Changes in the glucose-6-phosphatase complex in hepatomas

Hepatomas tend to have a decreased glucose-6-phosphatase activity. We have observed phenotypic stability for this change in Morris hepatomas transplanted in rats. To determine if this decrease is selective for translocase functions or the hydrolase activity associated with glucose-6-phosphatase, we have compared activities in liver and hepatomas with glucose-6-phosphate or mannose-6-phosphate as substrates and with intact or histone-disrupted microsomes. In five out of seven subcutaneously transplanted rat hepatoma lines, the microsomal mannose-6-phosphatase activity was lower than in preparations from liver of normal or tumor-bearing rats. With liver microsomes and with most hepatoma microsomes, preincubation with calf thymus histones caused a greater increase in mannose-6-phosphatase than in glucose-6-phosphatase activity. In studies with liver and hepatoma microsomes there were similar increases in mannose-6-phosphatase activity with total calf thymus histones and arginine-rich histones. A smaller increase was seen with lysine-rich histones. The effect of polylysine was similar to the action of lysine-rich histones. There was only a small effect with protamine at the same concentration (1 mg/ml). Rat liver or hepatoma H1 histones gave only about half the activation seen with core nucleosomal histones. Our data suggested that microsomes of rat hepatomas tend to have decreased translocase and hydrolase functions of glucose-6-phosphatase relative to activities in untransformed liver. (Mol Cell Biochem122: 17–24, 1993)     

Identification and characterisation of a new human glucose-6-phosphatase isoform

The liver endoplasmic reticulum glucose-6-phosphatase catalytic subunit (G6PC1) catalyses glucose 6-phosphate hydrolysis during gluconeogenesis and glycogenolysis. The highest glucose-6-phosphatase activities are found in the liver and the kidney; there have been many reports of glucose 6-phosphate hydrolysis in other tissues. We cloned a new G6Pase isoform (G6PC3) from human brain encoded by a six-exon gene (chromosome 17q21). G6PC3 protein was able to hydrolyse glucose 6-phosphate in transfected Chinese hamster ovary cells. The optimal pH for glucose 6-phosphate hydrolysis was lower and the K(m) higher relative to G6PC1. G6PC3 preferentially hydrolyzed other substrates including pNPP and 2-deoxy-glucose-6-phosphate compared to the liver enzyme.     

Comparative reactivity of carbamyl phosphate and glucose 6-phosphate with the glucose-6-phosphatase of intact microsomes

The ability of glucose 6-phosphate and carbamyl phosphate to serve as substrates for glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) of intact and disrupted microsomes from rat liver was compared at pH 7.0. Results support carbamyl phosphate and glucose 6-phosphate as effective substrates with both. Km values for carbamyl phosphate and glucose 6-phosphate were greater with intact than with disrupted microsomes, but Vmax values were higher with the latter. The substrate translocase-catalytic unit concept of glucose-6-phosphatase function is thus confirmed. The Km values for 3-O-methyl-D-glucose and D-glucose were larger when determined with intact than with disrupted microsomes. This observation is consistent with the involvement of a translocase specific for hexose substrate as a rate-influencing determinant in phosphotransferase activity of glucose-6-phosphatase.     

Pentamidine activates T1 the hepatic microsomal glucose 6-phosphate transport protein of the glucose-6-phosphatase system.

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by pentamidine has been investigated in both intact and fully disrupted microsomes. The major effect of pentamidine is a 4.7-fold reduction in the Km of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. The site of action of pentamidine is T1 the hepatic microsomal glucose 6-phosphate transport protein. The activation of T1 by pentamidine may contribute to the disturbed blood glucose homeostasis seen in many patients after the administration of the drug pentamidine.     

Pentamidine activates T1 the hepatic microsomal glucose 6-phosphate transport protein of the glucose-6-phosphatase system

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by pentamidine has been investigated in both intact and fully disrupted microsomes. The major effect of pentamidine is a 4.7-fold reduction in the K_m of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. The site of action of pentamidine is T₁ the hepatic microsomal glucose 6-phosphate transport protein. The activation of T₁ by pentamidine may contribute to the disturbed blood glucose homeostasis see in many patients after administration of the drug pentamidine.     

The kinetics of intact microsome glucose-6-phosphatase are sigmoid at physiologic glucose 6-phosphate concentrations

The kinetics of rat liver glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) were studied with intact and detergent-disrupted microsomes from normal and diabetic rats. Glucose-6-P concentrations employed (12 microM to 1.0 mM) spanned the physiologic range. With the enzyme of intact microsomes from both groups, plots of v versus [glucose-6-P] were sigmoid. Hanes plots (i.e. [glucose-6-P]/v versus [glucose-6-P]) were biphasic (concave upwards). A Hill coefficient of 1.45 was determined with substrate concentrations between 12 and 133 microM. Disruption of microsomal integrity abolished these departures from classic kinetic behavior, indicating that sigmoidicity may result from cooperative interaction of glucose-6-P with the glucose-6-phosphatase system at the substrate translocase specific for glucose-6-P. With the enzyme from normal rats the [glucose-6-P] at which the enzyme was maximally sensitive to variations in [glucose-6-P] (which we term "Smax"), determined from plots of dv/d [glucose-6-P] versus [glucose-6-P], was in the physiologic range. The Smax of 0.13 mM corresponded well with the normal steady-state hepatic [glucose-6-P] of 0.16 mM, consistent with glucose-6-phosphatase's function as a regulatory enzyme. With the diabetic enzyme, in contrast, values were 0.30 and 0.07 mM for the Smax and steady-state level, respectively. We suggest that the decreasing sensitivity of glucose-6-phosphatase activity to progressively diminishing glucose-6-P concentration, inherent in its sigmoid kinetics, constitutes a mechanism for the preservation of a residual pool of glucose-6-P for other hepatic metabolic functions in the presence of elevated concentrations of glucose-6-phosphatase such as in diabetes.     

Amiloride activation of hepatic microsomal glucose-6-phosphatase; activation of T1?

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by amiloride has been investigated in both intact and fully disrupted microsomes. The major effect of amiloride is a 4.5-fold reduction in the Km of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. Amiloride also decreased the Km of glucose-6-phosphatase activity in intact liver microsomes isolated from starved rats 2.5-fold. Kinetic calculations, direct enzyme assays and direct transport assays all demonstrated that the site of amiloride action was T1, the hepatic microsomal glucose 6-phosphate transport protein. This is, to our knowledge, the first report of an activation of any of the proteins of the multimeric hepatic microsomal glucose-6-phosphatase complex.     

Modulation of the activity of hepatic glucose-6-phosphatase by methylthioadenosine sulfoxide.

Methylthioadenosine sulfoxide (MTAS), an oxidized derivative of the cell toxic metabolite methylthioadenosine has been used in elucidating the relevance of an interrelationship between the catalytic behavior and the conformational state of hepatic glucose-6-phosphatase and in characterizing the transmembrane orientation of the integral unit in the microsomal membrane. The following results were obtained: (1) Glucose 6-phosphate hydrolysis at 37 degrees C is progressively inhibited when native microsomes are treated with MTAS at 37 degrees C. In contrast, glucose 6-phosphate hydrolysis of the same MTAS-treated microsomes assayed at 0 degrees C is not inhibited. (2) Subsequent modification of the MTAS-treated microsomes with Triton X-114 reveals that glucose-6-phosphatase assayed at 37 degrees C as well as at 0 degrees C is inhibited. (3) Although excess reagent is separated by centrifugation and the MTAS-treated microsomes diluted with buffer before being modified with Triton the temperature-dependent effect of MTAS on microsomal glucose-6-phosphatase is not reversed at all. (4) In native microsomes MTAS is shown to inhibit glucose-6-phosphatase noncompetitively. The subsequent Triton-modification of the MTAS-treated microsomes, however, generates an uncompetitive type of inhibition. (5) Preincubation of native microsomes with MTAS completely prevents the inhibitory effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) as well as 4,4'-diazidostilbene 2,2'-disulfonate (DASS) on glucose-6-phosphatase. (6) Low molecular weight thiols and tocopherol protect the microsomal glucose-6-phosphatase against MTAS-induced inhibition. (7) Glucose-6-phosphatase solubilized and partially purified from rat liver microsomes is also affected by MTAS in demonstrating the same temperature-dependent behavior as the enzyme of MTAS-treated and Triton-modified microsomes. From these results we conclude that MTAS modulates the enzyme catalytic properties of hepatic glucose-6-phosphatase by covalent modification of reactive groups of the integral protein accessible from the cytoplasmic surface of the microsomal membrane. The temperature-dependent kinetic behavior of MTAS-modulated glucose-6-phosphatase is interpreted by the existence of distinct catalytically active enzyme conformation forms. Detergent-induced modification of the adjacent hydrophobic microenvironment additionally generates alterations of the conformational state leading to changes of the kinetic characteristics of the integral enzyme.     

The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide

The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.

We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.     

Thermal stability of microsomal glucose-6-phosphatase

The thermal stability of glucose-6-phosphatase in rat liver microsomes was examined in untreated and cholate-treated microsomes. Activity of the enzyme was measured with both glucose-6-P and mannose-6-P as substrates. Heat treatment did not cause glucose-6-phosphatase activity to decline to zero with a single rate constant in untreated microsomes. Instead, heat treatment produced an enzyme with a small residual activity that was stable. The residual level of activity was not stimulated by addition of detergent. In untreated microsomes the energies of activation for the processes of decay were different for glucose-6-phosphatase and mannose-6-phosphatase activities, suggesting that the rate-limiting steps for the hydrolysis of these compounds were different. Treatment of microsomes with detergent increased the rate constants for the thermal decay of glucose-6-phosphatase by about 150 times, and, in contrast to untreated microsomes, glucose-6-phosphatase and mannose-6-phosphatase decayed to zero with a single rate constant in cholate-treated microsomes. Also, rate constants for thermal inactivation of glucose-6-phosphatase and mannose-6-phosphatase were the same in cholate-treated microsomes. Removal of cholate increased the stability of glucose-6-phosphatase but did not regenerate the form of the enzyme present in untreated microsomes. The data for the stability of glucose-6-phosphatase under different conditions provide evidence that the enzyme can exist in at least five different stable states that are enzymatically active.     

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide on the rat liver microsomal glucose-6-phosphatase system

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose-6-phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose-6-phosphatase, pyrophosphate:glucose and carbamoyl-phosphate : glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose-6-phosphate, pyrophosphate, and carbamoyl-phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that CMC inactivates the microsomal glucose-6-phosphate translocase, the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose-6-phosphate translocase; pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo.     

Protease inhibitors but not proteases inhibit the glucose-6-phosphatase of native rat liver microsomes.

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.     

Hexose metabolism in pancreatic islets: The glucose-6-phosphatase riddle

Summary Glucose-6-phosphatase activity was measured in rat liver or pancreatic islet crude homogenates and microsomes. The data recorded in the liver were comparable to those reported in prior studies. However, in the islets, the hydrolysis of D-glucose 6-phosphate by disrupted microsomes represented, when expressed relative to the protein content, less than 2% of the value recorded in liver microsomes. Moreover, no phosphotransferase activity was detected in the islets. These findings impose reservation on both the presence of glucose-6-phosphatase in rat islets and its participation to stimulus-secretion coupling.     

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.     

Hormone-induced effects on the rat liver microsomal glucose-6-phosphatase system in vitro

We studied the effects of various glucocorticoids, glucagon and insulin on the activity of rat liver microsomal glucose-6-phosphatase. Preincubation of microsomes with corticosterone, cortisone, cortisol and dexamethasone as well as glucagon increased the rate of glucose-6-phosphate hydrolysis by about 1.5 fold relative to the controls. The maximum activation occurred at about 10 nM steroids and 0.3 nM glucagon, respectively. On the other hand, increasing concentrations (8.3 – 50 nM) of insulin progressively inhibited glucose-6-phosphatase up to 26%; the activity of which, however, remains completely in a latent state within the microsomal membrane and can be released from it by Triton treatment. In terms of the substrate transport hypothesis, the results are interpreted as evidence that regulation of glucose-6-phosphate hydrolysis is achieved by direct interactions either of the hormones themselves or of a possible second messenger with the carrier moiety of the rat liver microsomal glucose-6-phosphatase system.     

Membrane effects on hepatic microsomal glucose-6-phosphatase.

1) Rat liver microsomes exhibit only a weak hydrolyzing activity towards galactose 6-phosphate. Disruption of the microsomal vesicles does not change the apparent Michaelis constant for this substrate but enhances the apparent maximum velocity. 2) The inhibition of microsomal glucose-6-phosphatase (EC 3.1.3.9) by galactose 6-phosphate is of the competitive type in intact and disrupted microsomal vesicles, suggesting that both substrates are hydrolyzed by the same enzyme. 3) The high degree of latency found for the hydrolysis of galactose 6-phosphate compared to glucose 6-phosphate indicates the presence of a carrier for glucose 6-phosphate in the microsomal membrane. 4) Since glucose as a product is not trapped inside the microsomal vesicles, this sugar probably is able to penetrate the microsomal membrane.     

The characteristics of liver glucose-6-phosphatase in the envelope of isolated nuclei and microsomes are identical

We have compared the characteristics of glucose-6-phosphatase (EC 3.1.3.9) in the envelope of purified nuclei and microsomes from rat liver. The latency of mannose-6-P hydrolysis, permeability to EDTA, and susceptibility of the enzyme to protease-mediated inactivation all indicated that the permeability barrier defined by the envelope in situ is significantly disrupted in isolated nuclei (i.e. in vitro). Latency of mannose-6-P hydrolysis was demonstrated to provide a quantitative measure of the degree of nuclear membrane disruption. Electron micrographs confirmed the existence of substantial regions of the envelope in vitro where the permeability barrier to EDTA was intact (i.e. an "intact component"). The kinetics of glucose-6-phosphatase catalyzed by the intact component was obtained by subtracting the contribution of enzyme in disrupted regions from the total enzymic activity of untreated nuclei. The characteristics of glucose-6-phosphatase in intact and fully disrupted membranes of nuclei were indistinguishable from microsomes with respect to (a) the kinetics of glucose-6-P hydrolysis, (b) the effects of incubations with mannose-6-P, N-ethylmaleimide, and protease from Bacillus amyloliquefaciens, (c) the extremely high latency of carbamyl phosphate:glucose phosphotransferase activity, and (d) both the patterns of response of activity and the change in latency of glucose-6-phosphatase induced by fasting, experimental diabetes, and cortisol injection. Our results show clearly that apparent differences in the glucose-6-phosphatase activity of untreated preparations of nuclei and microsomes are simply expressions of significant differences in the degree of intactness of their respective permeability barriers. Since flattened cisternae, characteristic of the rough endoplasmic reticulum in situ, are preserved in intact regions of the envelope of isolated nuclei, the present findings constitute the most direct and definitive evidence to date that the properties of glucose-6-phosphatase in the endoplasmic reticulum in situ are faithfully reproduced with intact microsomes.