Effect of alloxan-diabetes and subsequent treatment with insulin on kinetic properties of succinate oxidase activity from rat liver mitochondria

We evaluated early and late effects of alloxan-diabetes and subsequent insulin treatment on the kinetic properties of succinate oxidase (SO) in rat liver mitochondria. Diabetic state lowered the SO activity; insulin treatment was effective in restoring the activity only in one-week diabetic rats. The energies of activation in low and high temperature ranges (EH and EL) decreased significantly in diabetic animals; once again insulin treatment was partially effective only in the one-week diabetic group. The total phospholipids (TPL) and cholesterol (CHL) contents did not change in one-week groups. In one-month diabetic animals TPL decreased while CHL increased; insulin treatment induced further changes without restoring normality. The lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI) and phosphatidylserine (PS) content increased in the diabetic state while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased. Insulin treatment had a partial restorative effect. The changes in EH correlated negatively with SPM. The phase transition temperature, Tt, decreased in diabetic and insulin-treated groups. These changes correlated positively with the ratios of TPL/PI and TPL/PS. The membrane fluidity decreased in the diabetic state; insulin had a restorative effect only in the one-week group.     

Diabetic Modulation of the Temperature Kinetics Properties of Cytochrome Oxidase Activity in Rat Brain Mitochondria

The effects of alloxan-diabetes and subsequent treatment with insulin on temperature kinetics properties of cytochrome oxidase activity from rat brain mitochondria were examined. The enzyme activity decreased only at the late stage of diabetes which was not normalized by insulin treatment; however at early stage of diabetes hyper-stimulation occurred. In the control animals the Arrhenius plot was chair shaped with three energies of (E₁, E₂ and E₃) and two phase transition temperatures (T_t1 and T_t2). At early diabetic stage the Arrhenius plot became biphasic and E₁ and E₂ decreased_; insulin treatment reversed chair-shaped pattern with increase in E₂. These changes correlated with transient changes in the phospholipids profiles especially decreased acidic phospholipids. The temperature kinetics parameters were minimally affected at the late stage of diabetes or by insulin treatment. Thus at the late stage the brain tissue seems to have readjusted to its insulin homeostasis.     

Effect of Alloxan Diabetes and Subsequent Insulin Treatment on Temperature Kinetics Properties of Succinate Oxidase Activity in Rat Kidney Mitochondria

Early and late effects of alloxan diabetes and subsequent treatment with insulin on the temperature kinetics properties of succinate oxidase (SO) activity in rat kidney mitochondria were examined. In diabetic animals SO activity increased significantly and the increase was more pronounced at the late stage. Insulin treatment partially restored SO activity. However, the effect was temperature-dependent. In diabetic animals the energy of activation in the low temperature range (E_L) increased significantly while that in the high temperature range (E_H) decreased. The latter seems to be responsible for improving catalytic efficiency in the diabetic state. Insulin treatment normalized E_H only in the 1-month diabetic group. The phase transition temperature (Tt), decreased in diabetic animals. Insulin treatment caused an increase beyond the control value in Tt in 1-month diabetic animals. The results suggest that insulin status-dependent modulation of SO activity is a complex process.     

Effect of Insulin on ACE2 Activity and Kidney Function in the Non-Obese Diabetic Mouse

We studied the non-obese diabetic (NOD) mice model because it develops autoimmune diabetes that resembles human type 1 diabetes. In diabetic mice, urinary albumin excretion (UAE) was ten-fold increased at an “early stage” of diabetes, and twenty-fold increased at a “later stage” (21 and 40 days, respectively after diabetes diagnosis) as compared to non-obese resistant controls. In NOD Diabetic mice, glomerular enlargement, increased glomerular filtration rate (GFR) and increased blood pressure were observed in the early stage. In the late stage, NOD Diabetic mice developed mesangial expansion and reduced podocyte number. Circulating and urine ACE2 activity were markedly increased both, early and late in Diabetic mice. Insulin administration prevented albuminuria, markedly reduced GFR, blood pressure, and glomerular enlargement in the early stage; and prevented mesangial expansion and the reduced podocyte number in the late stage of diabetes. The increase in serum and urine ACE2 activity was normalized by insulin administration at the early and late stages of diabetes in Diabetic mice. We conclude that the Diabetic mice develops features of early kidney disease, including albuminuria and a marked increase in GFR. ACE2 activity is increased starting at an early stage in both serum and urine. Moreover, these alterations can be completely prevented by the chronic administration of insulin.     

Tissue cholinesterases. A comparative study of their kinetic properties

The substrate saturation and temperature-dependent kinetic properties of soluble and membrane-bound forms of acetylcholinestarase (AChE) from brain and butyrylcholinesterase (BChE) from heart and liver were examined. In simultaneous studies these parameters were also measured for AChE in erythrocyte membranes and for BChE in the serum from rat and humans. For both soluble and membrane-bound forms of the enzyme from the three tissues, two components were discernible. In the brain, Km of component I (high affinity) and component II (low affinity) was somewhat higher in membrane-bound form than that of the soluble form components, while the Vmax values were significantly higher by about five fold. In the heart, Km of component II was lower in membrane-bound form than in the soluble form, while Vmax for both the components was about four to six fold higher in the membrane-bound form. In the liver, Vmax was marginally higher for the two components of the membrane-bound enzyme; the Km only of component I was higher by a factor of 2. In the rat erythrocyte membranes three components of AChE were present showing increasing values of Km and Vmax. In contrast, in the human erythrocyte membranes only two components could be detected; the one corresponding to component II of rat erythrocyte membranes was absent. In the rat serum two components of BChE were present while the human serum was found to possess three components. Component I of the human serum was missing in the rat serum. Temperature kinetics studies revealed that the Arrhenius plots were biphasic for most of the systems except for human serum. Membrane binding of the enzyme resulted in decreased energy of activation with shift in phase transition temperature (Tt) to near physiological temperature.     

Comparison of biochemical properties of liver arginase from streptozocin-induced diabetic and control mice

Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent Km and Vmax values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent Km values for Mn2+ of the basic form of the enzyme were 25 and 33 microM for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent Km values were measured, the values were 8 and 197 microM for arginase from controls and 35 and 537 microM from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.     

Phosphoenolpyruvate carboxykinase activity patterns in the liver acinus of diabetic and diabetic and estrogen treated rats

Summary The acinar activity pattern of phosphoenolpyruvate carboxykinase (PEPCK) was investigated in livers of streptozotocin diabetic male and female rats and in addition in livers of diabetic males, which had undergone estrogen treatment. In all diabetic animals blood glucose levels were supranormal and liver PEPCK activity was increased. This increase in activity was greatest in estrogen treated diabetic males and lowest in diabetic females. Plasma insulin levels were reduced after the application of streptozotocin to otherwise normal male and female rats. Yet, in males treated in addition with estrogens the plasma insulin levels reached the normal range again. The PEPCK activity showed a heterotopic distribution along the acinus. The periportal to perivenous gradient was steeper in males compared to females in the untreated as well as in the diabetic state. The application of estrogens to males resulted in a further steepening of the gradient.     

The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.

The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.     

Phosphoenolpyruvate carboxykinase activity patterns in the liver acinus of diabetic and diabetic and estrogen treated rats

The acinar activity pattern of phosphoenolpyruvate carboxykinase (PEPCK) was investigated in livers of streptozotocin diabetic male and female rats and in addition in livers of diabetic males, which had undergone estrogen treatment. In all diabetic animals blood glucose levels were supranormal and liver PEPCK activity was increased. This increase in activity was greatest in estrogen treated diabetic males and lowest in diabetic females. Plasma insulin levels were reduced after the application of streptozotocin to otherwise normal male and female rats. Yet, in males treated in addition with estrogens the plasma insulin levels reached the normal range again. The PEPCK activity showed a heterotopic distribution along the acinus. The periportal to perivenous gradient was steeper in males compared to females in the untreated as well as in the diabetic state. The application of estrogens to males resulted in a further steepening of the gradient.     

Changes in liver gangliosides in streptozotocin-induced diabetic rats

Diabetes mellitus is associated with various structural and functional liver abnormalities that affect the glycogen and lipid metabolisms. The effects of streptozotocin-induced diabetes and of insulin supplementation to Sprague-Dawley diabetic rats on ganglioside patterns in liver were determined. Diabetic livers showed a tendency to hepatomegaly 3 weeks after STZ-induction of diabetes. The concentration of total gangliosides in diabetic and non-diabetic livers was similar, but the concentration of total gangliosides in the liver of insulin-stabilized rats was slightly increased. Bidimensional TLC chromatographic analysis of gangliosides isolated from normal diabetic and insulin-stabilized diabetic livers showed quantitative and qualitative changes. In comparison with normal controls, the densitometric analyses of diabetic liver ganglioside patterns had increased amounts of GM3, GM1, GD1b, and GT1b gangliosides, while GM2 could not be detected. The hepatic ganglioside pattern of insulin-stabilized diabetic rats was partially restored, resembling the profile of normal rats. The activity of GalNAcT, GalT-2 and SialT-4 transferases was measured in liver microsomal fractions of the different groups of animals. Diabetic rats showed an increased activity of GalNAcT and a decrease in the activity of GalT-2 and SialT-4 compared with the controls. The enzymatic activities found in insulin-treated rats showed a tendency to return to the values observed in normal control animals. The results evidenced that streptozotocin-induced diabetes affects the liver ganglioside pattern and the ganglioside synthesis enzyme activity. The alterations found in ganglioside metabolism could represent one of the earliest changes associated with the diabetic pathology.     

Effect of starvation, diabetes and insulin on the casein kinase 2 from rat liver cytosol.

Starvation, diabetes and insulin did not alter the concentration of casein kinases in rat liver cytosol. However, the Km for casein of casein kinase 2 from diabetic rats was about 2-fold lower than that from control animals. Administration of insulin to control rats did not alter this parameter, but increased the Km for casein of casein kinase 2 in diabetic rats. Starvation did not affect the kinetic constants of casein kinases. The effect of diabetes on casein kinase 2 persisted after partial purification of the enzyme by glycerol-density-gradient centrifugation and affected also its activity on other protein substrates such as phosvitin, high-mobility-group protein 14 and glycogen synthase. The results indicate that rat liver cytosol casein kinase 2 is under physiological control.     

Nitrogenase. VII. Effect of component ratio, ATP and H2 on the distribution of electrons to alternative substrates.

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL     

Decrease in the levels of a constitutive cytochrome P-450 (RLM5) in hepatic microsomes of diabetic rats

Cytochrome P-450 dependent hydroxylation of testosterone has been measured in hepatic microsomes of control, diabetic and insulin-treated diabetic rats. The observed decrease in testosterone 16 alpha-hydroxylase activity in diabetes, an activity previously shown to be largely due to RLM5, was accompanied by a dramatic decrease in immunodetectable RLM5. Diabetic rats which received insulin had elevated testosterone 16 alpha-hydroxylase activity relative to the diabetic animals, which was accompanied by a corresponding increase in the levels of RLM5. These results provide evidence that specific constitutive cytochrome P-450 enzymes are altered in the diabetic state and that these changes are not permanent since they can be overcome, at least partially, by insulin replacement therapy.     

Compendium of the antidiabetic effects of supranutritional selenate doses. In vivo and in vitro investigations with type II diabetic db/db mice

In recent years, a number of investigations on the antidiabetic effects of supranutritional selenate doses have been carried out. Selenate (selenium oxidation state +VI) was shown to possess regulatory effects on glycolysis, gluconeogenesis and fatty acid metabolism, metabolic pathways which are disturbed in diabetic disorders. An enhanced phosphorylation of single components of the insulin signalling pathway could be shown to be one molecular mechanism responsible for the insulinomimetic properties of selenate. In type II diabetic animals, a reduction of insulin resistance could be shown as an outcome of selenate treatment. The present study with db/db mice was performed to investigate the antidiabetic mechanisms of selenate in type II diabetic animals. Twenty-one young adult female db/db mice were randomly assigned to three experimental groups (selenium deficient=0Se, selenite-treated group=SeIV and selenate-treated group=SeVI) with seven animals each. Mice of all groups were fed a selenium-deficient diet for 8 weeks. The animals of the groups SeIV and SeVI were supplemented with increasing amounts of sodium selenite or sodium selenate up to 35% of the LD50 in week 8 in addition to the diet by tube feeding. Selenate treatment reduced insulin resistance significantly and reduced the activity of liver cytosolic protein tyrosine phosphatases (PTPs) as negative regulators of insulin signalling by about 50%. In an in vitro inhibition test selenate (oxidation state +VI) per se did not inhibit PTP activity. In this test, however, selenium compounds of the oxidation state +IV were found to be the actual inhibitors of PTP activity. Selenate administration in vivo further led to characteristic changes in the selenium-dependent redox system, which could be mimicked in an in vitro assay and provided further evidence for the intermediary formation of SeIV metabolites. The expression of peroxisome proliferator-activated receptor gamma (PPARgamma), another important factor in the context of insulin resistance and lipid metabolism, was significantly increased by selenate application. In particular, liver gluconeogenesis and lipid metabolism were influenced strongly by selenate treatment. In conclusion, our results showed that supranutritional selenate doses influenced two important mechanisms involved in insulin-resistant diabetes, namely, PTPs and PPARgamma, which, in turn, can be assumed as being responsible for the changes in intermediary metabolism, e.g., gluconeogenesis and lipid metabolism. The initiation of these mechanisms thereby seems to be coupled to the intermediary formation of the selenium oxidation state +IV (selenite state) from selenate.     

Altered phospholipid secretion in type II pneumocytes isolated from streptozotocin-diabetic rats

To study the effect of diabetes on pulmonary surfactant secretion, type II pneumocytes from adult streptozotocin-induced diabetic rats were placed in short-term culture. As opposed to a linear secretory rate by control type II cells, the secretory rate of type II cells from diabetic animals was biphasic reaching a minimum at 1.5 h. When exogenous surfactant containing radioactive phosphatidylcholine was added to the incubation media for 1.5 h, the cells from diabetic animals incorporated more exogenous phosphatidylcholine into lamellar bodies than control cells. This suggests that in the type II cell from diabetic animals, the rate of reutilization is greater than the rate of secretion until 1.5 h, at which time the rate of secretion becomes greater. The altered secretory pattern was reversed by in vivo insulin treatment 30 min prior to killing but not by the addition of insulin to the incubation media. When challenged by isoproterenol, a beta-adrenergic agonist, the secretory pattern of cells from diabetic animals was biphasic as observed with basal secretion; however, secretion was stimulated 30% as opposed to 100% increase in control cells. These data suggest that basal and stimulated secretion are altered in the cultured type II cell from diabetic animals and restored by in vivo but not in vitro insulin treatment.     

Effect of metformin on insulin receptor binding and glycaemic control in type II diabetes.

To investigate the effect of metformin on insulin receptor binding and diabetic control, eight obese type II diabetic patients were studied before treatment, after one and four weeks of taking metformin (500 mg thrice daily), and four weeks after withdrawal of the drug. After one and four weeks of treatment the number of erythrocyte insulin receptors had increased by 116% and 184% respectively. This was due almost entirely to an increase in the number of low affinity binding sites. The number of receptors was still raised four weeks after metformin had been withdrawn. Diabetic control as assessed by urinary glucose, glycosylated haemoglobin (HbA1), and glucose tolerance values was significantly improved during metformin treatment, while plasma insulin concentrations were not altered. These results indicate that metformin produces a rapid and protracted increase in low affinity insulin receptors in type II diabetes, associated with greater insulin sensitivity and improved diabetic control.     

Alteration of the apparent Ki of carnitine palmitoyltransferase for malonyl-CoA by the diabetic state and reversal by insulin

The effects of streptozotocin-induced diabetes and the subsequent treatment of diabetic animals with insulin were studied using a dose of streptozotocin that produces highly ketotic animals 48 h after injection. Carnitine palmitoyltransferase of diabetic animals had apparent Ki values for malonyl-CoA that were approximately 10 times greater than control animals, indicating a greatly decreased affinity for malonyl-CoA in the diabetic state. Subsequent treatment of diabetic animals with insulin for 5 days produced non-ketotic animals with normal blood glucose, and the affinity of carnitine palmitoyltransferase for malonyl-CoA was increased to the control level. Treatment of other groups of ketotic diabetic animals with insulin produced substantial changes in the carnitine palmitoyltransferase apparent Ki value for malonyl-CoA within 4 h. These results suggest that insulin modulates the ketotic state, at least in part, by increasing the affinity of carnitine palmitoyltransferase for malonyl-CoA to bring about inhibition of fatty acid oxidation and ketogenesis.     

Inhibition of hepatic fatty acid oxidation at carnitine palmitoyltransferase I by the peroxisome proliferator 2-hydroxy-3-propyl-4-[6-(tetrazol-5-yl) hexyloxy]acetophenone.

1. The kinetic properties of overt carnitine palmitoyltransferase (CPT I, EC 2.3.1.21) were studied in rat liver mitochondria isolated from untreated, diabetic and insulin-treated diabetic animals. A comparison was made of the time courses required for the changes in these properties of CPT I to occur and for the development of ketosis during the induction of chronic diabetes and its reversal by insulin treatment. 2. The development of hyperketonaemia over the first 5 days of insulin withdrawal from streptozotocin-treated rats was accompanied by parallel increases in the activity of CPT I and in the I0.5 (concentration required to produce 50% inhibition) of the enzyme for malonyl-CoA. 3. The rapid reversal of the ketotic state by treatment of chronically diabetic rats with 6 units of regular insulin was not accompanied by any change in the properties of CPT I over the first 4 h. Higher doses of insulin (15 units), delivered throughout a 4 h period, resulted in an increase in the affinity of CPT I for malonyl-CoA, but the sensitivity of the enzyme to the inhibitor was still significantly lower than in mitochondria from normal animals. 4. Conversely, when insulin treatment was continued over a 24 h period, full restoration of the sensitivity of the enzyme to malonyl-CoA was achieved. However, the activity of the enzyme was only decreased marginally. 5. These results are discussed in terms of the possibility that the major regulatory sites of the rate of hepatic oxidation may vary in different phases of the induction and reversal of chronic diabetes.     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

Effect of diabetes on calcium/calmodulin dependent protein kinase-II from rat brain.

Changes in the protein levels and activity of Ca2+/Calmodulin dependent protein kinase II (CaM kinase II) level were studied in cytosolic and particulate fractions from cerebral hemisphere, cerebellum, brain stem, thalamus and hypothalamus regions of rat brain after 4 and 12 weeks of induction of diabetes. Streptozotocin induced diabetes, resulted in pronounced increase of CaM kinase II activity as determined by the kinase activity assay. The total amount of enzyme protein (alpha-subunit specific) also showed increase as revealed by western blotting. Parallel studies were also made in age matched control rats and insulin treated diabetic rats. The increase in CaM kinase II activity was more pronounced in the 12 weeks diabetic group. Insulin treatment of diabetic rats, resulted in recovery of enzyme activity near to control values from majority of the brain regions studied. The expression of alpha-subunit specific CaM kinase II correlates with the enzyme activity in the diabetic rat brain.