Identification of phosphatidate phosphohydrolase purified from rat liver membranes on SDS-polyacrylamide gel electrophoresis.

Phosphatidate phosphohydrolase (PAP; EC 3.1.3.4) insensitive to N-ethylmaleimide was partially purified from rat liver membranes by a combination of chromatographic methods, immunoabsorption and glycerol gradient centrifugation. The specific activity was increased more than 600-fold over that of the membrane extract. Enzyme antibodies precipitating more than 80% of PAP were obtained and used for the identification of PAP protein on SDS-polyacrylamide gels employing the immunodetection method of Muilerman et al. [(1982) Anal. Biochem. 120, 46-51]. By this approach PAP was localized as a 31 kDa polypeptide.

Effects of hypercholesterolemia on the nucleotide content in human blood cells.

In hypercholesterolemia significant changes in the nucleotide pattern of erythrocytes and lymphocytes as determined by high performance liquid chromatography were found. The decrease in ATP of lymphocytes in hypercholesterolemia from 10.4 +/- 0.3 to 7.0 +/- 0.4 nmol mg-1 protein (n = 8) was associated with an increase in ADP from 2.2 +/- 0.2 to 4.0 +/- 0.2 nmol mg-1 protein (P less than 0.005). The pattern of guanosine phosphates likewise was found to be changed in hypercholesterolemia. Akin to lymphocytes, red blood cells displayed marked changes in nucleotide levels. No such changes were observed in platelets. Cultured lymphocytes incubated with human plasma low density lipoproteins (LDL) (140 mg cholesterol dl-1) displayed a reversible fall in ATP and an increase in ADP by about 40% and 160%, respectively, with high density lipoproteins (HDL) or very low density lipoproteins (VLDL) being essentially ineffectual. It is concluded that in hypercholesterolemia a significant change in the nucleotide pattern of blood cells is exerted by the increase in LDL. Possible pathophysiological implications are discussed.

Control of pyruvate carboxylase activity by the pyridine-nucleotide redox state in mitochondria from rat liver.

Pyruvate carboxylation by isolated mitochondria from rat liver is inhibited by t-butylhydroperoxide in a fully reversible manner. The rate of malate formation at 10 mM pyruvate was decreased by some 80% by 30 microM t-butylhydroperoxide. The effective peroxide concentration was dependent on the mitochondrial hydrogen supply, being increased to about 120 microM in the presence of 50 microM palmitoylcarnitine. Regarding the mechanism(s) of the t-butylhydroperoxide action, pyruvate transport and intramitochondrial energy or activator supply are unlikely involved, because the effect also took place with alanine as the substrate and was not accompanied by a change in the intramitochondrial levels of adenine nucleotides and acetyl-CoA respectively. However, t-butylhydroperoxide caused a rapid fall in the 3-hydroxybutyrate/acetoacetate ratio and a marked increase in the oxidized glutathione content. Therefore, experiments were designed to disclose the participation of the respective redox couples in the expression of pyruvate carboxylase activity. From measurements of NADPH, NADH, oxidized and reduced glutathione contents of mitochondria incubated under a variety of conditions, evidence has been obtained indicating that the mitochondrial NADH supply represents an important factor in the regulation of pyruvate carboxylase activity. The results presented seemingly provide a new basis for the understanding of the functional relationship between beta-oxidation and pyruvate carboxylation.

Stimulation by 3-hydroxybutyrate of pyruvate carboxylation in mitochondria from rat liver.

Isolated rat liver mitochondria incubated in the presence of 3-hydroxybutyrate display a markedly increased rate of pyruvate carboxylation as measured by malate and citrate production from pyruvate. The stimulation was demonstrable both with exogenously added pyruvate, even at saturating concentration, and with pyruvate intramitochondrially generated from alanine. The concentration of DL-3-hydroxybutyrate required for half-maximal stimulation amounted to about 1.5 mM. The intramitochondrial ATP/ADP ratio as well as the matrix acetyl-CoA level was found to remain unchanged by 3-hydroxybutyrate exposure, which, however, lowered the absolute intramitochondrial contents of the respective adenine nucleotides. The effects of 3-hydroxybutyrate were diminished by the concomitant addition of acetoacetate. Moreover, a direct relationship between mitochondrial reduction by proline and the rate of pyruvate carboxylation was observed. The results seem to indicate that the mitochondrial oxidation--reduction state might be involved in the expression of the 3-hydroxybutyrate effect. As to the physiological relevance of the findings, 3-hydroxybutyrate could be shown to activate pyruvate carboxylation in isolated hepatocytes.

Nonenzymatic glycation of immunoglobulins leads to an impairment of immunoreactivity.

Incubation of purified human and rabbit immunoglobulin G with glucose leads to covalent incorporation of the sugar into the protein, depending on glucose concentration, incubation time and pH. Furthermore, the level of glycated immunoglobulin G from normal and diabetic subjects has been determined using the thiobarbituric acid reaction. The median for glycated immunoglobulin G, expressed as mmol 5-hydroxymethylfurfural per mol IgG, obtained from 20 normal and 29 diabetic subjects was 62 and 107, respectively. Glucose incubation of immunoglobulin G purified from rabbit anti-human-transferrin serum, from human anti-varicella/zoster virus serum and from human anti-lues-spirochete serum, respectively, leads to a marked decrease in biological activity, as determined in a micro complement fixation test. Inactivation of specific antibody was dependent on incubation time and glucose concentration employed. Loss in complement-fixing activity was observed at glycation levels well comparable to those found in diabetics.

Decrease by glucagon in peroxide generation by isolated hepatocytes.

Exposure of isolated rat liver cells to glucagon or dibutyryl cyclic AMP leads to a prompt decrease in the rate of cellular peroxide generation as evidenced by (i) a reduced rate of [14C]formate oxidation and (ii) a lowered steady-state concentration of catalase Compound I.

Possible role of Pi supply in mitochondrial actions of glucagon.

Glucagon is able to diminish the net release of inorganic phosphate (Pi) occurring on incubation of isolated hepatocytes from 48-h-starved rats. Concomitantly the hormone increases the cellular Pi content. This is associated with a rise of Pi in the cytosolic fraction. Other hormonal effectors like phenylephrine, vasopressin and angiotensin II exert a smaller and transient effect as compared to glucagon. It is proposed that this increase in Pi availability to the mitochondria, by favouring substrate level phosphorylation at the succinyl-CoA synthetase step plays a role in the development of the metabolite pattern found in the mitochondrial matrix space after exposure of hepatocytes to glucagon or the above agents. With regard to the glutamate level this view is evidenced by the finding that its hormone-dependent decrease was inversely correlated to the respective increase in the cytosolic Pi concentration. Further evidence is provided by experiments with isolated mitochondria incubated under state-3 conditions at medium Pi concentrations corresponding to those metabolically active in the cytosolic compartment of control and glucagon-stimulated hepatocytes, being 2 mM and 3 mM, respectively. Increasing medium phosphate concentration from 2 mM to 3 mM caused a marked decrease in the level of succinyl-CoA and increased the rates of 2-oxoglutarate utilization and of malate and phosphoenolpyruvate production. Citrulline synthesis also was found to be stimulated at 3 mM Pi. Taken together our results suggest a role of Pi supply in mitochondrial actions of glucagon in intact hepatocytes. Moreover, they could contribute to a better interpretation of glucagon effects on isolated mitochondria from hormone-pretreated liver cells.

Concentration of free oxaloacetate in the mitochondrial compartment of isolated liver cells.

The concentration of metabolically active (i.e. 'free') oxaloacetate in the mitochondrial compartment of isolated liver cells was investigated by two independent approaches. On the basis of mitochondrial aspartate aminotransferase maintaining equilibrium and the direct measurements of mitochondrial aspartate, 2-oxoglutarate and glutamate, the concentration of free oxaloacetate was calculated to be 5 microM after incubation of hepatocytes in the presence of 1.5 mM-lactate and 0.05 mM-oleate. Gradually increasing oleate up to 0.5 mM decreased the free oxaloacetate to 2 microM. Very similar results were obtained when free oxaloacetate concentration was derived from the CO2 production of hepatocytes as a measure of citrate flux through the tricarboxylic acid cycle, and the kinetic data on citrate synthase in situ. The decrease in free oxaloacetate on increasing oleate concentration was associated with lowered rates of cycle-dependent CO2 output and O2 uptake, indicating a decrease in the disposal of acetyl-CoA into the tricarboxylic acid cycle. This decrease could explain 25-30% of the increase in ketone-body production occurring at elevated fatty acid supply. This work documents on a quantitative basis the role of free oxaloacetate in the regulation of ketogenesis.

Different actions of mono- and disaccharides on rat liver mitochondria.

Mitochondria, isolated with 0.3M disaccharide (sucrose, maltose, trehalose) solutions, showed significantly lower specific activities both in uncoupler-stimulated adenosinetriphosphatase and succinate dehydrogenase activities than organelles prepared in parallel from the same livers with isosomolar media based on mannitol, glucose or sorbitol. Furthermore, the glutamate content and the inulin impermeable space appeared markedly reduced by 0.3M disaccharides. These effects of the disaccharides were dependent on the concentration of the solute, and were not discernible at a concentration of 0.2M. On the basis of these results, one might suggest the avoidance of further use of sucrose in the preparation of liver mitochondria.

Influence of isolation media on the preservation of mitochondrial functions.

1) In the present study the influence of sucrose and mannitol-based isolation media on the degree of functional preservation of rat liver mitochondria has been investigated. Apparently intact mitochondria conventionally prepared with a 0.3M sucrose medium displayed significantly lower rates of state-3 respiration, pyruvate carboxylation, ATP hydrolysis and thiol group production than mitochondria prepared from the same livers with mannitol. 2) Extracts from the latter, furthermore, showed a significantly higher activity of succinate dehydrogenase activity, whereas no difference in glutamate dehydrogenase activity was demonstrable. 3) The low activities apparent with the sucrose medium could be brought to the level of the mannitol medium by the addition of potassium phosphate (4mM). A similar effect was exerted by K2SO4, whereas KCl and the respective sodium salts were significantly less effective. 4) Sucrose-prepared mitochondria display decreased contents of metabolites such as ATP, glutamate, citrate and malate. 5) Comparative studies with a variety of carbohydrates indicated that isolation media based on disaccharides are inferior to those based on monosaccharides in the preparation of functionally intact mitochondria from rat liver. 6) The results reported herein appear to be of general interest as sucrose-prepared mitochondria have been employed in the past in a great number of studies and are still widely used at present.

Inactivation of bovine kidney beta-N-acetyl-D-glucosaminidase by nonenzymatic glucosylation.

Evidence is presented that the incubation of beta-N-acetyl-D-glucosaminidase from bovine kidney with glucose leads to the covalent incorporation of the sugar into the enzyme protein. Concomitantly, the enzyme activity becomes markedly reduced depending on the time of incubation and the concentration of glucose employed. The separate investigation of the isoenzymes A and B, as obtained after DEAE-cellulose chromatography of the preparation, revealed that isoenzyme A had lost some 80% of its initial activity after 15 days at 37 degrees C at 44.4mM glucose, whereas isoenzyme B activity remained unchanged. The inactivation of A was associated (1) with a marked decrease in protein stain intensity after polyacrylamide gel and Cellogel electrophoresis and (2) with the formation of a small amount of apparent isoenzyme B, as indicated by the following observations: (a) appearance of enzyme activity at the position of isoenzyme B after DEAE-cellulose chromatography, (b) appearance of protein with enzyme activity in the region of authentic isoenzyme B after Cellogel electrophoresis, (c) increase in molecular mass from 130 kDa to 205 kDa, (d) similarity in heat-stability and Km for p-nitrophenyl N-acetyl-beta-glucosaminide between glucose-treated isoenzyme A and authentic isoenzyme B. As in diabetes mellitus the activity of beta-N-acetyl-D-glucosaminidase has been reported to be decreased in kidney and other tissues, the possibility that nonenzymatic glucosylation of the enzyme might occur in vivo is considered.

Essential role of coenzyme A in pyruvate dehydrogenase kinase activity.

The rate of phosphorylation and concomitant inactivation of purified pig heart muscle pyruvate dehydrogenase complex by intrinsic kinase (EC 2.7.1.99) is markedly accelerated by the addition of coenzyme A to the incubation medium, showing a half-maximum effect at 1.8 microM. The pantetheine moiety is the effective part of the coenzyme A molecule. The free thiol group is prerequisite for the stimulatory action, acetyl-CoA, benzoyl-CoA or CoAS-SCoA being ineffectual. The thiol's specificity is evidenced by showing that dithiothreitol, 2-mercaptoethanol or glutathione up to 5 mM failed to replace coenzyme A. The possibility is considered that coenzyme A might act as a physiological modifier of pyruvate dehydrogenase kinase activity.

Evidence that glucagon stabilizes rather than activates mitochondrial functions in rat liver.

The present study is concerned with the question as to whether the acute treatment of intact rats or hepatocytes with glucagon and dibutyryl cAMP, respectively, leads to a stabilization or an activation of mitochondrial functions, such as state-3 respiration, succinate dehydrogenase activity and pyruvate carboxylase activity. For this purpose, the influence of various parameters of mitochondria preparation (isolation medium, washing steps, storage) as well as of phospholipase A inhibitors (cinchocain, chloroquine) on the expression of the hormone effect was examined. With regard to the above mentioned functions, the values displayed by control mitochondria were found to be considerably higher if mannitol instead of sucrose had been used for isolation. Accordingly, only small effects of hormone treatment became apparent. The addition of cinchocain or chloroquine to the sucrose medium yielded results similar to those obtained with mannitol. Furthermore, the hormone effect on state-3 respiration and succinate dehydrogenase activity was only small if the mitochondria had been prepared faster than usual and had been used without washing. Regarding pyruvate carboxylase, a considerably smaller glucagon effect was observed when it was assayed at 25 degrees C and not (as usual) at 37 degrees C. Our results indicate that glucagon application stabilizes rather than activates mitochondrial functions.

Distinctive roles of oleate and glucagen in gluconeogenesis.

1. The effect of oleate on the subcellular distribution of ATP, 2-oxoglutarate, glutamate, citrate, malate and phosphoenolpyruvate was studied in hepatocytes from rats starved for 48 h by applying a modified digitonin method. The results markedly differ from those observed after glucagon [Siess, E. A., Brocks, D. G., Lattke, H. K., and Wieland, O. H. (1977) Biochem J. 166, 225-235]. Total cellular amounts and the distribution of ATP and 2-oxoglutarate remained unchanged. In the mitochondrial matrix glutamate was increased, while mitochondrial phospho-enolpyruvate was decreased. Citrate and malate were increased both in the mitochondrial and cytosolic space. 2. In contrast to the effect of glucagon, gluconeogenesis from dihydroxyacetone, fructose or glutamine was not stimulated by oleate. Gluconeogenesis from propionate was even inhibited by the fatty acid. 3. The stimulation by glucagon of glucose production from dihydroxyacetone or fructose was undiminished in biotin-deficient hepatocytes. Glucose formation from lactate, however, was stimulated only in biotin-substituted hepatocytes. 4. The results indicate that oleate stimulates gluconeogenesis by increasing pyruvate carboxylase activity (EC 6.4.1.1), whereas glucagon displays a more complex mode of action.

Early kinetics of glucagon action in isolated hepatocytes at the mitochondrial level.

The temporal relationship between the effect of glucagon on respiratory functions and the changes in metabolites related to gluconeogenesis has been studied. Mitochondria prepared from hepatocytes after incubation with glucagon for 1 min already displayed a maximal stimulation of state-3 respiration. The increase in succinate dehydrogenase activity was almost fully expressed 3 min after glucagon. With respect to the utilization of pyruvate, 2-oxoglutarate and glutamate, glucagon produced a significant effect within 1 min. The rate of this decrease was linear for about 3 min slowing down thereafter. The stimulation of glucose production from lactate became significant within 1 min and remained constant up to 15 min. The influence of glucagon on the mitochondrial redox state also was an early event. It was maximally shifted to the more reduced state within 2 min and declined within 15 min. Under the conditions employed no effect of glucagon on urea synthesis or branched-chain amino acid release up to 15 min incubation time was discernible. Glucagon influenced the respiratory parameters virtually independent of Ca2+, in contrast to its action on intermediary metabolism. As to the hormone specificity, no enhancement of state-3 respiration and succinate dehydrogenase activity was caused by phenylephrine or isoproterenol. From the time course studies presented, it appears that the mitochondrial effects of glucagon might be causally interrelated with the regulation of gluconeogenesis. Moreover, our results indicate that the stimulation of state-3 respiration represents the earliest, specific action of glucagon at the mitochondrial level.