Use of protein-mediated lipid exchange in the study of membrane-bound enzymes. The lipid dependence of glucose-6-phosphatase.

The ability of liver lipid-exchange proteins to introduce foreign phospholipids into microsomes was used in a study of the lipid dependence of glucose-6-phosphatase. Supplementation of intact rat liver and hepatoma microsomes with exogeneous aminophospholipids prevents the decline of glucose-6-phosphatase activity during incubation, whereas the introduction of exogeneous phosphatidylcholine has no protective effect. On the contrary with deoxycholate-disrupted hepatoma microsomes, introduction of additional phosphatidylcholine causes activation while phosphatidylethanolamine has only little effect. The results are explained by assuming that the transport unit and the catalytic moiety of the glucose-6-phosphatase system have different lipid requirements, the activity of the former protein depending mainly on phosphatidylethanolamine and phosphatidylserine and that of the catalytic protein depending on phosphatidylcholine. In deoxycholate-disrupted liver microsomes (in which both the glucose-6-phosphatase activity and the phosphatidylcholine content are much higher than in hepatoma microsomes) incubation with phosphatidylcholine and lipid-exchange proteins alters neither the phospholipid composition nor the enzyme activity. THis suggests that the diminished activity of glucose-6-phosphatase in hepatomas may be partly due to a low level of phosphatidylcholine.     

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.     

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)     

The effects of growth hormone, thyroxine and insulin on the activities of reduced nicotinamide adenine dinucleotide phosphate dehydrogenase, glucose-6-phosphatase and glycogen phosphorylase in fetal rat liver.

Growth hormone (GH), thyroxine (T4) and insulin were injected, in utero into 20.5 day-old rat fetuses to study the effects of these hormones on the activities of liver NADPH dehydrogenase, glucose-6-phosphatase and glycogen phosphorylase. It was found that at 21.5 days of gestation, GH increases the fetal liver glucose-6-phosphatase activity and decreases the liver glycogen phosphorylase activity. T4 treatment augments the activity of NADPH dehydrogenase even at 0.3% of the dose shown previously to produce premature elevation of activity. Prior to this experiment T4 in large doses has been shown to be capable of elevating glucose-6-phosphatase. However, at the lower T4 dose used, no treatment effect was observed. The fetal rat liver is responsive to insulin at 21.5 days and insulin was able to depress glucose-6-phosphatase activity. Thereby, showing that the influence of insulin on this enzyme begins prior to birth instead of just subsequent to birth.     

Liver glucose-6-phosphatase activity is modulated by physiological intracellular Ca2+ concentrations

The effect of varying concentrations of free Ca2+ on the formation of Pi from mannose-6-P or of Pi and [U-14C]glucose from [U-14C]glucose-6-P was investigated in isolated fasted rat hepatocytes made permeable by freezing and in liver microsomes. Free Ca2+ concentration was adjusted by the use of Ca-EGTA buffers. In permeabilized cells, glucose-6-phosphatase (EC 3.1.3.9) activity was inhibited up to 50% and in intact microsomes up to 70% by increasing free Ca2+ concentrations from 0.01 to 10 microM. The inhibition was reversible and competitive with respect to glucose-6-P. Treatment of microsomes with 0.4% deoxycholate exposed 90% of latent mannose-6-phosphatase activity which was insensitive to Ca2+. The results indicate that Ca2+ affects the glucose-6-P translocase rather than the phosphohydrolase component. It is concluded that the glucose-6-phosphatase system is modulated by changes in Ca2+ concentrations in the range of those occurring in the liver cell upon hormonal stimulation.     

Role of Glucose-6-phosphatase in Hepatic and Renal Gluconeogenesis

Comparison of the effects of a high fat and high protein diet on the capacity for glucose formation from pyruvate and glycerol was investigated in vivo and in vitro. Ratios of radioactivity incorporated from either pyruvate-3-¹⁴C or glycerol-l-¹⁴C into blood glucose to those into expired CO₂ were higher in both groups fed the high fat and the high protein diet than those in a group fed a high carbohydrate diet. Gluconeogenesis from pyruvate and glycerol by liver slices were both increased significantly in rats fed the high fat diet, while feeding the high protein diet caused increase of renal gluconeogenesis from pyruvate and glycerol. The activities of hepatic and renal glucose-6-phosphatase(s) were changed in a similar fashion to changes in hepatic and renal gluconeogenesis, respectively.

In addition, the response of the activity of hepatic glucose-6-phosphatase with high dietary fat was more rapid than that of the activity of renal glucose-6-phosphatase with high dietary protein. Furthermore, the intraperitoneal injection of actinomycin-D to rats resulted in decrease of the activities of renal glucose-6-phosphatase of both groups fed the high fat and the high protein diet, but no significant change of the activity of hepatic glucose-6-phosphatase was observed among dietary groups.

These findings suggested that the increases in the overall flow of metabolites towards glucose formation by feeding the high fat and the high protein diet might be based on the action of different mechanisms which regulate the activities of glucose-6-phosphatase(s) of the liver and kidney.     

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.     

Noradrenaline and glycogen content and the activity of several enzymes of carbohydrate metabolism in normal, embryonic, and partly denervated livers and in hepatomas of the rat]

The noradrenaline and glycogen contents as well as hexokinase, glucokinase and glucose-6-phosphatase activities were determined in normal, embryonic and partially denervated (bilateral dissection of the Nervus splanchnicus or Nervus vagus) rat liver and in two transplantable hepatomas. In embryonic liver and hepatomas a strong decrease or complete loss of noradrenaline and glycogen levels and glucokinase and glucose-6-phosphatase activities is demonstrable as compared to the livers of adult animals, while the hexokinase activity is enhanced. Following bilateral splanchnicotomy the glycogen content and hexokinase activity are enhanced; the glucose-6-phosphatase activity is reduced, and the liver does not contain any noradrenaline. Bilateral vagotomy causes decrease of the glycogen content, of the hexokinase and glucokinase activities and an enhancement of glucose-6-phosphatase activity. The results lend support to the idea of antagonistic action of the sympathetic and parasympathetic nervous systems upon several partial reactions of carbohydrate metabolism of liver. In addition, it can be assumed that the alterations of the carbohydrate metabolism demonstrable in hepatomas as compared to normal liver are not solely attributable to disturbance or breakdown of the nervous regulation.     

Enzymatic characterization of the pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP)

Glucose is the main physiological stimulus for insulin biosynthesis and secretion by pancreatic beta-cells. Glucose-6-phosphatase (G-6-Pase) catalyzes the dephosphorylation of glucose-6-phosphate to glucose, an opposite process to glucose utilization. G-6-Pase activity in pancreatic islets could therefore be an important factor in the control of glucose metabolism and, consequently, of glucose-dependent insulin secretion. While G-6-Pase activity has been shown to be present in pancreatic islets, the gene responsible for this activity has not been conclusively identified. A homolog of liver glucose-6-phosphatase (LG-6-Pase) specifically expressed in islets was described earlier; however, the authors could not demonstrate enzymatic activity for this protein. Here we present evidence that the previously identified islet-specific glucose-6-phosphatase-related protein (IGRP) is indeed the major islet glucose-6-phosphatase. IGRP overexpressed in insect cells possesses enzymatic activity comparable to the previously described G-6-Pase activity in islets. The K(m) and V(max) values determined using glucose-6-phosphate as the substrate were 0.45 mm and 32 nmol/mg/min by malachite green assay, and 0.29 mm and 77 nmol/mg/min by glucose oxidase/peroxidase coupling assay, respectively. High-throughput screening of a small molecule library led to the identification of an active compound that specifically inhibits IGRP enzymatic activity. Interestingly, this inhibitor did not affect LG-6-Pase activity, while conversely LG-6-Pase inhibitors did not affect IGRP activity. These data demonstrate that IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors to this molecule can be obtained. IGRP inhibitors may be an attractive new approach for the treatment of insulin secretion defects in type 2 diabetes.     

Topographical localization and characterization of microsomal glucose-6-phosphatase binding sites accessible to 4,4'-diazidostilbene 2,2'-disulfonic acid

The effect of the photoactivated reagent 4,4'-diazidostilbene 2,2'-disulfonic acid (DASS) on rat liver microsomal glucose-6-phosphatase has been investigated in order to analyze the accessibility and the chemical nature of functional sites of the integral enzyme protein. The following results were obtained. (i) When native rat liver microsomes are irradiated with the photoactive reagent, the activity of glucose-6-phosphatase is progressively inhibited. However, complete reactivation is obtained by modification of the DASS-labeled microsomes with Triton X-114. (ii) Inhibition of glucose-6-phosphatase is also reversed when the DASS-labeled microsomes are treated with p-mercuribenzoate or dithiothreitol. (iii) When native microsomes are labeled with DASS an intensely fluorescent adduct is formed whose emission and excitation maximum corresponds with those obtained when cysteine or 3-mercaptopropionic acid are irradiated in the presence of the photolabile reagent. (iv) The data from fluorescence measurements show that p-mercuribenzoate and dithiothreitol reduce fluorescence labeling of the microsomes whereas Triton modification of the DASS-labeled membranes does not affect the DASS-induced fluorescence. (v) Glucose 6-phosphate hydrolysis of the partially purified glucose-6-phosphatase is also inhibited as observed with native microsomes. The DASS-induced inhibition is reversed and prevented by p-mercuribenzoate; however, the partially purified enzyme cannot be reactivated by Triton X-114. (vi) When glucose-6-phosphatase is partially purified from the DASS-labeled microsomes this enzyme preparation is fluorescence labeled and inhibited. From these results we conclude that DASS directly reacts with the integral phosphohydrolase mainly by chemical modification of essential sulfhydryl groups of the enzyme protein accessible from the cytoplasmic surface of the native microsomal membrane. The Triton-induced reactivation of the glucose-6-phosphatase of DASS-labeled microsomes is explained in terms of conformational changes of the integral protein elicited during modification of the surrounding membrane by detergent.     

Glucose-6-phosphatase in the insulin secreting cell line INS-1

The glucose-6-phosphatase system of the glucose sensitive insulin secreting rat insulinoma cells (INS-1) was investigated. INS-1 cells contain easily detectable levels of glucose-6-phosphatase enzyme protein (assessed by Western blotting) and have a very significant enzymatic activity. The features of the enzyme (Km and Vmax values, sensitivity to acidic pH, partial latency, and double immunoreactive band) are similar to those of the hepatic form. On the other hand, hardly detectable levels of glucose-6-phosphatase activity and protein were present in the parent glucose insensitive RINm5F cell line. The mRNA of the glucose-6-phosphate transporter was also more abundant in the INS-1 cells. The results support the view that the glucose-6-phosphatase system of the beta-cell is associated with the regulation of insulin secretion.     

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.     

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.     

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.     

The purification of a detergent-soluble glucose-6-phosphatase from rat liver.

A highly active and soluble glucose-6-phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5'-phosphate and NaBH4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700-fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose-6-phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose-6-phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5'-phosphate from the cytoplasmic side.     

Cytoplasmic changes in level and distribution of glucose-6-phosphatase activities from rat liver during diethylnitrosamine-induced carcinogenesis.

Glucose-6-phosphatase (EC 3.1.3.9) activities were determined in isolated microsomes, cytoplasmic smooth and rough membranes, ribosomes and free cytosol from rat liver undergoing carcinogenesis by diethylnitrosamine (DENA) and compared with cytoplasmic fractions isolated in parallel from healthy animals from the same age.With continuous administration of a low dose of DENA (2.6 mg/kg rat per day for 20 weeks in the drinking water) livers of carcinogen treated rats became heavier than the control livers but the body weight decreased. About 70% of total glucose-6-phosphatase activity could be detected in the microsomal fraction. While there was no significant difference in this activity in both animal groups up to the 4th week, glucose-6-phosphatase of cancerous liver showed a distinct decrease of activity compared with normal liver.During cancer induction this enzyme became more soluble, confirmed by the observation that it was detached from firmer structures of cytoplasm as rough membranes and polysomes and translocated to smooth membranes and the soluble cytoplasmic fraction successively. The corresponding increase in glucose-6-phosphatase activity in the 105 000 g supernatant appears to be due to the loss of enzyme activity in a distinct cytoplasmic membrane fraction. These data strongly suggest that in parallel with alteration of cytoplasmic membrane structures during carcinogen feeding glucose-6-phosphatase is detached from heavier components of the cytoplasm while total activity decreased. Possible mechanisms of these findings are discussed.     

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum.

The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5'-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphatase (r larger than or equal to 0.98) and negatively with the plasma membrane marker 5'-nucleotidase (r ranging between -0.88 and -0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.     

On the mechanism of a radiation-induced change in enzymic differentiation during development

In experiments on glucose-6-phosphatase and tyrosine aminotransferase it was shown that radiation induces changes in enzymic differentiation in perinatal rat liver. A study was made of the probable reasons for the observed changes. It was shown that the macromolecular system of the protein enzyme synthesis was not damaged by the radiation doses used. The observed decrease in glucose-6-phosphatase activity during late embryogenesis, after pre-irradiation at early organogenesis, is eliminated by administration of exogenous thyroxine. A radiation-induced rise in the tyrosine aminotransferase activity during the perinatal period correlated with the cyclic AMP system status. It is proposed that modification of enzymic differentiation after irradiation results from the change in the amount of inductors.