Effects of streptozotocin-induced diabetes on acetylcholine metabolism in rat brain

The main objective of this study was to determine whether uncontrolled hyperglycemia, as a consequence of diabetes, altered the metabolism of acetylcholine (ACh) in rat brain. To accomplish this, rats received injections of streptozotocin (STZ, 60 mg/kg, i.v.) or vehicle, and were maintained for up to 7 weeks after the injections. Various indices of ACh metabolism were determined in striatum and hippocampus, two brain regions densely innervated by cholinergic neurons. STZ induced diabetes in 96% of the rats injected, as evidenced by glucose spillage into the urine within 48 hours. Serum glucose levels increased to 326% of control values by 1 week and remained at this level for the duration of the study. The steady-state concentrations of ACh and choline, determined in brain tissue from animals killed by head-focused microwave irradiation, did not differ between the control and STZ-injected groups. However, the synthesis and release of neurotransmitter by striatal slices, measured in vitro, decreased in a time-dependent manner. Although the basal release of ACh was unaltered at 1 week, neurotransmitter release decreased significantly by 21% at 5 weeks and by 26% at 7 weeks. The release of ACh evoked by incubation with 35 mM KCl was inhibited significantly by 20% at all time points studied. ACh synthesis by slices incubated under basal conditions decreased by 13% and 27% at 5- and 7-weeks, respectively, the latter significantly less than controls. Synthesis by striatal slices incubated with 35 mM KCl was inhibited by 17% at 7 weeks. Although the synthesis and release of ACh by hippocampal slices from diabetic animals tended to be less than controls, these alterations were not statistically significant. Investigations into the mechanism(s) mediating the deficit in ACh synthesis exhibited by striatal slices indicated that it did not involve alterations in precursor choline availability, nor could it be attributed to alterations in the activities of the synthetic or hydrolytic enzymes choline acetyltransferase or acetylcholinesterase; rather, the decreased turnover of ACh may be secondary to other STZ-induced, hyperglycemia-mediated neurochemical alterations.     

Properties of thiamine-binding proteins isolated from rat brain,liver and kidneys

The study of thiamine-binding proteins (ThBP) isolated from liver and kidneys of rats was held in order to find out the peculiarities and physiological role of the ThBP isolated earlier from the rat brain. It was demonstrated that ThBP from liver and kidneys of rats as well as ThBP from rat brain described earlier, were bifunctional: on an equal footing with ability to bind thiamine specifically, they show an ability to hydrolyse the phosphoric esters of thiamine selectively. The ThBP of these tissues (liver, kidneys and brain) didn't differ by the molecular weight, but differed by the enzymatic activities. The molecular weight of ThBP was estimated to be 100 kDa by gel-filtration; 63 kDa and 35 kDa by sodium dodecylsulfate gel electrophoresis. Specific thiamine-binding activity increases as follows: ThBP from rat brain < ThBP from rat liver < ThBP from rat kidneys.     

Malate-aspartate shuttle enzymes in rat brain regions, liver and heart during alloxan diabetes and insulin replacement

Regionally selective and time-dependent variations were observed in the activity of brain aspartate aminotransferase at early phases of diabetes. Malate dehydrogenase activity showed an opposite pattern of changes in soluble and particulate fractions of cerebral hemispheres and brain stem, with cerebellum showing consistent increase in the activity. The activity of both the enzymes increased significantly in liver, in contrast to heart where malate dehydrogenase activity decreased in particulate fraction. Insulin treatment to diabetic animals restored the enzymes to near control levels at early stages of diabetes, except in liver. The results indicate that malate-aspartate shuttle is probably stimulated under diabetic conditions to enable glycolysis to continue and ATP levels to be restored partially, particularly in cerebellum and liver.     

Deficit of coenzyme Q in heart and liver mitochondria of rats with streptozotocin-induced diabetes

Mitochondrial dysfunction and oxidative stress participate in the development of diabetic complications, however, the mechanisms of their origin are not entirely clear. Coenzyme Q has an important function in mitochondrial bioenergetics and is also a powerful antioxidant. Coenzyme Q (CoQ) regenerates alpha-tocopherol to its active form and prevents atherogenesis by protecting low-density lipoproteins against oxidation. The aim of this study was to ascertain whether the experimentally induced diabetes mellitus is associated with changes in the content of endogenous antioxidants (alpha-tocopherol, coenzymes Q9 and Q10) and in the intensity of lipoperoxidation. These biochemical parameters were investigated in the blood and in the isolated heart and liver mitochondria. Diabetes was induced in male Wistar rats by a single intravenous injection of streptozotocin (45 mg x kg(-1)), insulin was administered once a day for 8 weeks (6 U x kg(-1)). The concentrations of glucose, cholesterol, alpha-tocopherol and CoQ homologues in the blood of the diabetic rats were increased. The CoQ9/cholesterol ratio was reduced. In heart and liver mitochondria of the diabetic rats we found an increased concentration of alpha-tocopherol, however, the concentrations of CoQ9 and CoQ10 were decreased. The formation of malondialdehyde was enhanced in the plasma and heart mitochondria. The results have demonstrated that experimental diabetes is associated with increased lipoperoxidation, in spite of the increased blood concentrations of antioxidants alpha-tocopherol and CoQ. These changes may be associated with disturbances of lipid metabolism in diabetic rats. An important finding is that heart and liver mitochondria from the diabetic rats contain less CoQ9 and CoQ10 in comparison with the controls. We suppose that the deficit of coenzyme Q can participate in disturbances of mitochondrial energy metabolism of diabetic animals.     

Effects of streptozotocin-induced diabetes on neuronal sigma receptors in the rat brain

To study the effect of diabetes mellitus on the density of sigma receptors, in vitro binding experiments were conducted in whole brain homogenate membranes of 5-week and 10-week control rats and streptozotocin (STZ)-induced diabetic rats. sigma-1 Receptors were labelled with [3H](+)-pentazocine while sigma-2 receptors were labelled with [3H] 1,3-di-o-tolylguanidine (DTG) in the presence of 0.5 microM (+)-pentazocine to mask sigma-1 sites. Non-specific binding was determined in the presence of 20 microM haloperidol. Scatchard analysis revealed a 27% (p<0.01) decreased in sigma-1 receptor density and a 33% (p<0.01) decreased in sigma-2 receptor density in whole brain of 10-week STZ-diabetic rats. No statistically significant difference was found in the sigma receptor content of 5-week STZ-diabetic rats. These results provide evidence that neuronal sigma receptors are reduced in 10-week STZ-diabetic rats and suggest that changes in sigma receptors may play a role in diabetes related abnormalities. Further evaluation is required to determine whether changes observed in the brain are homogeneous for either or both sigma receptor subtypes as well as potential links between other CNS receptor changes previously observed in STZ-induced diabetic rats.     

The effect of chronic alloxan- and streptozotocin-induced diabetes on isolated rat heart performance

Cardiac disease is a common secondary complication appearing in chronic diabetics. Isolated perfused working hearts obtained from both acute and chronic diabetic rats have also been shown to exhibit cardiac functional abnormalities when exposed to high work loads. We studied cardiac performance at various time points after induction of diabetes in rats to determine exactly when functional alterations appeared and whether these alterations progressed with the disease state. Female Wistar rats were made diabetic by a single i.v. injection of either alloxan (65 mg/kg) or streptozotocin (STZ 60 mg/kg). Cardiac performance was assessed at 7, 30, 100, 180, 240, and 360 days after induction of diabetes using the isolated perfused working heart technique. No changes were observed in the positive and negative dP/dt development at various atrial filling pressures in the diabetic hearts 7 days after treatment. Alloxan diabetic rat hearts exhibited depressed left ventricular pressure and positive and negative dP/dt development when perfused at high atrial filling pressures, at 30, 100, and 240 days after treatment. STZ diabetic rat hearts exhibited depressed cardiac performance at high atrial filling pressures, at 100, 180, and 360 days after treatment, but not at 30 days after treatment. Control hearts exhibited slight but significant depressions in cardiac function with age. These results suggest that cardiac functional alterations appear in diabetic rats about 30 days after induction and progress with the disease. These alterations may indicate the development of a cardiomyopathy.     

The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabetes was controlled by daily injections of insulin. These results are discussed in relation to the hormonal control of phenylalanine hydroxylase gene expression.     

Bicarbonate measurements in rat liver, brain, heart, and skeletal muscle.

A method is described for measuring total CO₂ and HCO₃^? in tissues rapidly frozen in liquid nitrogen. The method is a modification of the procedure of D. D. Van Slyke and J. M. Neill (1924, J. Biol. Chem.61, 523–573) for use with freeze-clamped tissue where anoxic changes have not occurred. The HCO₃^? content exclusive of tissue CO₂ in fed rats was found to be: liver, 19.4 ± 1.0; brain, 20.2 ± 0.9; thigh, 16.2 ± 0.8; and heart, 15.4 ± 1.4 μmol/g.     

Influence of alloxan diabetes and insulin treatment on the activity of alanine aminotransferase in rat brain regions, liver and heart

Studies with brain alanine aminotransferase showed higher activity of the enzyme in the soluble fraction of cerebellum. Among the tissues, the liver soluble fraction was the richest source of the enzyme. Alloxan-induced diabetes caused both regional and time-dependent variations in the activity of brain alanine aminotransferase. Significant among these changes were the decrease in both soluble and particulate enzyme from cerebral hemispheres and an increase in the soluble enzyme activity from cerebellum at early stages of diabetes. Brain stem did not show any marked change in enzyme activity. Liver and heart enzyme, however, increased significantly after 1-2 weeks of diabetes. Insulin treatment to diabetic animals caused an 'over-shoot' in soluble alanine aminotransferase activity, particularly in cerebellum and liver.     

Effect of streptozotocin-induced diabetes on epidermal growth factor receptors in rat liver plasma membrane

The effect of streptozotocin-induced diabetes on 125I-labeled epidermal growth factor (EGF) binding was studied in microsomal membranes from rat liver. The binding of EGF in membranes from diabetic animals was significantly low, the value being about 60% of the control level. Scatchard analysis of the binding data clearly showed that the decrease in EGF binding was due to a decrease in the number of receptors. Treatment of diabetic animals with insulin restored EGF receptors to control levels, whereas the treatment with triiodothyronine had no effect. Serum EGF concentrations measured were almost the same among the control, diabetic, and insulin-treated diabetic groups. These results suggest that insulin deficiency in vivo causes a decrease in hepatic EGF receptors.     

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.     

The effect of streptozotocin-induced diabetes and of insulin supplementation on glycogen metabolism in rat liver.

The effects of streptozotocin-induced diabetes and of insulin supplementation to diabetic rats on glycogen-metabolizing enzymes in liver were determined. The results were compared with those from control animals. The activities of glycogenolytic enzymes, i.e. phosphorylase (both a and b), phosphorylase kinase and protein kinase (in the presence or in the absence of cyclic AMP), were significantly decreased in the diabetic animals. The enzyme activities were restored to control values by insulin therapy. Glycogen synthase (I-form) activity, similarly decreased in the diabetic animals, was also restored to control values after the administration of insulin. The increase in glycogen synthase(I-form) activity after insulin treatment was associated with a concomitant increase in phosphoprotein phosphatase activity. The increase in phosphatase activity was due to (i) a change in the activity of the enzyme itself and (ii) a decrease in a heat stable protein inhibitor of the phosphatase activity.     

Alterations in atrial natriuretic peptide and its receptors in streptozotocin-induced diabetic rat kidneys

In this study the effect of diabetes mellitus on atrial natriuretic peptide (ANP) receptors in streptozotocin- (STZ-) induced diabetic rat kidneys was studied. Moreover, plasma ANP concentration was evaluated in diabetic and control rats by using radioimmunoassay. In addition, the expression of ANP in the kidneys of control and diabetic rats was evaluated by immunohistochemistry. Body-weight loss and increased glucose levels were used as indices of diabetes mellitus in the STZ-induced rats. There was a significant loss in the body weight of the diabetic rats compared to controls. The efficacy of STZ administration was confirmed by rising blood glucose levels, which were significantly higher in diabetic rats compared to controls. Plasma ANP concentration was significantly greater in the diabetic rats in comparison with controls. Moreover, our immunohistochemical results show that the expression of ANP in diabetic rats was higher than that in age-matched controls. ANP was observed in the cells lining the proximal convoluted tubules in the cortex. The distribution and levels of ANP receptors in the kidneys of diabetic rats and age-matched controls were investigated using quantitative receptor autoradiography. Our results demonstrate significant decrease in ANP receptors in the kidneys of the diabetic rats compared to controls. The significant decrease was found in the juxtaglomerular medulla, inner medulla, and the papillae. The decrease in ANP receptors observed in the diabetic kidneys could have pathological consequences resulting in renal resistance to ANP in diabetes.     

N-acetyl-beta-D-glucosaminidase activity in serum, kidney and liver of streptozotocin-induced diabetic rat

Serum N-acetyl-beta-D-glucosaminidase (NAG) activity in streptozotocin-induced diabetic rats was significantly increased. There was neither a difference in total NAG activity in kidney and liver, nor in optimal pH of NAG in serum, kidney and liver between diabetic and control rats. The ratio of the thermounstable fraction of NAG increased in diabetic kidney and liver, while there was no difference in thermostability of between diabetic and control rats. Isoelectricfocusing of diabetic serum NAG indicated an increase in the neutral form. That of kidney and liver NAG indicated an increase in the acid form. These results may suggest that NAG clearance from the serum is decreased diabetic state.     

Guanylate cyclase activity in rat liver and other tissues with starvation and streptozotocin-induced diabetes mellitus.

Guanylate cyclase activities in supernatant and particulate fractions of homogenates from various rat tissues were examined in fed and fasted normal animals and in those with diabetes mellitus induced with streptozotocin. With fasting guanylate cyclase activity in supernatant fractions increased in liver and epididymal fat, decreased in kidney and lung, and was unchanged in cerebral cortex and skeletal muscle. Lung particulate activity also decreased with fasting while particulate activities in other tissues were unchanged. In diabetic animals soluble but not particulate activity was less in several tissues and the effect of fasting on soluble liver guanylate cyclase was absent. The effect of fasting on soluble liver guanylate cyclase reversed with refeeding animals and was associated with a decrease in the apparent K_m for GTP as well as an increase in V. An inhibitory material was found in livers from fed but not fasted animals. The inhibitory material had properties of a nucleotide and inhibited guanylate cyclase in a competitive manner. Thus, soluble and particulate guanylate cyclase activities can be influenced independently of one another in the same and different tissues with fasting, refeeding, and diabetes mellitus. Some of these effects may be attributable to altered levels of small heat-stable inhibitory materials such as nucleotides.     

Alterations in atrial natriuretic peptide and its receptors in streptozotocin-induced diabetic rat kidneys

In this study the effect of diabetes mellitus on atrial natriuretic peptide (ANP) receptors in streptozotocin- (STZ-) induced diabetic rat kidneys was studied. Moreover, plasma ANP concentration was evaluated in diabetic and control rats by using radioimmunoassay. In addition, the expression of ANP in the kidneys of control and diabetic rats was evaluated by immunohistochemistry. Body-weight loss and increased glucose levels were used as indices of diabetes mellitus in the STZ-induced rats. There was a significant loss in the body weight of the diabetic rats compared to controls. The efficacy of STZ administration was confirmed by rising blood glucose levels, which were significantly higher in diabetic rats compared to controls. Plasma ANP concentration was significantly greater in the diabetic rats in comparison with controls. Moreover, our immunohistochemical results show that the expression of ANP in diabetic rats was higher than that in age-matched controls. ANP was observed in the cells lining the proximal convoluted tubules in the cortex. The distribution and levels of ANP receptors in the kidneys of diabetic rats and age-matched controls were investigated using quantitative receptor autoradiography. Our results demonstrate significant decrease in ANP receptors in the kidneys of the diabetic rats compared to controls. The significant decrease was found in the juxtaglomerular medulla, inner medulla, and the papillae. The decrease in ANP receptors observed in the diabetic kidneys could have pathological consequences resulting in renal resistance to ANP in diabetes. (Mol Cell Biochem 261: 3–8, 2004)     

S-Adenosylmethionine decarboxylase in liver, heart and pancreas of pyridoxine-deficient chickens

S-adenosylmethionine decarboxylase activity has been measured in liver, heart and pancreas of pyridoxine-deficient chickens: in liver and heart muscle it is increased, while in pancreas the activity is unchanged with respect to control animals. Insulin induced activity in liver and in heart muscle of normal as well as of pyridoxine-deficient chickens, while in the pancreas an induction was observed in the control animals and a decrease in the deficient ones. These data appear to rule out any involvement of pyridoxal phosphate in the reaction catalyzed by S-adenosylmethionine decarboxylase.