Jadwiga Bryla

Melatonin attenuates diabetes-induced oxidative stress in rabbits

Abstract:  Oxidative stress is considered to be the main cause of diabetic complications. As the role of antioxidants in diabetes therapy is still underestimated, the aim of the present investigation was to study the antioxidative action of melatonin in comparison with N‐acetylcysteine (NAC) under diabetic conditions. Alloxan‐diabetic rabbits were treated daily with either melatonin (1 mg/kg, i.p.), NAC (10 mg/kg, i.p.) or saline. Blood glutathione redox state and serum hydroxyl free radicals (HFR), creatinine and urea levels were monitored. After 3 wk of treatment animals were killed and HFR content, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio as well as the activities of glutathione reductase, glutathione peroxidase and γ‐glutamylcysteine synthetase were estimated in both liver and kidney cortex. Diabetes evoked a several‐fold increase in HFR levels accompanied by a significant decline in GSH/GSSG ratio in serum and the examined organs. In contrast to NAC, melatonin (at 1/10 the dose of NAC) attenuated diabetes‐induced alterations in glutathione redox state and HFR levels, normalized creatinine concentration and diminished urea content in serum. Moreover, the indole resulted in an increase in glutathione reductase activity in both studied organs and in a rise in glutathione peroxidase and γ‐glutamylcysteine synthetase activities in the liver. In contrast to NAC, melatonin seems to be beneficial for diabetes therapy because of its potent antioxidative and nephroprotective action. The indole‐induced increase in the activities of the enzymes of glutathione metabolism might be of importance for antioxidative action of melatonin under diabetic conditions.

The stimulatory effect of glucagon and dibutyryl cyclic AMP on ureogenesis and gluconeogenesis in relation to the mitochondrial ATP contnet

The stimulation by glucagon of both ureogenesis and gluconeogenesis has been known for some years [1,2]. Recently Yamazaki reported that pretreatment of the rats with glucagon yields mitochondria having (i) An increased phosphorylation rate [3] (ii) An increased rate of citruline synthesis [4] (iii) An enhanced ATPase activity in presence of uncoupler and an increased rate of K’ uptake [5] . Garrison and Haynes [6] in their description of the hormone-induced stimulation of pyruvate carboxylation concluded that changes of mitochondrial content did not appear to account for the effect, which they attributed to an increased uptake and metabolism of pyruvate. The results presented here show that the stimulatory effect of both glucagon and cyclic AMP (CAMP) on ureogenesis and gluconeogenesis in isolated rat liver cells was accompanied by an increase of the mitochondrial ATP/ADP ratio which consequently furnishes more energy for the mitochondrial steps of these two processes. Higher rates of citrulline production in liver mitochondria isolated from rats pretreated with glucagon or CAMP were associated with a raised mitochondrial ATP content and an enhanced adenine nucleotide translocase.

Hypoglycaemic, antioxidative and nephroprotective effects of taurine in alloxan diabetic rabbits

The therapeutic potential of taurine was investigated under diabetic conditions. Alloxan diabetic rabbits were treated daily for three weeks with 1% taurine in drinking water. The following parameters were measured: 1) serum glucose, urea, creatinine and hydroxyl free radical (HFR) levels; 2) blood glutathione redox state; 3) urine albumin concentration; 4) hepatic and renal HFR levels, GSH/GSSG ratios and the activities of catalase, superoxide dismutase and the enzymes of glutathione metabolism; 5) renal NADPH oxidase activity; 6) the rates of renal and hepatic gluconeogenesis. Histological studies of kidneys were also performed. Taurine administration to diabetic rabbits resulted in 30% decrease in serum glucose level and the normalisation of diabetes-elevated rate of renal gluconeogenesis. It also decreased serum urea and creatinine concentrations, attenuated diabetes-evoked decline in GSH/GSSG ratio and abolished hydroxyl free radicals accumulation in serum, liver and kidney cortex. Animals treated with taurine exhibited elevated activities of hepatic gamma-glutamylcysteine syntetase and renal glutathione reductase and catalase. Moreover, taurine treatment evoked the normalisation of diabetes-stimulated activity of renal NADPH oxidase and attenuated both albuminuria and glomerulopathy characteristic of diabetes. In view of these data, it is concluded that: 1) diminished rate of renal gluconeogenesis seems to contribute to hypoglycaemic effect of taurine; 2) taurine-induced increase in the activities of catalase and the enzymes of glutathione metabolism is of importance for antioxidative action of this amino acid and 3) taurine nephroprotective properties might result from diminished renal NADPH oxidase activity. Thus, taurine seems to be beneficial for the therapy of both diabetes and diabetic nephropathy.

Inhibition of gluconeogenesis by vanadium and metformin in kidney-cortex tubules isolated from control and diabetic rabbits.

Effect of vanadyl acetylacetonate (VAc) and metformin on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules of rabbit. Glucose formation from alanine+glycerol+octanoate, pyruvate or dihydroxyacetone was inhibited by 50-80% by 100 microM VAc or 500 microM metformin in renal tubules of control and alloxan-diabetic animals, while the inhibitory action of these compounds in hepatocytes was less pronounced (by about 20-30%). In contrast to VAc, metformin increased the rate of lactate formation by about 2-fold in renal tubules incubated with alanine+glycerol+octanoate. In view of VAc-induced changes in intracellular gluconeogenic intermediates and gluconeogenic enzyme activities, it is likely that this compound may decrease fluxes through pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase. In contrast to VAc, metformin-induced decrease in renal gluconeogenesis may result from a decline of cytosolic oxaloacetate level and consequently PEPCK activity. Following 6 days of VAc administration (1.275 mg Vkg(-1) body weight daily) the blood glucose level in alloxan-diabetic rabbits was normalised while blood glucose changes in control animals were not observed. On the contrary, in diabetic animals treated for 6 days with metformin (200 mg kg(-1) body weight day(-1)) a high blood glucose level was maintained. Unfortunately, VAc-treated control and diabetic rabbits exhibited elevated serum urea and creatinine levels. In VAc-treated animals vanadium was accumulated in kidney-cortex up to 7.6+/-0.6 microg Vg(-1) dry weight. In view of a potential vanadium nephrotoxicity a therapeutic application of vanadium compounds needs a critical re-evaluation.

Diabetes-induced changes in glucose synthesis, intracellular glutathione status and hydroxyl free radical generation in rabbit kidney-cortex tubules

Diabetes-induced changes in glucose formation, intracellular and mitochondrial glutathione redox states as well as hydroxyl free radicals (HFR) generation have been investigated in rabbit kidney-cortex tubules. In contrast to renal tubules of control animals, diabetes-evoked increase in glucose formation in the presence of either aspartate + glycerol + octanoate or malate as gluconeogenic precursors (for about 50%) was accompanied by a diminished intracellular glutathione reduced form (GSH)/glutathione oxidised one (GSSG) ratio by about 30–40%, while the mitochondrial GSH/GSSG ratio was not altered. However, a relationship between the rate of gluconeogenesis and the intracellular glutathione redox state was maintained in renal tubules of both control and diabetic rabbits, as concluded from measurements in the presence of various gluconeogenic precursors. Moreover, diabetes resulted in both elevation of the glutathione reductase activity in rabbit kidney-cortex and acceleration of renal HFR generation (by about 2-fold). On the addition of melatonin, the hormone exhibiting antioxidative properties, the control values of HFR production were restored, suggesting that this compound might be beneficial during diabetes therapy. In view of the data, it seems likely that diabetes-induced increase in HFR formation in renal tubules might be responsible for a diminished intracellular glutathione redox state despite elevated glutathione reductase activity and accelerated rate of gluconeogenesis, providing glucose-6-phosphate for NADPH generation via pentose phosphate pathway. (Mol Cell Biochem 261: 91–98, 2004)

Effects of oleate, palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells.

Abstract The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3- OHB AcAc ) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.

Differential effects of vanadium, tungsten and molybdenum on inhibition of glucose formation in renal tubules and hepatocytes of control and diabetic rabbits: Beneficial action of melatonin and N-acetylcysteine

Effect of vanadyl acetylacetonate (VAc), tungstate and molybdate on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules. In renal tubules of control and alloxan-diabetic animals, the rank order of the metal-compounds-induced (i) inhibition of glucose formation from alanine + glycerol + octanoate or aspartate + glycerol + octanoate, (ii) decrease in the mitochondrial membrane potential (ΔΨm), (iii) increase in the hydroxyl free radicals (HFR) generation and (iv) decline in glucose-6-phosphatase activity was the following: VAc > tungstate > molybdate. Moreover, in contrast to VAc, both tungstate and molybdate at 100 μM concentration did not practically decrease glucose production in hepatocytes isolated from diabetic rabbits, and significantly increased the rate of lactate formation in renal tubules. N-acetylcysteine at 2 mM concentration partially attenuated vanadium-induced alterations in glucose formation, ΔΨm and the cellular glutathione redox state, whereas 0.1 mM melatonin did not abolish vanadium-induced changes in gluconeogenesis despite attenuation of vanadium effects on HFR formation and ΔΨm decline. However, similarly to control rabbits, following 6 days of intraperitoneal administration of both VAc (1.275 mg V/kg body weight daily) and melatonin (1 mg/kg body weight daily) to alloxan-diabetic animals, vanadium-induced elevated serum creatinine and urea levels were decreased, indicating the beneficial effect of melatonin on diabetes- and vanadium-induced nephrotoxicity in rabbits. As serum glucose levels were also significantly diminished by vanadium + melatonin treatment of diabetic animals, the combination therapy of vanadium compounds and melatonin needs a careful evaluation. (Mol Cell Biochem 261: 9–21, 2004)

The inhibition of gluconeogenesis by chloroquine contributes to its hypoglycaemic action

The effect of chloroquine on gluconeogenesis in isolated hepatocytes and kidney-cortex tubules of rabbit has been studied. The inhibitory action of 200 microM chloroquine was the highest in hepatocytes and renal tubules incubated with glutamine and glutamate+glycerol+octanoate, respectively, while in the presence of other substrates the drug action was less pronounced. With amino acids as substrates, the inhibition of gluconeogenesis was accompanied by a decreased glutamine production, resulting from a decline of glutamate dehydrogenase activity. A decrease in the urea production by hepatocytes incubated with chloroquine in the presence of glutamine but not NH4Cl as the source of ammonium is in agreement with this suggestion. The degree of inhibition by chloroquine of the rate of gluconeogenesis in renal tubules isolated from control rabbits was similar to that determined in diabetic animals. Chloroquine-induced changes in levels of intracellular gluconeogenic intermediates indicate a decrease in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities probably due to increased concentration of 2-oxoglutarate, an inhibitor of these two enzymes. In view of the data, it is likely that inhibition by chloroquine of glucose formation in liver and kidney may contribute to the hypoglycaemic action of this drug. The importance of the inhibitory effect of chloroquine on glutamate dehydrogenase activity in the antihyperglycaemic action of the drug is discussed.

Relationship between pyruvate carboxylation and citrulline synthesis in rat liver mitochondria: The effect of ammonia and energy

Abstract 1. 1. Pyruvate carboxylation and citrulline synthesis were studied in rat liver mitochondria incubated in Slate 4 and State 3 in order to vary the intramitochondrial ATP/ADP ratio. 2. 2. 1 mM NH 4 Cl diminished the rate of pyruvate carboxylation in the presence and absence of fatty acids. 3. 3. The addition of NH 4 Cl resulted in a decrease of V max value of pyruvate carboxylation but had no effect on the K m value for ATP. 4. 4. In mitochondria incubated in State 4 and State 3 citrulline synthesis resulted in a decrease of pyruvate carboxylation, suggesting a competition for energy between carbamoylphosphate synthase (ammonia) and pyruvate carboxylase. 5. 5. When exogenous ATP was used as an energy source an inhibition of the rate of pyruvate carhoxylation by citrulline synthesis was not observed.

Melatonin-induced modulation of glucose metabolism in primary cultures of rabbit kidney-cortex tubules.

Abstract:  The effect of melatonin on glucose metabolism in the presence and absence of insulin has been investigated in the primary cultures of renal tubules grown in a defined medium. In the absence of glucose in the medium containing 5 μg/mL of insulin and 2 mm alanine + 5 mm glycerol + 0.5 mm octanoate, 100 nm melatonin stimulated both glucose and lactate synthesis, while in the medium devoid of insulin melatonin action was negligible. Melatonin‐induced increase in glucose and lactate synthesis was accompanied by an enhancement of alanine and glycerol consumption. In view of measurements of [U‐14C]l‐alanine and [U‐14C]l‐glycerol incorporation into glucose, it is likely that melatonin increased alanine utilization for glucose production, while accelerated lactate synthesis was because of an enhanced glycerol consumption. As (i) 10 nm luzindole attenuated the stimulatory action of melatonin on glucose formation and (ii) the indole induced a decrease in intracellular cAMP level, it seems likely that in renal tubules melatonin binds to ML1 membrane receptor subtype. In view of a decline of intracellular fructose‐1,6‐bisphosphate content accompanied by a significant rise in hexose‐6‐phosphate and glucose levels, melatonin might result in an acceleration of flux through fructose‐1,6‐bisphosphatase probably because of an increase in the active, dephosphorylated form of this enzyme. Thus, the administration of melatonin in combination with insulin might be beneficial for diabetic therapy because of protection against hypoglycemia.