a calcium- and a protein kinase C-dependent pathway.

In the present study, we investigated the role of calcium and protein kinase C (PKC) in the activation of mitogen-activated protein kinase (MAPK) in isolated rat hepatocytes. We found that the glycogenolytic hormone norepinephrine (NE), acting through the alpha1-adrenergic receptor and the G protein Gq, was able to induce a dose- and time-dependent activation of MAPK in hepatocytes. Vasopressin, which acts through a different receptor but also through stimulation of the Gq-dependent pathway, also caused a twofold activation of MAPK. Activation of MAPK by both agonists required the presence of free extracellular calcium and was blocked by the specific PKC inhibitor, Ro 31-8220. MAPK activation was also induced by phorbol myristate acetate (PMA), confirming that a PKC-dependent pathway exists for MAPK activation in liver. Furthermore, calcium-mobilizing agents such as thapsigargin and ionomycin were able to induce an activation of MAPK by a PKC-independent pathway that was totally abolished by preincubation of cells with EGTA. A second pathway for MAPK activation that relies solely on calcium may therefore exist. Ro 31-8220 did not affect phosphorylase activation by NE, vasopressin, thapsigargin, and ionomycin, indicating that PKC inhibition did not interfere with the signaling pathway leading to inositol-1,4,5-triphosphate (IP3)-induced calcium mobilization or with changes in calcium fluxes. The role of MAPK activation by NE and vasopressin in the regulation of hepatic carbohydrate metabolism is discussed.

Glucose transport and glucose 6-phosphate hydrolysis in intact rat liver microsomes.

Glucose transport was investigated in rat liver microsomes in relation to glucose 6-phosphatase (Glu-6-Pase) activity using a fast sampling, rapid filtration apparatus. 1) The rapid phase in tracer uptake and the burst phase in glucose 6-phosphate (Glu-6-P) hydrolysis appear synchronous, while the slow phase of glucose accumulation occurs during the steady-state phase of glucose production. 2) [14C]Glucose efflux from preloaded microsomes can be observed upon addition of either cold Glu-6-P or Glu-6-Pase inhibitors, but not cold glucose. 3) Similar steady-state levels of intramicrosomal glucose are observed under symmetrical conditions of Glu-6-P or vanadate concentrations during influx and efflux experiments, and those levels are directly proportional to Glu-6-Pase activity. 4) The rates of both glucose influx and efflux are characterized by t1/2 values that are independent of Glu-6-P concentrations. 5) Glucose efflux in the presence of saturating concentrations of vanadate was not blocked by 1 mM phloretin, and the initial rates of efflux appear directly proportional to intravesicular glucose concentrations. 6) It is concluded that glucose influx into microsomes is tightly linked to Glu-6-Pase activity, while glucose efflux may occur independent of hydrolysis, so that microsomal glucose transport appears unidirectional even though it can be accounted for by diffusion only over the accessible range of sugar concentrations.

Evidence for a membrane exchangeable glucose pool in the functioning of rat liver glucose-6-phosphatase.

We have investigated the kinetics of tracer uptake into rat liver microsomes in relation to [14C]glucose 6-phosphate (Glu-6-P) hydrolysis by glucose 6-phosphatase (Glu-6-Pase). 1) The steady-state levels of intravesicular tracer accumulated during the rapid (AMP1) and slow (AMP2) phases of uptake both demonstrate Michaelis-Menten kinetics relative to outside Glu-6-P concentrations with Km values similar to those observed for the initial burst (Vi) and steady-state (VSS) rates of Glu-6-P hydrolysis. 2) The AMP1/AMP2 ratio is constant (mean value = 0.105 +/- 0.018) over the whole range of outside Glu-6-P concentrations and is equal to the AMP1max/AMP2max ratio (0.109 +/- 0.032). 3) Linear relationships are observed between the initial rates of glucose transport during the slow uptake phase (V alpha 2) and [AMP1], and between [VSS] and [AMP2]. 4) The value of Vss max exceeds by more than 10-fold that of V alpha 2 max. 5) It is concluded that the substrate transport model is incompatible with those results and that AMP1 represents a membrane exchangeable glucose pool. 6) We propose a new version of the conformational model in which the catalytic site lies deep within a hydrophilic pocket of an intrinsic membrane protein and communicates with the extra- and intravesicular spaces through channels with different glucose permeabilities.