Effect of thyroid hormones on the levels of metabolic intermediates in diabetic rat liver

Experimental diabetes results in an inhibition of the glycolytic and lipogenic pathways in rat liver, while treatment of diabetic rats with T3 for four days increases the activity of a number of enzymes linked to lipogenesis. Hepatic metabolites were estimated in control (untreated), control + T3-treated, alloxan-diabetic and alloxan-diabetic + T3-treated rats. Diabetes resulted in the expected decrease in the content of fructose 2,6-bisphosphate and an increase in the content of cyclic AMP and citrate, changes consistent with an inhibition of hepatic glycolysis. Treatment of diabetic rats with T3 did not reverse these changes. There was a marked accumulation of both acetyl CoA and citrate in the diabetic rat liver, which was of even greater magnitude in diabetic and in the T3-treated group. In addition, T3 treatment significantly increased the free CoA content of liver in both normal and diabetic groups. Of the parameters measured which influence lipogenesis, including long chain acyl CoA, the energy charge and redox state of the nicotinamide nucleotides, the raised hepatic citrate content correlated most closely with the known increase in lipogenesis in diabetic rats treated with T3 for a four day period.     

Effect of alloxan diabetes on (Na+ + K+)-activated ATPase in brush border membrane of the mucosal cell of rat small intestine]

In order to elucidate a possible relationship between (Na+ + K+)-activated ATPase and intestinal absorption of actively transported monosaccharides enzyme activity was measured in mucosal cells from alloxan diabetic rats. The general effect of increasing capacity of active, Na+-dependent transport processes in diabetes mellitus is associated with a significantly enhanced (Na+ +K+)-activated ATPase activity in mucosal homogenate from diabetic animals. To study the localization of these effects within the cell we isolated purified brush borders and their substructures. To enable a comparison to be made between preparation procedures of diabetic and control animals the fractions were controlled by electronmicroscopy and by measuring the sucrase activity. In the purified brush border fraction of alloxan treated rats there was no significant increase in (Na+ + K+)-activated ATPase activity. Based on these results we conclude that the (Na+ + K+)-activated ATPase in the basolateral membranes was increased in alloxan diabetes, and it seems very likely that this enzyme is involved in the regulation of Na+-dependent transport processes.     

Oral administration of orthovanadate and Trigonella foenum graecum seed powder restore the activities of mitochondrial enzymes in tissues of alloxan-induced diabetic rats

The effect of oral administration of sodium orthovanadate (SOV) and Trigonella foenum graecum seed powder (TSP), a medicinal plant used extensively in Asia, on the mitochondrial metabolism in the alloxan diabetic rats has been investigated. Rats were injected with alloxan monohydrate (20 mg/100 g body wt) or vehicle (Na-acetate buffer), the former were treated with either 2 IU insulin i.p., 0.6 mg/ml SOV ad libitum, 5% TSP ad libitum, and a combination of 0.2% SOV and 5% TSP ad libitum for 21 days. Selected rate-limiting enzymes of the tricarboxylic acid cycle, hydrogen shuttle system, ketone body metabolism, amino acid metabolism and urea cycle were measured in the mitochondrial and cytosolic fractions of liver, kidney and brain tissues of the experimental rats. Majority of the mitochondrial enzymes in the tissues of the diabetic rats had significantly higher activities compared to the control rats. Similarly, the activities of mitochondrial and cytosolic aminotransferases and arginase were significantly higher in liver and kidney tissues of the diabetic rats. The separate administrations of SOV and TSP to diabetic rats were able to restore the activities of these enzymes to control values. The lower dose of SOV (0.2%) administered in combination with TSP to diabetic rats lowered the enzyme activities more significantly than when given in a higher dose (0.6%) separately. This is the first report of the effective combined action of oral SOV and TSP in ameliorating the altered mitochondrial enzyme activities during experimental type-1 diabetes. Our novel combined oral administration of SOV and TSP to diabetic rats thus conclusively proves as a possible method to minimize potential vanadate toxicity without compromising its positive effects in the therapy of experimental type-1 diabetes.     

AP-PCR detection of Streptococcus mutans and Streptococcus sobrinus in caries-free and caries-active subjects

Dyslipidemia is common in patients with type 2 diabetes. Statins are used as the first choice in treatment of diabetic dyslipidemia. Atorvastatin represents a first-line treatment option, alongside other hydroxyl methylglutaryl coenzyme A reductase inhibitors. Repaglinide is a short-acting, oral, insulin secretagogue that is used in the treatment of type 2 diabetes mellitus. Both the category of drugs undergo extensive metabolism with cytochrome enzyme system. This may lead to drug-drug interaction problems with altered repaglinide activity which is cautious. Repaglinide/atorvastatin/atorvastatin + repaglinide were administered orally to normal, diabetic rats, and to normal rabbits. Blood samples were collected at different time intervals and were analyzed for blood glucose by GOD-POD method using commercial glucose kits and repaglinide estimation in plasma by HPLC method. Diabetes was induced by alloxan 100 mg/kg body weight administered by I.P route. In the presence of atorvastatin, repaglinide activity was increased and maintained for longer period in diabetic rats compared with repaglinide matching control. The present study concludes co-administration of atorvastatin was found to improve repaglinide responses significantly in diabetic rats and improved glucose metabolism of atorvastatin played an important role and increased repaglinide levels by competitive CYP 3A4 enzyme inhibition by atorvastatin could be added advantage for anti hyperglycemic activity.     

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.     

Hepatic and pancreatic effects of polyenoylphosphatidylcholine in rats with alloxan-induced diabetes

Polyenoylphosphatidylcholine (PPC: 100 or 300 mg kg^?1 b.w., by gastric intubation for 30 days) produced a clearcut protection of the liver of rats treated with alloxan (150 mg kg^?1 b.w., i.p.). The liver of rats treated with alloxan was characterized by hydropic dystrophy and lymphocytic infiltrations. Treatment with alloxan increased serum γ-GT and ALAT activities. The liver structure of rats treated with PPC did not differ from the liver of control animals. PPC normalized the biochemical abnormalities caused by the diabetes. The number of pancreatic islets and β/α; cell ratio decreased in the diabetic rats. A number of β-cells in this group did not contain granules. PPC prevented the decrease in the number of islets and the β/α; cell ratio in the pancreas of the diabetic rats. The intensity of staining of β-cell granules in the pancreas of PPC-treated rats had a position intermediate between the control and diabetic groups. Alloxan increased the blood glucose content where treatment with PPC decreased this. The results suggest that PPC acts as a cytoprotector in the liver and pancreas of rats with experimental diabetes induced by alloxan.     

Antioxidant enzyme alterations in experimental and clinical diabetes

Previous studies from our laboratory have demonstrated the presence of complex alterations in the activities of antioxidant enzymes in various tissues of rats with streptozotocin (STZ)-induced diabetes. In the present investigation, it is shown that rats made diabetic with alloxan (ALX), an agent differing from STZ both chemically and in its mechanism of diabetogenesis, show virtually identical tissue antioxidant enzyme changes which, as is the case with STZ, are preventable by insulin treatment. The finding that the patterns of antioxidant enzyme alterations in chemically-induced diabetes are independent of the diabetogenic agent used and the presence of similar abnormalities in tissues of spontaneously diabetic (BB) Wistar rats (particularly when diabetic control is less than optimal) suggest that the changes observed are a characteristic feature of the uncontrolled diabetic state and that these may be responsible for (or predispose to) the development of secondary complications in clinical diabetes. Comparative studies involving red cells of diabetic rats and human diabetics revealed a number of common changes, namely an increase in glutathione reductase activity, a decreased susceptibility to oxidative glutathione depletion (which was related to the presence of hyperglycemia) and an increased production of malondialdehyde (an indirect index of lipid peroxidation) in response to in vitro challenge with hydrogen peroxide. In the diabetic patients, the extent of this increase in susceptibility of red cell lipids to oxidation paralleled the severity of diabetic complications. Our results suggest that increased (or uncontrolled) oxidative activity may play an important role in the pathogenesis of complications associated with the chronic diabetic state.This work was supported by grants from the British Columbia Health Care Research Foundation and the Canadian Diabetes Association.     

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.     

Changes in insulin receptor,hexokinase and NADPH producing enzymes in Choroid plexus during experimental diabetes

The activities of insulin receptor and the enzymes hexokinase (EC 2.7.1.1) and NADP-dependent malic enzyme (EC1.1.1.40), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in rat choroid plexus in alloxan induced diabetes. A significant decrease was observed in the activities of all the enzymes except isocitrate dehydrogenase and also the choroid plexus insulin receptor activity was decreased. A reversal of the efect was observed with insulin administration to diabetic rats. It may be concluded that the enzymes of choroid plexus together with insulin receptor are directly controlled by-the concentration of insulin.     

Combined treatment of sodium orthovanadate and Momordica charantia fruit extract prevents alterations in lipid profile and lipogenic enzymes in alloxan diabetic rats

Momordica charantia Linn., commonly called bitter gourd, is a medicinal plant used in the Ayurvedic system of medicine for treating various diseases including diabetes mellitus. Sodium orthovanadate (SOV) is also well-known insulin mimetic and an antidiabetic compound. Our laboratory has been using reduced doses of SOV along with administration of herbal extracts to alloxan diabetic rats and has established this combination as a good antihyperglycemic agent. The present study was undertaken to investigate the effects of treatment of Momordica fruit extract (MFE) and sodium orthovanadate, separately and in combination, on serum and tissue lipid profile and on the activities of lipogenic enzymes in alloxan induced diabetic rats. The results show that there was a significant (p < 0.01) increase in serum total lipids, triglycerides and total cholesterol levels after 21 days of alloxan diabetes. In the liver and kidney of diabetic rats the levels of total lipids and triglycerides also increased significantly (p < 0.01) while levels of total cholesterol decreased significantly (p < 0.01 and p < 0.05, respectively). The lipogenic enzymes showed decreased activity in the diabetic liver, while in kidney they showed an increased activity. When compared with the controls these changes were significant. The treatment of alloxan diabetic rats with MFE and SOV prevented these alterations and maintained all parameters near control values. Most effective prevention was however observed in a combined treatment of Momordica with a reduced dose of SOV (0.2%). The results suggest that Momordica fruit extract and SOV exhibit hypolipidemic as well as hypoglycemic effect in diabetic rats and their effect is pronounced when administered in combination. (Mol Cell Biochem 268: 111–120, 2005)     

Silymarin increases antioxidant enzymes in alloxan-induced diabetes in rat pancreas

The aim of this study was to analyze the effect of the flavonoid silymarin, a free radical scavenger that prevents lipoperoxidation, on the pancreatic activity of superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase (CAT) in rats with alloxan-induced diabetes mellitus. Alloxan intoxicated rats were treated with silymarin in two manners, simultaneously (four or eight doses) or 20 days after alloxan administration for 9 weeks. Alloxan elicited a transient increase in the activity of the three enzymes, which decreased after 5 days of treatment. On its own, silymarin significantly increased the activity of these enzymes. Simultaneous treatment with alloxan and silymarin also induced an increment in the activity of the enzymes followed by a delayed decrease (four doses). However, a longer treatment with silymarin (eight doses) induced a more sustained effect. Interestingly, silymarin treatment recovered to control values for the activity of the three-antioxidant enzymes that were significantly diminished after 20 days of alloxan administration. It is suggested that the protective effect of silymarin on pancreatic damage induced by alloxan may be due to an increase in the activity of antioxidant enzymes that, in addition to the glutathione system, constitute the more important defense mechanisms against damage by free radicals.     

Role of ubiquitin-proteasome-dependent proteolytic process in degradation of muscle protein from diabetic rabbits.

The activity of ATP, ubiquitin (Ub)-dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ub-dependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ub-dependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ub-proteasome-dependent proteolysis have an important role in the control of muscle protein degradation and may be regulated by insulin.     

A comparison of the effects of diabetes induced with either alloxan or streptozotocin and of starvation on the activities in rat liver of the key enzymes of gluconeogenesis

1. Measurements of the activities in rat liver of the four key enzymes involved in gluconeogenesis, i.e. pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), have been carried out, all four enzymes being measured in the same liver sample. Changes in activities resulting from starvation and diabetes have been studied. Changes in concentration (activity/unit wet weight of tissue) were compared with changes in the hepatic cellular content (activity/unit of DNA). 2. Each enzyme was found to increase in concentration during starvation for up to 3 days, but only glucose 6-phosphatase and phosphoenolpyruvate carboxykinase showed a significant rise in content. Fructose 1,6-diphosphatase appeared to decrease in content somewhat during the early stages of starvation. 3. There was a marked increase in the concentration of all four enzymes in non-starved rats made diabetic with alloxan or streptozotocin, for the most part similar responses being found for the two diabetogenic agents. On starvation, however, the enzyme contents in the diabetic animals tended to fall, often with streptozotocin-treated animals to values no greater than for the normal overnight-starved rat. Deprivation of food during the period after induction of diabetes with streptozotocin lessened the rise in enzyme activity. 4. The results are compared with other published values and factors such as substrate and activator concentrations likely to influence activity in vivo are considered. 5. Lack of correlation of change in fructose 1,6-diphosphatase with the other enzymes questions whether it should be included in any postulation of control of gluconeogenic enzymes by a single gene unit.     

Action of insulin on liver carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) activity

Carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) (EC 6.3.5.5) (synthetase II), is the first and rate-limiting enzyme in the de novo UMP biosynthetic pathway. The present investigation showed that insulin has a regulatory action on hepatic synthetase II activity. When diabetes was induced with injection of different doses of alloxan the plasma insulin concentrations decreased in a dose-dependent fashion to 72, 38, 31 and 28% and concurrently the liver synthetase II activity decreased to 75, 43, 29 and 22% of the normal values. In diabetic rats dose response studies showed that with insulin injections of 4, 6, 8 or 10 U/day for 48 h the hepatic synthetase II activity increased to 81, 95, 99 and 103% of the control liver values. In the diabetic rats the insulin-induced rise in liver synthetase II activity was prevented by treatment of the rats with actinomycin.     

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     

Increased degradation of dermal collagen in diabetic rats.

The effect of alloxan induced diabetes on the dermal collagen content of albino rats was studied in relation to few lysosomal enzymes. Diabetes decreased the dermal collagen content. The specific activities of the lysosomal enzymes studied in the diabetic rat skin were elevated. It has been established that lysosomal enzymes degrade the connective tissue components. Thus, it may be suggested that the increase in the lysosomal enzymes studied should have facilitated the decrease in dermal collagen content of diabetic rats by increasing the degradation of dermal collagen.     

Alterations of elastin and elastase-like activities in aortae of diabetic rats

The elastin content of the aortic muscle and the elastase-like activity of the extracts of aortic muscle were studied in spontaneously diabetic BB rats and in rats made diabetic by a single bolus i.v. injection of alloxan. In both modes of diabetes, the total alkaline-insoluble aortic elastin content was significantly reduced in diabetic rats compared to that in the corresponding control rats. This reduction in aortic elastin was also accompanied by a consistent increase in the elastase-like activities of the aortic extracts prepared from the same tissues. Such a reciprocal relationship between aortic elastin content and elastase-like activity has previously been reported in rats with malignant hypertension. Since the rats used in this study were not hypertensive, the altered elastin metabolism observed in this work is likely to be a manifestation of diabetic disease and may in part account for the vascular changes associated with diabetes mellitus.     

Influence of antioxidant (L- ascorbic acid) on tolbutamide induced hypoglycaemia/antihyperglycaemia in normal and diabetic rats

BACKGROUND: Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. Increased oxidative stress and decreased antioxidant levels are the leading cause of diabetes and diabetic complications. So it is felt that supplementation of antioxidants may be useful in controlling the glucose levels and to postpone the occurrence of diabetic complications. The objective of our study is to find the influence of antioxidant supplementation (L-ascorbic acid) on tolbutamide activity in normal and diabetic rats. METHODS: L- ascorbic acid/tolbutamide/L-ascorbic acid + tolbutamide were administered orally to 3 different groups of albino rats of either sex in normal and diabetic condition. Blood samples were collected from retro-orbital puncture at different time intervals and were analyzed for blood glucose by GOD-POD method. Diabetes was induced by alloxan 100 mg/kg body weight administered by I.P route. RESULTS: L-ascorbic acid/ tolbutamide produced hypoglycaemic activity in a dose dependant manner in normal and diabetic condition. In the presence of L-ascorbic acid, tolbuatmide produced early onset of action and maintained for longer period compared to tolbutamide matching control. CONCLUSION: Supplementation of antioxidants like L-ascorbic acid was found to improve tolbutamide response in normal and diabetic rats.     

Effects of diabetes and glycogen on the distribution between soluble and particulate fractions of activities of enzymes involved in hepatic glycogen metabolism.

The large decreases in hepatic glycogen associated with alloxan diabetes in fed rats were accompanied by apparent decreases in total activities of glycogen synthase, phosphorylase, protein kinase and synthase phosphatase determined on 8000 × g supernatants of liver homogenates. Inclusion of 4% glycogen in the extraction buffer normalized total soluble activities of synthase in the diabetic. Whereas inclusion of 4% glycogen in the extraction buffer doubled total soluble phosphorylase, total activity remained lower in the diabetic than in the normal. Extraction and assay of soluble protein kinase were unaffected by added glycogen. When activities were determined on whole homogenates, total glycogen synthase activities were the same in normal and diabetic liver. Although the decreases in total activities of phosphorylase, kinase and phosphatase were less when determined on whole homogenates of livers from diabetic rats, the diabetes-related decreases in total activities remained significant. Therefore, it appears that while alloxan diabetes results in absolute decreases in total hepatic activities of phosphorylase, kinase and phosphatase, it may also result in redistribution of hepatic synthase and phosphorylase between soluble and particulate fractions, a phenomenon possibly related to tissue glycogen concentrations. Such a redistribution might be involved in the lack of control of hepatic glycogenesis observed in alloxan diabetic rats.     

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats. (Mol Cell Biochem141: 97–102, 1994)