Effects of Aminoguanidine on Lipid and Protein Oxidation in Diabetic Rat Kidneys

Nonenzymatic glycation of tissue and plasma proteins may stimulate the production of oxidant and carbonyl stress in diabetes. The aim of this study was to evaluate the effects of aminoguanidine (AG) on lipid peroxidation, protein oxidation and nitric oxide (NO) release in diabetic rat kidneys. After induction of diabetes with streptozotocin, female Wistar rats were divided into 2 groups. Group DAG (n=9) rats were given AG hydrogen carbonate (1 g/L) in drinking water and group D (n=8) was diabetic control rats given only tap water. Group H (n=8) was followed as healthy controls. At the end of an 8 week period, NO release, lipid and protein oxidation were determined in kidney tissues. NO release was significantly lower in diabetic rats compared with healthy controls (p<0.05). Lipid peroxidation was significantly high in group D (3.9 ± 0.3 nmol MDA/g tissue) compared with the group DAG (2.6 ± 0.1 nmol MDA/g tissue, p<0.01) and group H (2.4 ± 0.2 nmol MDA/g tissue). Protein oxidation was significantly higher in diabetics than healthy controls (563.8 ± 23.9, 655.8 ± 7.2 , 431.5 ± 8.8 mmol carbonyl / g tissue for group DAG, D and H, respectively, p< 0.05). A positive correlation between albuminuria and thiobarbituric acid reactive substance (TBARS) levels (r= 0.54,p<0.005) and carbonyl content (r=0.70, p<0.0005) in kidney homogenate were observed. Although AG treatment had no effect on NO release, it significantly decreased lipid peroxidation in diabetic rat cortices. Consequently increased lipid peroxidation -as well as- protein oxidation could be involved in the pathogenesis of diabetic albuminuria.     

Biochemical properties of porcine white adipose tissue mitochondria and relevance to fatty acid oxidation

The capacity of white adipose tissue mitochondria to support a high beta-oxidative flux was investigated by comparison to liver mitochondria. Based on marker enzyme activities and electron microscopy, the relative purity of the isolated mitochondria was similar thus allowing a direct comparison on a protein basis. The results confirm the comparable capacity of adipose tissue and liver mitochondria for palmitoyl-carnitine oxidation. Relative to liver, both citrate synthase and α-ketoglutarate dehydrogenase were increased 7.87- and 10.38-fold, respectively. In contrast, adipose tissue NAD-isocitrate dehydrogenase was decreased (2.85-fold). Such modifications in the citric acid cycle are expected to severely restrict citrate oxidation in porcine adipose tissue. Except for cytochrome c oxidase, activities of the enzyme complexes comprising the electron transport chain were not significantly different. The decrease in adipose cytochrome c oxidase activity could partly be attributed to a decreased inner membrane as suggested by lipid and enzyme analysis. In addition, Western blotting indicated that adipose and liver mitochondria possess similar quantities of cytochrome c oxidase protein. Taken together these results indicate that not only is the white adipose tissue protoplasm relatively rich in mitochondria, but that these mitochondria contain comparable enzymatic machinery to support a relatively high beta-oxidative rate.     

An immunological study of the uncoupling protein of brown adipose tissue mitochondria

1. Ewes were injected with purified 32,000-Mr uncoupling protein from mitochondria of brown adipose tissue of cold-adapted rats in order to raise antibodies. 2. The existence of antibodies in the plasma of ewes and the cross-reactivity of plasmas were demonstrated and studied by 125I-labelled antigen-antibody reaction, double immunodiffusion, the inhibition of GDP binding to the 32,000 Mr protein and by immunohistochemistry. 3. The antibodies raised against the homogeneous protein yielded a single immunoprecipitation band with detergent-solubilized mitochondrial membranes of brown adipose tissue from rat, hamster, guinea-pig, rabbit and with the purified uncoupling protein of these animals. No immunoprecipitation was obtained with the protein purified from brown adipose tissue of term lamb foetus. 4. The GDP-binding activity of the uncoupling protein (isolated or in solubilized membranes) was largely inhibited by the antiserum. 5. The anti-(rat uncoupling protein) could not cross-react with solubilized membranes from liver or muscle, nor with the purified beef heart or rat liver ADP/ATP translocator.     

Prolonged beta-adrenoceptor stimulation increases the amount of GDP-binding protein in brown adipose tissue mitochondria

Cold acclimation increases the thermogenic capacity of brown adipose tissue (BAT) and this is associated with both a general hypertrophy of the tissue and a selective increase in the proportion of a specific 32,000 molecular weight, GDP-binding protein in the mitochondrial membrane. This protein regulates the activity of the major thermogenic pathway of BAT. Although noradrenaline mediates the acute thermogenic response to cold, repeated injections of this hormone fail to mimic cold acclimation in that they do not increase the proportion of 32,000 molecular weight (32K) protein. However, noradrenaline is rapidly metabolised in vivo and effective levels would only be maintained over a short period of time following administration of the hormone. We have therefore investigated the effects of two long-acting sympathomimetic compounds: fenoterol, a beta-adrenoceptor agonist, and ephedrine, which causes release of noradrenaline from nerve terminals. Chronic treatment with both of these compounds increased the GDP-binding ability of BAT mitochondria and the proportion of 32K protein as measured by gel electrophoresis. We suggest that prolonged stimulation of beta-adrenoceptors, probably in BAT, but possibly also in other tissues, is sufficient to produce a selective increase in GDP-binding protein in BAT, and it is unnecessary to invoke any factor that is not a consequence of beta-adrenoceptor stimulation to explain the effect of cold.     

Brown adipose tissue (Na+-K+)-ATPase activity and substrate uptake during the breeding cycle of rats

Brown adipose tissue (Na+-K+)-ATPase activity, in vitro glucose and 2-aminoisobutyric acid uptake, as well as mitochondrial GDP-binding and succinate dehydrogenase activity were determined in order to study the relationship between these parameters and the thermogenic status. Analysis were carried out on control animal, pregnant rats, dams and pups during lactation, GDP-binding, (Na+-K+)-ATPase and glucose uptake were found to be decreased in brown adipose tissue from pregnant rats and dams, and increased in pups, 2-aminoisobutyric acid uptake was only increased in pups, but no changes were observed in the other experimental groups tested. GDP-binding and (Na+-K+)-ATPase activity showed a parallelism which suggests that the enzyme is a good index of thermogenic status of the animal.     

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.     

Development of the uncoupling protein in the rat brown-adipose tissue during the perinatal period. Its relationship with the mitochondrial GDP-binding and GDP-sensitive ion permeabilities and respiration.

In this paper we report the development of the immunologically detected uncoupling protein (UCP) in brown adipose tissue during the perinatal period in the rat and its relationship with its functional activity expressed in terms of GDP-binding capacity, GDP-sensitive permeabilities and GDP-sensitive respiration. Immunologically detected UCP increased during the last 2 days of foetal life (under euthermic conditions) and after birth (after postnatal hypothermia) during the early suckling period, reaching its maximum value on day 10 after birth. This increase in UCP is accompanied by parallel increases in the GDP-binding capacity, GDP-sensitive permeabilities to protons and chloride ions and GDP-inhibitable respiration. During the suckling--weaning transition, there was a regression of the parameters related to the functional activity of the UCP (GDP-binding capacity and nucleotide-sensitive permeabilities and respiration) without changes in the immunologically detected UCP. These results suggest that the involution of this tissue in the rat commences in this period; the first parameters affected are those related to the functional activity of the UCP while the UCP is still present in its highest level. This seems to support the idea that, in this period of development of the rat, the UCP may exist in the brown fat mitochondria in a functional (unmasked) form and a non-functional (masked) form.     

Brown adipose tissue activity in hypocaloric-diet fed lactating rats

Hypocaloric diet feeding reduced the mitochondrial protein content and whole tissue GDP-binding in interscapular brown adipose tissue from both virgin and lactating rats. A reduction in brown fat lipoprotein lipase activity was also detected in underfed virgin and lactating animals. These results indicate that lactation in the rat, even though it produces a reduction in brown fat activity, does not impair the capacity of the tissue to respond to a diminished caloric intake by lowering its activity further.     

Sulphated proteins secreted by rat mammary epithelial cells

The main sulphated proteins secreted by rat mammary gland tissue have Mr of approximately 32 000, 27 000 and 25 000 Da. In addition, there are high Mr components which have a diffuse electrophoretic mobility (Mr > 200 000) and most likely corresponded to proteoglycans. The sulphate groups in the proteins with discrete Mr are most likely all linked to carbohydrates. These sulphated molecules were partially purified and identified to isoforms of rat alpha-lactalbumin for the 25-27 kDa bands and to kappa-casein for the 32 kDa band. This pattern of protein sulphation is, as far as we know, quite specific to rat mammary epithelial cells.     

Exercise suppression of thermoregulatory thermogenesis in warm- and cold-acclimated rats

An evaluation was made of the effects of an acute exercise bout on nonshivering thermogenesis (NST) in cold-acclimated rats (4 degrees C for 6 weeks) and shivering thermogenesis in 24 degrees C-acclimated rats (24 degrees C for 6 weeks). Assessment techniques included indirect calorimetry during treadmill running and brown adipose tissue (BAT) mitochondrial guanosine diphosphate (GDP) binding immediately following a treadmill run. Calorimetric results for 24 degrees C-acclimated rats running at 4 degrees C indicated total substitution of shivering thermogenesis by exercise-derived heat. No difference in GDP-binding, an index of BAT nonshivering thermogenic activity, was observed between exercised and nonexercised 24 degrees C-acclimated rats. Calorimetric results for cold-acclimated rats running at 4 degrees C indicated a total suppression in the energy cost associated with NST, exercise-derived heat replacing or substituting for NST. Examining BAT properties in the exercised cold-acclimated rats revealed a significant 40% decrease in BAT mitochondrial GDP-binding. These results suggest that during running, metabolic heat due to the exercise totally replaces shivering in 24 degrees C-acclimated rats and totally replaces BAT nonshivering thermogenesis in cold-acclimated rats.     

Purification and characterization of a long chain, fatty-acid-binding protein supplying the mitochondrial beta-oxidative system in the heart.

A fatty-acid-binding protein with a molecular weight of approximately 12 000 was purified from rat heart and the binding investigated by electron spin resonance. The stearic acid bound to the protein was found to be transferred to the mitochondrial beta-oxidative system, suggesting a role as transcytoplasmic fatty acid carrier for this protein. For the first time a physiological cytoplasmic protein was used as a carrier supplying the mitochondrial beta-oxidative system. A new mechanism of action is proposed to explain the control exerted by this type of protein in some membrane-linked enzymatic processes.     

Protein kinase and its endogenous substrates in coated vesicles

Coated vesicles prepared from bovine brains contained a protein kinase activity which catalyzed the phosphorylation of endogenous structural proteins, Mr 150 000, 120 000, 48 000 and 32 000. An endogenous protein, Mr 48 000 was most strongly phosphorylated by this kinase. This protein kinase also phosphorylated exogenous proteins, phosvitin intensely and casein slightly but not histone or protamine. The enzyme activity was independent of cyclic nucleotides or Ca2+/calmodulin. Mg2+ stimulated the kinase activity. Some divalent cations were substituted for Mg2+; the potency decreased in the order Mn2+, Mg2+, Co2+, Ca2+, Zn2+. Two separate subfractions, the outer coat and the inner vesicle (core), were prepared from coated vesicles by a urea treatment followed by sucrose density gradient centrifugation and dialysis. The kinase activity was found predominantly in the coat subfraction.     

Effects of antioxidant stobadine on protein carbonylation, advanced oxidation protein products and reductive capacity of liver in streptozotocin-diabetic rats: role of oxidative/nitrosative stress

BACKGROUND: Increased oxidative/nitrosative stress is important in the pathogenesis of diabetic complications, and the protective effects of antioxidants are a topic of intense research. The purpose of this study was to investigate whether a pyridoindole antioxidant stobadine (STB) have a protective effect on tissue oxidative protein damage represented by the parameters such as protein carbonylation (PC), protein thiol (P-SH), total thiol (T-SH) and non-protein thiol (Np-SH), nitrotyrosine (3-NT), and advanced oxidation protein products (AOPP) in streptozotocin-diabetic rats. METHODS: Diabetes was induced in male Wistar rats by intraperitonal injection of streptozotocin (55 mg/kg). Some of the non-diabetic (control) and diabetic rats treated with STB (24.7 mg/kg/day) during 16 weeks, and the effects on blood glucose, PC, AOPP, 3-NT, P-SH, T-SH and Np-SH were studied. Biomarkers were assayed by enzyme-linked immunosorbent assay (ELISA) or by colorimetric methods. RESULTS: Administration of stobadine to diabetic animals lowered elevated blood glucose levels by approximately 16% relative to untreated diabetic rats. Although stobadine decreased blood glucose, poor glycemic control was maintained in stobadine treated diabetic rats during the treatment period. Biochemical analyses of liver proteins showed significant diminution of sulfhydryl groups, P-SH, T-SH, Np-SH, and elevation of carbonyl groups in diabetic animals in comparison to healthy controls. As a biomarker of nitrosative stress, 3-NT levels did not significantly change by diabetes induction or by stobadine treatment when compared to control animals. However, the treatment with stobadine resulted in a significant decrease in PC, AOPP levels and normalized P-SH, T-SH, Np-SH groups in liver of diabetic animals. CONCLUSIONS: The results are in accordance with the pro-oxidant role of chronic hyperglycemia, and the ability of stobadine to attenuate protein oxidation and improving tissue reductive capacity may account, at least partly for its observed beneficial effects on tissue function in diabetes.     

Evaluation of alpha(1)-adrenoceptor antagonist on diabetes-induced changes in peripheral nerve function, metabolism, and antioxidative defense.

The role for nerve blood flow (NBF) vs. other factors in motor nerve conduction (MNC) slowing in short-term diabetes was assessed by evaluating alpha(1)-adrenoceptor antagonist prazosin on NBF, MNC, as well as metabolic imbalances and oxidative stress in the neural tissue. Control and diabetic rats were treated with or without prazosin (5 mg.kg(-1).d(-1) for 3 wk). NBF was measured by hydrogen clearance. Both endoneurial vascular conductance and MNC velocity were decreased in diabetic rats vs. controls, and this decrease was prevented by prazosin. Free NAD(+):NADH ratios in mitochondrial cristae, matrix, and cytosol assessed by metabolite indicator method, as well as phosphocreatine levels and phosphocreatine/creatine ratios, were decreased in diabetic rats, and this reduction was ameliorated by prazosin. Neither diabetes-induced accumulation of two major glycation agents, glucose and fructose, as well as sorbitol and total malondialdehyde plus 4-hydroxyalkenals nor depletion of myo-inositol, GSH, and taurine or decrease in (Na/K)-ATP-ase activity were affected by prazosin. In conclusion, decreased NBF, but not metabolic imbalances or oxidative stress in the neural tissue, is a key mechanism of MNC slowing in short-term diabetes. Further experiments are needed to estimate whether preservation of NBF is sufficient for prevention of nerve dysfunction and morphological abnormalities in long-standing diabetes or whether the aforementioned metabolic imbalances closely associated with impaired neurotropism are of greater importance in advanced than in early diabetic neuropathy.     

Preliminary characterization of a heat-stable protein from rat adipose tissue whose phosphorylation is stimulated by insulin.

Exposure of 32P-labelled isolated rat adipocytes or epididymal fat-pads to insulin resulted in an increase in the phosphorylation of a heat-stable acid-soluble protein of Mr 22 000. The phosphorylation of this protein was unaffected by isoprenaline (isoproterenol) in intact cells, nor was its phosphorylation catalysed by exposure in vitro to the cyclic AMP-dependent protein kinase or smooth-muscle myosin light-chain kinase. The properties of the Mr-22 000 protein include: heat-stability; solubility in 1% trichloroacetic acid; pI 4.9; elution at apparent Mr 37 500 on gel filtration; and it contains both phosphoserine and phosphothreonine. It can be distinguished from the heat-stable phosphatase inhibitor 1 of adipose tissue (inhibitor 1A) and the phosphorylated form of adipose-tissue myosin light chain by several criteria. Its identity, and the possible functional significance of the insulin-stimulated phosphorylation, remain problems for future study.     

Schistosoma mansoni surface glycoproteins. Analysis of their expression and antigenicity

Surface glycoproteins from newly transformed schistosomula of Schistosoma mansoni have been identified by surface radioiodination and lectin-affinity chromatography. From the glycoconjugates bound by the three lectins used, concanavalin A, peanut agglutinin and fucose-binding protein, only in the concanavalin-A-bound fractions were glycoproteins identified. Changes in concanavalin-A-binding glycoproteins were detected after transformation and early maturation of the schistosomula. Some glycoproteins disappeared (Mr 38 000, 29 000 and 25 000), some appeared independently of host molecules (Mr 19 000), others only appeared after culture in human serum (Mr 45 000). Two major glycoproteins of Mr 32 000 and 16 000 were detected on all stages examined. Within the total set of surface glycoproteins identified on 3-h schistosomula only the strong Mr-38 000-32 000 complex was found to be antigenic. Thus many major low-molecular-mass surface glycoproteins of the parasite are not recognised as antigens by immune animals. The separation of only the Mr-38 000-32 000 antigens by concanavalin A affinity chromatography indicates the feasibility of using this method in conjunction with immunoaffinity columns to purity these molecules.     

The energetics of early platelet responses. Energy consumption during shape change and aggregation with special reference to protein phosphorylation and the polyphosphoinositide cycle.

Among the different platelet responses, secretion requires the greatest amount of metabolic energy. The velocities of dense, alpha- and acid hydrolase granule secretion vary in parallel with the increase in energy consumption seen in thrombin-stimulated cells. This covariance is preceded by a phase in which energy consumption is increased without the extracellular appearance of secretion markers. By treating the platelets with thrombin and hirudin we have stimulated the platelets for short intervals and succeeded in separating shape change, single platelet disappearance and secretion to a great extent. In this report we show that the early increase in energy consumption reflects the energy requirement of aggregation but not of shape change. The cost of 100% of single platelet disappearance is 2.8 mumol of ATPeq. X (10(11) platelets)-1. Concurrent analysis of phosphorylation of Mr 20 000 and 47 000 proteins and of 32P-labelled phosphatidylinositol metabolites led to the following observations. Firstly, shape change is neither accompanied by an increase in protein phosphorylation nor by changes in the steady state levels of 32P-labelled phosphatidylinositol metabolites. Secondly, when aggregation occurs both proteins are phosphorylated, but the phosphatidylinositol metabolites do not change. Thirdly, when secretion follows, more phosphorylation of the Mr 47 000 protein occurs and initially only phosphatidic acid accumulates. At a later stage of the secretion responses, more protein phosphorylation and phosphatidic acid accumulation become evident, and are now accompanied by alterations in the steady state levels of 32P-labelled (poly)phosphoinositides. Hence, the early increase in energy consumption coincides with protein phosphorylation and, at a later stage, with alterations in (poly)phosphoinositides metabolites. This demonstrates that metabolic energy is directly involved in stimulus-response coupling in aggregating platelets.     

Uncoupling protein in human brown adipose tissue mitochondria. Isolation and detection by specific antiserum

A protein of Mr 32 000 has been isolated from human infant brown adipose tissue mitochondria following the procedure used to purify the uncoupling protein from rat brown adipose tissue mitochondria. A specific antiserum has been raised against the human 32 kDa protein, and used to detect it by probing mitochondrial proteins separated by SDS-PAGE. The protein is present in large amounts in brown adipose tissue but is undetectable in human liver, heart or white adipose tissue. It has strong immunological cross-reactivity with rat brown adipose tissue uncoupling protein.     

Brown fat activity in fasted and refed rats

Four days of fasting in the rat reduced brown-adipose-tissue (BAT) mass, mitochondrial protein, and tissue protein content. Specific binding of guanosine diposphate (GDP) to BAT mitochondria was depressed by 55% in 4d-fasted rats. Rats fasted for 3 d, and then refed a single carbohydrate meal (40 kJ), showed a significant increase in specific GDP-binding (27% above fasted) 24 h later, and a large increase in total binding. Specific activities of cytochrome oxidase and -glycerophosphate dehydrogenase in BAT mitochondria were not significantly affected by fasting or refeeding. These results suggest that BAT may be partly responsible for the fall in metabolic rate associated with fasting and the delayed increase after carbo-hydrate refeeding. These effects may be due to changes in the mitochondrial proton-conductance pathway in brown fat.     

Insulin binds to and promotes the phosphorylation of a Mr 210 000 component of its receptor in detergent extracts of rat liver microsomes

Insulin in the presence of Mn2+ and [gamma 32P]ATP promoted the phosphorylation of two proteins of Mr 95 000 and Mr 210 000 in detergent extracts of rat liver microsomes. The Mr 210 000 protein was identified as a component od the insulin receptor by immunoprecipitation. It also bound [125I]insulin specifically, was phosphorylated largely on a tyrosine residue and could not be cleaved to smaller subunits under extreme reducing conditions. The Mr 210 000 protein appears to be a component of a sub-population of liver membrane insulin receptors in which insulin-binding and insulin-stimulated tyrosine kinase phosphorylation site(s) reside in a single polypeptide chain.