Arginine rich coconut kernel protein modulates diabetes in alloxan treated rats

Diabetes mellitus is a syndrome characterized by the loss of glucose homeostasis due to several reasons. In spite of the presence of known anti-diabetic medicines in the pharmaceutical market, remedies from natural resources are used with success to treat this disease. The present study was undertaken to investigate the effect of coconut kernel protein (CKP) on alloxan induced diabetes in Sprague-Dawley rats. Diabetes was induced by injecting a single dose of alloxan (150mg/kg body weight) intraperitoneally. After inducing diabetes, purified CKP isolated from dried coconut kernel was administered to rats along with a semi synthetic diet for 45 days. After the experimental period, serum glucose, insulin, activities of different key enzymes involved in glucose metabolism, liver glycogen levels and the histopathology of the pancreas were evaluated. The amount of individual amino acids of CKP was also determined using HPLC. Results showed that CKP has significant amount of arginine. CKP feeding attenuated the increase in the glucose and insulin levels in diabetic rats. Glycogen levels in the liver and the activities of carbohydrate metabolizing enzymes in the serum of treated diabetic rats were reverted back to the normal levels compared to that of control. Histopathology revealed that CKP feeding reduced the diabetes related pancreatic damage in treated rats compared to the control. These results clearly demonstrated the potent anti-diabetic activity of CKP which may be probably due to its effect on pancreatic β cell regeneration through arginine.     

Isolation and characterization of nuclei from rice embryos.

A method has been developed to isolate pure preparations of nuclei in high yield from commercially available viable rice embryos (germ), employing extraction with buffer solution containing glycerol (without detergent) and polyamine, followed by centrifugation on a 30% Percoll cushion. The intactness of the isolated nuclei was confirmed by light microscopy as well as electron microscopy. The protein profiles of both whole nuclei and nuclear extracts obtained by SDS-PAGE, organellar marker enzyme activities, DNA and RNA analyses, and in vitro RNA synthesis, all indicate that the highly purified nuclei are isolated from rice embryos.     

Synaptosomes isolated from Goto-Kakizaki diabetic rat brain exhibit increased resistance to oxidative stress: role of vitamin E

Increased oxidative stress is believed to be an important factor in the development of diabetic complications. In this study, the effect of diabetes on the susceptibility of synaptosomes to oxidative stress, induced by the oxidizing system ascorbate/Fe2+, on the activity of antioxidant enzymes and on the levels of glutathione and vitamin E was investigated. Synaptosomes were isolated from brain of 29-weeks-old Goto-Kakizaki (GK) rats, a model of non-insulin dependent diabetes mellitus and from normal Wistar rats. Synaptosomes isolated from GK rats displayed a lower susceptibility to lipid peroxidation, as assessed by quantifying thiobarbituric acid reactive substances (TBARS), than normal rats (5.33 +/- 0.79 and 7.58 +/- 0.7 nmol TBARS/mg protein, respectively). In the absence of oxidants, no significant differences were found between the levels of peroxidation in synaptosomes of diabetic or control rats. Superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase activities were unaltered in the brain of diabetic rats. There were no statistically significant differences in fatty acid composition of total lipids and reduced glutathione levels in synaptosomes of diabetic and control rats. The decreased susceptibility to membrane lipid peroxidation of diabetic rats synaptosomes correlated with a 1.3-fold increase in synaptosomal vitamin E levels. Vitamin E levels in plasma were also higher in diabetic rats (21.32 micromol/l) as compared to normal rats (15.13 micromol/l). We conclude that the increased resistance to lipid peroxidation in GK rat brain synaptosomes may be due to the increased vitamin E content, suggesting that diabetic animals might develop enhanced defense systems against brain oxidative stress.     

Nuclear thiols: technical limitations on the determination of endogenous nuclear glutathione and the potential importance of sulfhydryl proteins

Significant discrepancies were found between the values for glutathione levels determined by the Tietze enzymatic assay and those measured by labeling with monobromobimane followed by HPLC analysis when these methods were applied to proliferating and quiescent cells of the 66 murine mammary tumor line depleted of glutathione by buthionine sulfoximine or to nuclei prepared from these cells by permeabilization with Nonident detergent. The probable origin of the discrepancy was traced to the presence of acid-soluble sulfhydryl proteins in the extracts which are thought to lead to erroneous values in the Tietze assay method. Using the monobromobimane-HPLC method it was found that the low-molecular-weight thiol levels in nuclei prepared by detergent permeabilization equilibrate in less than 1 min with the permeabilizing medium, indicating that (i) endogenous nuclear glutathione levels cannot be determined reliably using conventional methods of cellular disruption and (ii) the endogenous nuclear glutathione level is likely to be the same as the cytoplasmic value. The levels of protein sulfhydryl associated with the nuclear preparations were found to be of the same magnitude as the cytoplasmic GSH level and must therefore be considered a potentially significant source of thiol capable of repairing DNA radicals.     

The Protective Effect of Vanadium Against Diabetic Cataracts in Diabetic Rat Model

The present study was designed to investigate the effect of vanadium in alloxan-induced diabetes and cataract in rats. Different doses of vanadium was administered once daily for 8 weeks to alloxan-induced diabetic rats. To know the mechanism of action of vanadium, lens malondialdehyde (MDA), protein carbonyl content, activity of superoxide dismutase (SOD), activities of aldose reductase (AR), and sorbitol levels were assayed, respectively. Supplementation of vanadium to alloxan-induced diabetic rats decreased the blood glucose levels due to hyperglycemia, inhibited the AR activity, and delayed cataract progression in a dose-dependent manner. The observed beneficial effects may be attributed to polyol pathway activation but not decreased oxidative stress. Overall, the results of this study demonstrate that vanadium could effectively reduce the alloxan-induced hyperglycemia and diabetic cataracts in rats.     

Effect of diabetes and Sorbinil treatment on phospholipid metabolism in rat glomeruli

To explore the hypothesis that changes in membrane phospholipids accompany tissue myo-inositol depletion and reduced (Na+ + K+)-ATPase activity in diabetes, we examined phospholipid concentrations in glomeruli isolated from control and streptozotocin-diabetic rats and the effect of diabetes on myo-[3H]inositol incorporation in vitro into glomerular phosphatidylinositol. Since the aldose reductase inhibitor, Sorbinil, prevents the fall in myo-inositol and the decrease in (Na+ + K+)-ATPase activity associated with diabetes, phospholipid and phosphatidylinositol content were also examined in glomeruli isolated from Sorbinil-treated diabetic rats. Total phospholipids (microgram phosphorus/mg dry weight) did not differ in the three groups of animals. The concentration of phosphatidylcholine was elevated in preparations from diabetic rats, both untreated and Sorbinil-treated. Phosphatidylethanolamine was reduced in glomeruli from Sorbinil-treated rats. Neither acute experimental diabetes nor Sorbinil treatment produced detectable changes in the glomerular concentration of phosphatidylinositol. In vitro incubations with glomeruli isolated from control and diabetic animals resulted in increased levels of incorporation of myo-[3H]inositol into phospholipids of diabetic glomeruli. The specific activity of [3H]phosphatidylinositol in glomeruli from diabetic rats was significantly greater than that in control samples. The findings do not support the postulate invoking correspondent changes in myo-inositol and phosphatidylinositol contents as contributory to diminished glomerular (Na+ + K+)-ATPase activity in diabetes, but are compatible with depletion of glomerular intracellular myo-inositol in diabetes.     

Effect of streptozotocin diabetes on motor and inhibitory transmission in rat anococcygeus.

The effect of streptozotocin diabetes of 4-week duration on the adrenergic motor transmission and on the nonadrenergic, noncholinergic, inhibitory transmission in the rat anococcygeus was investigated by recording contractile and relaxant activity of isolated muscle preparations taken from diabetic and age-matched control animals. The neurogenic contractile responses to electrical field stimulation were significantly reduced in the preparations from diabetic rats. The inhibitory transmission remained unaffected in the diabetic rats. Concentration--response curves showed no change in sensitivity of the diabetic anococcygei to noradrenaline. The maximum tension generated was also similar in preparations from diabetic and nondiabetic animals. The contractile responses to electrical field stimulation were significantly greater in preparations from diabetic rats treated for 4 weeks with either sorbinil (20 mg.kg-1.day-1 orally) or myo-inositol (667 mg.kg-1.day-1 orally) when compared with the untreated diabetic controls; the sensitivity to noradrenaline was identical in all three groups. It is concluded that streptozotocin diabetes causes a significant reduction of adrenergic contractile responses of the anococcygeus to electrical field stimulation by a prejunctional mechanism, and the reduction can be prevented by treating the animals with the aldose reductase inhibitor sorbinil or with myo-inositol.     

Decreased susceptibility to lipid peroxidation of Goto-Kakizaki rats: relationship to mitochondrial antioxidant capacity.

The respiratory function and the antioxidant capacity of liver mitochondrial preparations isolated from Goto-Kakizaki non-insulin dependent diabetic rats and from Wistar control rats, with the age of 6 months, were compared. It was found that Goto-Kakizaki mitochondrial preparations presented a higher coupling between oxidative and phosphorylative systems, compared to non-diabetic preparations. Goto-Kakizaki mitochondria presented a lower susceptibility to lipid peroxidation induced by ADP/Fe2+, as evaluated by the formation of thiobarbituric acid substances. The decreased susceptibility to peroxidation in diabetic rats was correlated with an increase in mitochondrial vitamin E (alpha-tocopherol) content and GSH/GSSG ratio. Moreover, the glutathione reductase activity was significantly increased, whereas the glutathione peroxidase was decreased. Superoxide dismutase activity was unchanged in diabetic rats. Fatty acid analyses showed that the content in polyunsaturated fatty acids of Goto-Kakizaki mitochondrial membranes was significantly higher compared to controls. These results indicate that the lower susceptibility to lipid peroxidation of mitochondria from diabetic rats was related to their antioxidant defense systems, and may correspond to an adaptative response of the cells against oxidative stress in the early phase of diabetes.     

Alanine and glutamine synthesis and release from skeletal muscle. III. Dietary and hormonal regulation.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Alanine release from skeletal muscle was increased by fasting (65%), cortisone (145%), thyroxine (200%), and diabetes (185%). Glutamine release was decreased by cortisone (37%) and diabetes (23%) but not significantly affected by fasting or thyroxine. Tissue levels of alanine were unchanged but tissue glutamine levels were markedly reduced (30 to 60%) in all treatment groups. Insulin added in vitro did not affect amino acid release even with preparations obtained from diabetic animals. Inhibition of glycolysis with 0.2 mM iodoacetate had no effect on the rate of alanine and glutamine formation in any treatment group. Pyruvate generation was increased by all treatments even in the presence of the inhibitor. Total skeletal muscle alanine, aspartate, and branched chain aminotransferase, glutamate dehydrogenase, and malic enzyme activities were not significantly altered in any treatment groups. The addition of 10 mM aspartate, cysteine, branched chain amino acids, and serine significantly increased alanine formation, whereas the maximal rate of glutamine formation in the presence of stimulating amino acids was reduced in each treatment groups--the most marked effects were noted with cortisone and diabetic preparations. Although accelerated muscle proteolysis is an important factor regulating alanine formation in skeletal muscle, the redirection of carbon flow from glutamine toward alanine formation observed in fasting, cortisone, thyroxine-treated, and diabetic rats, indicates that factors other than proteolysis also participate in the control of amino acid release from muscle.     

Alterations in adenylate cyclase activity due to streptozotocin-induced diabetic cardiomyopathy

The effect of chronic experimental diabetes on the adenylate cyclase system (AC) in the rat heart was investigated. Rats were made diabetic by an intravenous injection of streptozotocin (65 mg/kg), hearts were removed 8 weeks later and washed cell particles were isolated. AC activity was measured in the absence and presence of different concentrations of forskolin, NaF, GTP analogue [Gpp(NH)p] or epinephrine. A significant depression in the epinephrine stimulated AC activity was observed in diabetic hearts. Basal AC activity and stimulation of AC with forskolin, NaF and Gpp(NH)p were not significantly different between control and diabetic preparations. These results indicate no apparent alterations in the regulatory or catalytic properties of AC in hearts from chronic diabetic rats. The observed depression in epinephrine stimulated AC activity may account for the depressed inotropic action of catecholamines in the diabetic cardiomyopathy.     

Ameliorating effect of eugenol on hyperglycemia by attenuating the key enzymes of glucose metabolism in streptozotocin-induced diabetic rats

Epidemiological studies have demonstrated that diabetes mellitus is a serious health burden for both governments and healthcare providers. This study was hypothesized to evaluate the antihyperglycemic potential of eugenol by determine the activities of key enzymes of glucose metabolism in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced into male albino Wistar rats by intraperitoneal administration of STZ (40 mg/kg body weight (b.w.)). Eugenol was administered to diabetic rats intragastrically at 2.5, 5, and 10 mg/kg b.w. for 30 days. The dose 10 mg/kg b.w. significantly reduced the levels of blood glucose and glycosylated hemoglobin (HbA1c) and increased plasma insulin level. The altered activities of the key enzymes of carbohydrate metabolism such as hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and liver marker enzymes (AST, ALT, and ALP), creatine kinase and blood urea nitrogen in serum and blood of diabetic rats were significantly reverted to near normal levels by the administration of eugenol. Further, eugenol administration to diabetic rats improved body weight and hepatic glycogen content demonstrated the antihyperglycemic potential of eugenol in diabetic rats. The present findings suggest that eugenol can potentially ameliorate key enzymes of glucose metabolism in experimental diabetes, and it is sensible to broaden the scale of use of eugenol in a trial to alleviate the adverse effects of diabetes.     

Vanadyl Sulfate Administration Protects the Streptozotocin-Induced Oxidative Damage to Brain Tissue in Rats

Diabetes mellitus manifests itself in a wide variety of complications and the symptoms of the disease are multifactorial. The present study was carried out to investigate the effects of vanadyl sulfate on biochemical parameters, enzyme activities and brain lipid peroxidation, glutathione and nonenzymatic glycosylation of normal- and streptozotocin-diabetic rats. Streptozotocin (STZ) was administered as a single dose (65 mg/kg) to induce diabetes. A dose of 100 mg/kg vanadyl sulfate was orally administered daily to STZ-diabetic and normal rats, separately until the end of the experiment, at day 60. In STZ-diabetic group, blood glucose, serum sialic and uric acid levels, serum catalase (CAT) and lactate dehydrogenase (LDH) activities, brain lipid peroxidation (LPO) and nonenzymatic glycosylation (NEG) increased, while brain glutathione (GSH) level and body weight decreased. In the diabetic group given vanadyl sulfate, blood glucose, serum sialic and uric acid levels, serum CAT and LDH activities and brain LPO and NEG levels decreased, but brain GSH and body weight increased.The present study showed that vanadyl sulfate exerted antioxidant effects and consequently may prevent brain damage caused by streptozotocin-induced diabetes.     

Selenium treatment protects diabetes-induced biochemical and ultrastructural alterations in liver tissue

We have shown that a single dose of streptozotocin (STZ) (50 mg/kg body weight) injected into rats caused significant changes in some antioxidant enzyme activities, such as glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities, and acid-soluble sulfhydryl levels of the liver tissue with respect to the control rats. Furthermore, these alterations in the activities of the antioxidant enzymes were accompanied by significant changes in the ultrastructure of the liver tissue; mainly intercellular biliary canaliculi were distended and contained stagnant bile, swollen mitochondria in hepatocytes and disoriented and disintegrating cristae, dilatation of the rough endoplasmic reticulum (rER) with detachment of ribosomes, and dissociation of polysomes. Both diabetic and normal rats were treated with sodium selenite (5 micromol/kg/d, intra peritoneally) for 4 wk following 1 wk of diabetes induction. This treatment of diabetic rats improved significantly diabetes-induced alterations in liver antioxidant enzymes. Moreover, treating of diabetic rats with sodium selenite prevented primarily the variation in staining quality of hepatocytes nuclei, increased density and eosinophilia of the cytoplasm, focal sinusoidal dilatation and congestion, and increased numbers of mitochondria with different size and shape. In summary, treatment of diabetic rats with sodium selenite has beneficial effects on both antioxidant system and the ultrastructure of the liver tissue. These findings suggest that diabetes-induced oxidative stress can be responsible for the development of diabetic complications and antioxidant treatment can protect the target organs against diabetes.     

Sodium Tungstate Administration Ameliorated Diabetes-Induced Electrical and Contractile Remodeling of Rat Heart without Normalization of Hyperglycemia

Recently, sodium tungstate was suggested to improve cardiac performance of diabetic rats in perfused hearts based on its insulinomimetic activity. In this study, we aimed to investigate the cellular and molecular mechanisms underlying this beneficial effect of sodium tungstate. Tungstate was administered (100 mg/kg/day) to diabetic and control rats intragastrically for 6 weeks. Blood glucose levels increased, whereas body weight, heart weight and plasma insulin levels decreased significantly in diabetic animals. Interestingly, none of these parameters was changed by tungstate treatment. On the other hand, fractional shortening and accompanying intracellular Ca(2+) [Ca(2+)](i) transients of isolated ventricular myocytes were measured, and sodium tungstate was found to improve the peak shortening and the amplitude of [Ca(2+)](i) transients in diabetic cardiomyocytes. Potassium and L-type Ca(2+) currents were also recorded in isolated ventricular cells. Significant restoration of suppressed I (to) and I (ss) was achieved by tungstate administration. Nevertheless, L-type calcium currents did not change either in untreated or treated diabetic rats. Tissue biochemical parameters including TBARS, protein carbonyl content, xanthine oxidase (XO) and xanthine dehydogenase (XDH) were also determined, and diabetes revealed a marked increase in TBARS and carbonyl content which were decreased significantly by tungstate treatment. Conversely, although XO and XDH activities didn't change in untreated diabetic rats, a remarkable but insignificant decrease was detected in treated animals. In conclusion, tungstate treatment improved diabetes-induced contractile abnormalities via restoration of dysregulated [Ca(2+)](i) and altered ionic currents. This beneficial effect is due to antioxidant property of sodium tungstate rather than normalization of hyperglycemia.     

Efficacy of lower doses of vanadium in restoring altered glucose metabolism and antioxidant status in diabetic rat lenses

Vanadium compounds are potent in controlling elevated blood glucose levels in experimentally induced diabetes. However the toxicity associated with vanadium limits its role as therapeutic agent for diabetic treatment. A vanadium compound sodium orthovanadate (SOV) was given to alloxan-induced diabetic Wistar rats in lower doses in combination withTrigonella foenum graecum, a well-known hypoglycemic agent used in traditional Indian medicines. The effect of this combination was studied on lens morphology and glucose metabolism in diabetic rats. Lens, an insulin-independent tissue, was found severely affected in diabetes showing visual signs of cataract. Alterations in the activities of glucose metabolizing enzymes (hexokinase, aldose reductase, sorbitol dehydrogenase, glucose-6-phosphate dehydrogenase) and antioxidant enzymes (glutathione peroxidase, glutathione reductase) besides the levels of related metabolites, [sorbitol, fructose, glucose, thiobarbituric acid reactive species (TBARS) and reduced glutathione (GSH)]were observed in the lenses from diabetic rats and diabetic rats treated with insulin (2 IU/day), SOV (0.6 mg/ml),T. f. graecum seed powder (TSP, 5%) and TSP (5%) in combination with lowered dose of vanadium SOV (0.2 mg/ml), for a period of 3 weeks. The activity of the enzymes, hexokinase, aldose reductase and sorbitol dehydrogenase was significantly increased whereas the activity of glucose-6-phosphate dehydrogenase, glutathione peroxidase and glutathione reductase decreased significantly in lenses from 3 week diabetic rats. Significant increase in accumulation of metabolites, sorbitol, fructose, glucose was found in diabetic lenses. TBARS measure of peroxidation increased whereas the levels of antioxidant GSH decreased significantly in diabetic condition. Insulin restored the levels of altered enzyme activities and metabolites almost to control levels. Sodium orthovanadate (0.6 mg/ml) andTrigonella administered separately to diabetic animals could partially reverse the diabetic changes, metabolic and morphological, while vanadate in lowered dose in combination withTrigonella was found to be the most effective in restoring the altered lens metabolism and morphological appearance in diabetes. It may be concluded that vanadate at lowered doses administered in combination withTrigonella was the most effective in controlling the altered glucose metabolism and antioxidant status in diabetic lenses, these being significant factors involved in the development of diabetic complications, that reflects in the reduced lens opacity     

Inhibitory effects of astragaloside IV on diabetic peripheral neuropathy in rats

Astragaloside IV (AGS-IV), a new glycoside of cycloartane-type triterpene isolated from the root of Astragalus membranaceus (Fisch.) Bunge, has been used experimentally for its potent immune-stimulating, anti-inflammatory, and antioxidative actions. A recent study has shown AGS-IV to be an aldose-reductase inhibitor and a free-radical scavenger. This study examined the effects of AGS-IV on motor nerve conduction velocity (MNCV), tailflick threshold temperature, biochemical indexes, and the histology of the sural nerve after diabetes was induced in rats with 75 mg/kg streptozotocin (STZ). AGS-IV (3, 6, 12 mg/kg, twice a day) was administered by oral gavage for 12 weeks after diabetes was induced. Compared with control (nondiabetic) rats, obvious changes in physiological behaviors and a significant reduction in sciatic MNCV in diabetic rats were observed after 12 weeks of STZ administration. Morphological analysis showed that AGS-IV suppressed a decrease in myelinated fiber area, an increase in myelinated fiber density, and an increase in segmental demyelination in diabetic rats. The protective mechanism of AGS-IV involved a decrease in declining blood glucose concentration and HbA1C levels, and an increase in plasma insulin levels. AGS-IV increased the activity of glutathione peroxidase in nerves, depressed the activation of aldose reductase in erythrocytes, and decreased the accumulation of advanced glycation end products in both nerves and erythrocytes. Moreover, AGS-IV elevated Na+,K+-ATPase activity in both the nerves and erythrocytes of diabetic rats. These results indicate that AGS-IV exerts protective effects against the progression of peripheral neuropathy in STZ-induced diabetes in rats through several interrelated mechanisms.     

Streptozotocin-induced alterations in rat liver Golgi complexes are ameliorated by BMOV [Bis(maltolato)oxovanadium(IV)] activity.

Twenty years ago, we detected the interdependence between structure and function of rat liver Golgi complexes that are characteristic for streptozotocin diabetes, which served us in further investigations as a useful indicator of the effectiveness of drugs we were testing. This work presented results obtained in eight groups of rats (four control and four diabetic) that were administered orally either bis(maltolato)oxovanadium(IV) [BMOV] or maltol alone. The activities of the rat liver Golgi marker enzyme, galactosyltransferase [GalT], as well as the morphology of Golgi complexes were studied in situ using an electron microscope; parallel estimations of vanadium concentration and phospholipid percentage were made in Golgi-rich preparations isolated from the liver. Our main findings were normalization in diabetic animals orally treated with 1.8 mmol BMOV in 0.09 mol NaCl solutions over seven days, which demonstrated an accompanying increase in phosphatidic acid (PA) percentage (p < 0.05) compared to controls. In the diabetic groups, Pearson's test showed a positive double correlation between GalT activity, vanadium concentration, and PA percentage in Golgi-rich membrane preparations from the liver. Additionally, a negative correlation was found between vanadium concentration and phosphatidylcholine percentage in the fractions.     

Activation of liver tryptophan oxygenase by hydrocortisone, hematin and tryptophan in streptozotocin-diabetic rats

This study compared changes in liver tryptophan oxygenase (TPO) activity in response to hydrocortisone, hematin and tryptophan administration to non-diabetic and diabetic (streptozotocin) rats. Hydrocortisone caused similar increases in apoenzyme (inactive), holoenzyme (heme-saturated) and total (holoenzyme + apoenzyme) TPO activities in non-diabetic and diabetic rats. The ability of hematin to increase total TPO activity was significantly less in diabetic rats. The largest differences between diabetic and non-diabetic rats were found with tryptophan which increased total TPO and holoenzyme activities 300% and 650% respectively in non-diabetic rats. However, tryptophan increased both apoenzyme (unchanged in non-diabetic rats) and holoenzyme activities by 300% in diabetic rats. These results indicate that in the diabetic state, the TPO-heme conjugation process is impaired, especially substrate mediated TPO-heme saturation.