Intact Rat Liver Microsomes Research Articles

Gluconeogenesis inhibition and phytochemical composition of two Cecropia species

Aim of the study Cecropia obtusifolia and Cecropia peltata are plants highly used by the Mexican diabetic population to treat type 2 diabetes. Previous studies have assessed their hypoglycemic effect in animal models and in type 2 diabetic patients. Both plants contain cholorogenic acid, an inhibitor of glucose-6-phosphate translocase. In this work, we found a mechanism by which to understand how these plants could produce the observed hypoglycemic effect according to their traditional use. To test the hypothesis that targeting gluconeogenesis with an inhibitor of Gl-6-P translocase could result in a reduction of hepatic glucose production, we examined the effects of Cecropia obtusifolia and Cecropia peltata on gluconeogenesis (in vivo) and the activity of the enzyme (in vitro). Materials and methods The extracts of the two plants were analyzed by HPLC to confirm their phytochemical composition. To test the inhibition of gluconeogenesis in vivo, a pyruvate tolerance test (2 g/kg) was performed in 18-h fasted n5-STZ rats. The effect of the extracts ( Cecropia obtusifolia and Cecropia peltata 150 mg/kg) on glucose-6-phosphatase activity was assayed in vitro with intact rat liver microsomes. Results Using HPLC-DAD, we confirmed that the main components of both species are chlorogenic acid and isoorientin. Diabetic rats treated with the extracts showed a lower glucose curve. The tested extracts were able to reduce the increase in the glucose blood level, and they inhibited the glucose-6-P activity with IC 50s of 224 μg/ml for Cecropia obtusifolia aqueous, 160 μg/ml for Cecropia obtusifolia butanolic, 146 μg/ml for Cecropia peltata aqueous and 150 μg/ml for Cecropia peltata butanolic. Conclusions The results of the experiments presented here suggest that the administration of both plants can improve glycemic control by blocking the hepatic glucose output, especially in the fasting state. These data support the traditional use of the plants as “agua de uso”, a cold infusion of the plant consumed over the course of a day.

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 T1/2 values that proved insensitive to unlabeled G6P or 100 μM vanadate, and could not be activated over background levels by intravesicular phosphate in the complete absence of G6P hydrolysis. [32P]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.

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.

Preparation and use of a photoactivatable glucose-6-phosphate analogue

A benzophenone-containing derivative of glucose-6-phosphate, 6-[(3-([2,3-3H2]-p-benzoyl dihydrocinnamidoylpropyl-1-oxy)phosphoryl]-d-glucopyranose ([3H]BZDC-Glc-6-P) was synthesized and employed to photoaffinity label proteins on intact rat liver microsomes. The use of a non-photoactivatable, UV-transparent desoxy analogue of BZDC, named p-benzyldihydrocinnamoyl (BnDC), is introduced as a general method to achieve competition when hydrophilic ligands are modified with hydrophobic photophores.

Co-purification of Microsomal Epoxide Hydrolase with the Warfarin-Sensitive Vitamin K 1 Oxide Reductase of the Vitamin K Cycle

Vitamin K 1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K 1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level of epoxide hydrolase activity against two xenobiotic epoxide substrates. The K m values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25–35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K 1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K 1 oxide reduction in rat liver microsomes.

Chlorogenic acid and synthetic chlorogenic acid derivatives: novel inhibitors of hepatic glucose-6-phosphate translocase.

The enzyme system glucose-6-phosphatase (EC 3.1.3.9) plays a major role in the homeostatic regulation of blood glucose. It is responsible for the formation of endogenous glucose originating from gluconeogenesis and glycogenolysis. Recently, chlorogenic acid was identified as a specific inhibitor of the glucose-6-phosphate translocase component (Gl-6-P translocase) of this enzyme system in microsomes of rat liver. Glucose 6-phosphate hydrolysis was determined in the presence of chlorogenic acid or of new synthesized derivatives in intact rat liver microsomes in order to assess the inhibitory potency of the compounds on the translocase component. Variation in the 3-position of chlorogenic acid had only poor effects on inhibitory potency. Introduction of lipophilic side chain in the 1-position led to 100-fold more potent inhibitors. Functional assays on isolated perfused rat liver with compound 29i, a representative of the more potent derivatives, showed a dose-dependent inhibition of gluconeogenesis and glycogenolyosis, suggesting glucose-6-phosphatase as the locus of interference of the compound for inhibition of hepatic glucose production also in the isolated organ model. Gl-6-P translocase inhibitors may be useful for the reduction of inappropriately high rates of hepatic glucose output often found in non-insulin-dependent diabetes.

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.

Time-dependent inhibition of glucose 6-phosphatase by 3-mercaptopicolinic acid

3-Mercaptopicolinate (3-MP) inhibits d-glucose-6-phosphate (G6P) phosphohydrolase activity of the glucose-6-phosphatase system (Bode et al. (1993) Biochem. Cell Biol. 71, 113–121). We therefore attempted to maximize the inhibition by varying the physical state of microsomes, the concentration of 3-MP, and the time of preliminary incubation of 3-MP with the enzyme. The inhibition was irreversible and time- and inhibitor-concentration-dependent, with G6P phosphohydrolase activity of intact rat liver microsomes, but there was no inhibition with detergent-treated microsomes. The effectiveness of 3-MP as a time-dependent inhibitor of glucose 6-phosphatase was demonstrated in situ by measuring glycogenolysis in isolated, perfused livers from fed rats. We first exposed the livers to 2 mM 3-MP for 40 min, and then assessed the inhibitory effects on glycogenolysis. It was lowered by 50%. These observations establish that 3-MP at the mM level may be useful as an experimental probe in the study of the role(s) of G6P in the regulation of glycogenolysis as well as glycogenesis. Further, they validate the use of much lower (μM) concentrations of 3-MP to block gluconeogenesis (at the phospho enolpyruvate carboxykinase step) without interfering with glucose 6-phosphatase. We also explored the mechanism of 3-MP inhibition. The time-dependent inhibition of carbamoyl-phosphate:glucose phosphotransferase activity with microsomes incubated with 1 mM 3-MP for 60 or 90 min and then assayed with 1 mM carbamoyl phosphate and 180 mM glucose was modest compared with inhibition of G6P phosphohydrolase. When G6P production by carbamoyl-phosphate:glucose phosphotransferase was reduced by decreasing glucose concentration to 60 mM, no inhibition by 3-MP was discernible. There was no inhibition of inorganic pyrophosphatase activity. These studies support the model of time-dependent, irreversible reaction of 3-MP with the G6P translocase component of the glucose-6-phosphatase system.

Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases.

A new approach to determining the active site orientation of microsomal glycosyltransferases is presented which utilizes the photoaffinity analogs [32P]5-Azido-UDP-glucose ([32P]5N3UDP-Glc) and [32P]5-Azido-UDP-glucuronic acid ([32P]5N3UDP-GlcA). It was previously shown that both photoprobes could be used to photolabel UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), as well as the family of UDP-glucuronosyltransferases in rat liver microsomes. The effects of detergents, proteases, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the photolabeling of these enzymes were examined in intact rat liver microsomes. Photolabeling of Glc-P-Dol synthase by either photoprobe was the same in intact or disrupted vesicles, was susceptible to trypsin digestion, and was inhibited by the nonpenetrating inhibitor DIDS. Photolabeling of the UDP-glucuronosyltransferases by [32P]5N3UDP-GlcA was stimulated 1.3-fold in disrupted vesicles as compared to intact vesicles, whereas photolabeling of these enzymes by [32P]5N3UDP-Glc showed a 14-fold increase when vesicles were disrupted. Photolabeled UDP-glucuronosyltransferases were only susceptible to trypsin digestion in disrupted vesicles, and this was further verified by Western blot analyses. The results indicate a cytoplasmic orientation for access of UDP-sugars to Glc-P-Dol synthase and a lumenal orientation of most UDP-glucuronosyltransferases.

Metabolic intermediates as potential regulators of glucose-6-phosphatase

Twenty-five metabolites of glucose, gluconeogenic substrates, and related compounds were examined as potential inhibitors of glucose-6-phosphatase (EC 3.1.3.9) catalytic unit and substrate transport function, using disrupted and intact rat liver microsomes. Inhibitions (competitive) were noted with six. Calculated per cent inhibitions with presumed near-physiologic concetrations of inhibitor and substrate were small. However, when hepatic fructose-1-P concentration is elevated in response to a fructose load, inhibition of glucose-6-phosphatase by fructose-1-P may play a regulatory role, along with fructose-1-P-associated deinhibition of glucokinase, by directing glucose-6-P away from glucose formation and towards glycogen synthesis and glycolysis.

Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies.

Fifteen peptides, ranging in length from 6 to 31 amino acids and corresponding in sequence to portions of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P-450 PB-4), were previously synthesized chemically and used to prepare site-specific rabbit antibodies (Frey, A. B., D.J. Waxman, and G. Kreibich, 1985, J. Biol. Chem., 260:15253-15265). The antipeptide antibodies were affinity purified using Sepharose resins derivatized with the respective peptides and 14 preparations were obtained that in an ELISA assay showed affinities to immobilized P-450 judged to be adequate for binding studies on intact rat liver microsomes. The binding of these antibodies to rough microsomes from the livers of phenobarbital treated rats was assessed using 125I-labeled IgG and by immunoelectron microscopy employing protein A-gold as a marker. It was found that many of the antibodies bound to the cytoplasmic surface of the membrane but none bound to the luminal face of ruptured or inverted microsomal vesicles or to contaminating membranes of other organelles present in the preparations. These observations eliminate previously proposed models for the transmembrane disposition of P-450 that postulate the existence of multiple transmembrane domains and the exposure of several polar segments of the polypeptide on the luminal side of the membrane. The fact that an antibody raised to the first 31 residues of P-450 bound well to the purified P-450 but very poorly to rough microsomes, whereas an antibody to a peptide comprising residues 24-38 showed relatively strong binding to intact microsomes, is consistent with the proposal that the amino terminal segment of P-450 extending approximately to residue 20 is embedded in the phospholipid bilayer and the immediately following segment is exposed on the cytoplasmic surface of the membrane. All these results favor a model in which the cytochrome P-450 molecule is largely exposed on the cytoplasmic surface of the endoplasmic reticulum membrane to which it is anchored by its short amino terminal hydrophobic segment.

Superoxide generation by NADPH-cytochrome P-450 reductase: The effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by Superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe 3+ and EDTA-Fe 3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O 2 ⨪ production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O 2 ⨪ generation by xanthine oxidase. Although both EDTA-Pe 3+ and DTPA-Fe 3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe 3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe 3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O 2 ⨪ generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe 3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O 2 ⨪ generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe 3+ was demonstrated to be independent of O 2 ⨪ generation by the reductase.

Identification of a rat liver microsomal polypeptide involved in the transport of glucose 6-phosphate. Labeling with 4,4'-diisothiocyano-1,2-diphenyl[3H]ethane-2,2'-disulfonic acid.

Transport of glucose-6-P in intact rat liver microsomes is inhibited by 4,4'-diisothiocyano-1,2-diphenyl[3H]ethane-2,2'-disulfonic acid ([3H]H2DIDS). The concentration of [3H]H2DIDS that inhibits transport activity by 50% is 35 microM. Glucose-6-P protected against the inhibition of transport activity caused by [3H]H2DIDS; mannose-6-P, 2-deoxyglucose-6-P, galactose-6-P, fructose-6-P, or glycerol-2-P did not. [3H]H2DIDS-treated microsomes were solubilized in sodium dodecyl sulfate and the microsomal polypeptides were separated by polyacrylamide gel electrophoresis. Labeled polypeptides were identified by autoradiography. Treatment of microsomes with concentrations of [3H]H2DIDS (50-100 microM) that resulted in maximal inhibition of transport activity allowed the identification of two microsomal polypeptides that contained most of the incorporated radioactivity. Their molecular weights were 54,000 and 59,000. The labeling of the former, but not the latter polypeptide was saturable and correlated linearly with the level of inhibition of transport activity. Concomitantly with its protective effect on translocase activity, the presence of glucose-6-P during the reaction with [3H]H2DIDS resulted in a significant increase in the amount of label incorporated into this peptide. This stimulation of labeling was specific for glucose-6-P. These results implicate the 54,000-dalton polypeptide as an obligatory component of the rat liver microsomal glucose-6-P translocase.

Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase.

Methyl sterol oxidase of microsomal synthesis of cholesterol from lanosterol is a mixed-function oxidase that is dependent upon reduced pyridine nucleotide. The methyl sterol oxidase, as well as NADH-cytochrome c reductase, in intact rat liver microsomes are inhibited by anti-cytochrome b5 immunoglobulin, but NADPH-cytochrome c reductase is not affected. There is a decreased time lag prior to onset of reoxidation of steady state levels of reduced cytochrome b5 when 4-methyl sterol oxidase substrates are present. Trypsin treatment of microsomes destroys cytochrome b5 with loss of methyl sterol oxidase activity. Activity is restored by addition of purified cytochrome b5 to trypsin-treated microsomes. Initial attempts to solubilize and purify 4-methyl sterol oxidase have been only partially successful due to the extreme lability of the oxidase. However, DEAE-cellulose column chromatography of a detergent extract of microsomes yields a fraction that contains the oxidase, lipids, and NADH-cytochrome b5 reductase but is free of cytochrome b5. Oxidation of 4 alpha [30-3H] methyl-5 alpha-cholest-7-en-3 beta-ol by methyl sterol oxidase in this isolated fraction can be fully restored by the addition of purified liver microsomal cytochrome b5. These results strongly support the suggestion that membrane-bound cytochrome b5 of rat liver microsomes is an obligatory electron carrier from NADH to 4-methyl sterol oxidase.

Resolution and reconstitution of soluble components of rat liver microsomal vitamin D 3-25-hydroxylase

Solubilized components of the vitamin D 3-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D 3 in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b 5 or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K m(NADPH) = 3.14 μM, K m(NADH) = 31.25 μM, and K m(cyt c) = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, 3H-labeled vitamin D 3, and soluble cytosolic supernatant, full vitamin D 3-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D 3-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D 3 to 25-hydroxyvitamin D 3.

The distinct enzymic lipid peroxidation systems from liver microsomes in the presence of ADP— or EDTA—iron complexes

It is known that the ADP-Fe complex is the most effective and widely used cofactor for lipid peroxidation in rat liver microsomes [ 1,2] . Many authors have considered the iron ions as initiators of this freeradical reaction [3,4] and ADP as a component which prevents the precipitation of iron at neutral pH [4]. However, the latest data on the reconstruction of the microsomal lipid peroxidation system indicate that besides ADP-Fe the presence of the EDTA-Fe complex is also necessary [5]. In this report data are presented about the interchange ability of ADP-Fe and EDTA-Fe in NADPHdependent lipid peroxidation in intact rat liver microsomes.

The requirement for membrane integrity in the inhibition of hepatic glucose 6-phosphatase by sulfhydryl reagents and taurocholate

Exposures of intact rat liver microsomes to various sulfhydryl reagents or to concentrations of taurocholate below 1 mM inhibited glucose 6-phosphate phosphohydrolase activity. The inhibitions were abolished by subsequent disruption of membrane integrity by higher concentrations of taurocholate or 1M NH 4OH. Also the reagents were ineffective when tested directly with disrupted microsomes. Thus, a requirement for an intact membrane structure in the action of these reagents is indicated. Moreover, the involvement of a second component of the endoplasmic reticulum in the catalysis of glucose 6-phosphate hydrolysis is suggested.