Regional variations in intestinal brush border membrane fluidity and function during diabetes and the role of oxidative stress and non-enzymatic glycation

The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and -glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.     

Decreased fluidity of isolated erythrocyte membranes in type 1 and type 2 diabetes. The effect of resorcylidene aminoguanidine.

Changes in the physico-chemical properties of erythrocyte membranes induced by nonenzymatic glycation as well as the possible prevention of their rise were studied. Using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescence anisotropy values were determined in erythrocyte membranes isolated from type 1 and type 2 diabetic patients with and without complications. The mean anisotropy values for the groups of diabetic patients were significantly higher than those for the control group (p < 0.01). This indicated pathologically decreased fluidity in cell membranes in the diabetics regardless of the type of diabetes or the presence of complications. The fluorescence anisotropy positively correlated (p < 0.01) with clinical parameters, such as glycohaemoglobin and plasma cholesterol content, which are important for the monitoring of the compensation status of the diabetic patient. Our results support the suggestion that protein crosslinking and oxidative stress induced by nonenzymatic glycation contribute to changes in the physico-chemical properties of erythrocyte membranes. In vitro testing of a new potential drug resorcylidene aminoguanidine (RAG) showed its ability to increase significantly (p < 0.001), to various extent (p < 0.01), the fluidity of both diabetic and control erythrocyte membranes. Upon the administration of RAG, reduced fluorescence anisotropy values for the groups of diabetic patients approached the normal values obtained for the controls. This may play an important role in the improvement of impaired cell functions found in diabetes that are controlled by the cell membrane.     

Implication of BBM lipid composition and fluidity in mitigated alkaline phosphatase activity in renal cell carcinoma

Previous study has documented reduced alkaline phosphatase (ALP) activity in brush border membrane (BBM) isolated from renal cell carcinoma (RCC). Diminished activity of ALP is associated with alteration in both increased K (m) as well as decreased V (max) of enzyme suggests that there may be a change in the conformation of enzyme as well as decreased number of ALP active molecules. The present study was conducted to find out any role of BBM lipid composition and its fluidity in diminished activity of alkaline phosphatase in renal cell carcinoma. Total phospholipids and glycolipids were significantly augmented in BBM from RCC as compared to control. Fractional analysis of total phospholipids revealed significantly increased phosphatidylethanolamine. Decreased fractions of sphingomyelin and phosphatidylinositol were observed. Cholesterol-to-total phospholipid molar ratios in tumor BBM was a significantly lower in tumor BBM. A significant reduction in polarization and microviscosity was found in BBM from RCC. Therefore, we conclude that alteration in membrane lipid composition and fluidity may play a substantial role in reduced activity of ALP in RCC.     

Action of pelargonidin on hyperglycemia and oxidative damage in diabetic rats: implication for glycation-induced hemoglobin modification

Glycation-modified hemoglobin in diabetes mellitus has been suggested to be a source of enhanced catalytic iron and free radicals causing pathological complications. The present study aims to verify this idea in experimental diabetes. Pelargonidin, an anthocyanidin, has been tested for its antidiabetic potential with emphasis on its role against pathological oxidative stress including hemoglobin-mediated free radical reactions. Male wistar rats were grouped as normal control, streptozotocin-induced diabetic control, normal treated with pelargonidin and diabetic treated with pelargonidin. Pelargonidin-treated rats received one time i.p injection of the flavonoid (3 mg/kg bodyweight). Biochemical parameters were assayed in blood samples of different groups of rats. Liver was used for histological examinations. Pelargonidin treatment normalized elevated blood glucose levels and improved serum insulin levels in diabetic rats. Glucose tolerance test appeared normal after treatment. Decreased serum levels of SOD and catalase, and increased levels of malondialdehyde and fructosamine in diabetic rats were reverted to their respective normal values after pelargonidin administration. Extents of hemoglobin glycation, hemoglobin-mediated iron release, iron-mediated free radical reactions and carbonyl formation in hemoglobin were pronounced in diabetic rats, indicating association between hemoglobin glycation and oxidative stress in diabetes. Pelargonidin counteracts hemoglobin glycation, iron release from the heme protein and iron-mediated oxidative damages, confirming glycated hemoglobin-associated oxidative stress in diabetes.     

Resveratrol protects diabetic kidney by attenuating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via Nrf2-Keap1 signaling

Hyperglycemia-mediated oxidative stress plays a crucial role in the progression of diabetic nephropathy. Hence, the present study was hypothesized to explore the renoprotective nature of resveratrol by assessing markers of oxidative stress, proinflammatory cytokines and antioxidant competence in streptozotocin-nicotinamide-induced diabetic rats. Oral administration of resveratrol to diabetic rats showed a significant normalization on the levels of creatinine clearance, plasma adiponectin, C-peptide and renal superoxide anion, hydroxyl radical, nitric oxide, TNF-α, IL-1β, IL-6 and NF-κB p65 subunit and activities of renal aspartate transaminase, alanine transaminase and alkaline phosphatase in comparison with diabetic rats. The altered activities of renal aldose reductase, sorbitol dehydrogenase and glyoxalase-I and elevated level of serum advanced glycation end products in diabetic rats were also reverted back to near normalcy. Further, resveratrol treatment revealed a significant improvement in superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase activities and vitamins C and E, and reduced glutathione levels, with a significant decline in lipid peroxides, hydroperoxides and protein carbonyls levels in diabetic kidneys. Similarly, mRNA and protein analyses substantiated that resveratrol treatment notably normalizes the renal expression of Nrf2/Keap1and its downstream regulatory proteins in the diabetic group of rats. Histological and ultrastructural observations also evidenced that resveratrol effectively protects the kidneys from hyperglycemia-mediated oxidative damage. These findings demonstrated the renoprotective nature of resveratrol by attenuating markers of oxidative stress in renal tissues of diabetic rats.     

Inhibition of intestinal and renal Na+-glucose cotransporter by naringenin

Reduction in glucose uptake constitutes a possible means of controlling diabetic hyperglycemia. Using purified intestinal brush border membrane vesicles and everted intestinal sleeves, we have demonstrated that naringenin, a flavonoid present in citrus fruits and juices, significantly inhibited glucose uptake in the intestine. In addition, naringenin also elicited inhibitory actions towards glucose uptake in renal brush border membrane vesicles. Naringin, a glycoside of naringenin, was totally inactive in these aspects. Naringenin exhibited moderate inhibitory action on glucose uptake in rabbit intestinal brush border membrane vesicles, and showed strong inhibitory action in rat everted intestinal sleeves. The IC(50) values were 205.9 and 2.4 micromol/l, respectively. Lineweaver-Burk analysis demonstrated that naringenin inhibited glucose uptake in rat everted intestinal sleeves in a competitive manner with a K(i) value of 1.1 micromol/l. Glucose uptake activities in both the intestinal and renal brush border membrane vesicles of diabetic rats were significantly higher than in normal rats. Naringenin (500 microM) reduced glucose uptake by more than 60% in both the intestinal and renal brush border membrane vesicles of diabetic rats to a level similar to that of the normal rats. The IC(50) values of naringenin in the renal brush border membrane vesicles of normal and diabetic rats were 323.9 and 166.1 micromol/l, respectively. These results suggest that inhibition of intestinal glucose uptake and renal glucose reabsorption explains, in part at least, the in vivo antihyperglycemic action of naringenin and its derivatives. The possible application of these natural compounds in controlling hyperglycemia warrants further investigations.     

Influence of dietary partially hydrogenated fat high in trans fatty acids on lipid composition and function of intestinal brush border membrane in rats

The effect of dietary hydrogenated fat (Indian vanaspati) high in trans fatty acids (6 en%) on lipid composition, fluidity and function of rat intestinal brush border membrane was studied at 2 and 8 en% of linoleic acid. Three groups of weanling rats were fed rice-pulse based diet containing 10% fat over a ten week period: Group I (groundnut oil), Group II (vanaspati), Group III (vanaspati + safflower oil). The functionality of the brush border membrane was assessed by the activity of membrane bound enzymes and transport of D-glucose and L-leucine. The levels of total cholesterol and phospholipids were similar in all groups. The data on fatty acid composition of membrane phospholipids showed that, at 2 en% of linoleic acid in the diet, trans fatty acids lowered arachidonic acid and increased linoleic acid contents indicating altered polyunsaturated fatty acid metabolism. Alkaline phosphatase activity was increased while the activities of sucrase, gamma-glutamyl transpeptidase and transport of D-glucose and L-leucine were not altered by dietary trans fatty acids. However at higher intake of linoleic acid in the diet, trans fatty acids have no effect on polyunsaturated fatty acid composition and alkaline phosphatase activity of intestinal brush border membrane. These data suggest that feeding dietary fat high in trans fatty acids is associated with alteration in intestinal brush border membrane polyunsaturated fatty acid composition and alkaline phosphatase activity only when the dietary linoleic acid is low.     

Advanced glycation end products (AGEs), free radicals and diabetes

Nonenzymatic glycation, i.e. binding of monosaccharides to amino groups of proteins, gives rise to complex components called "advanced glycation end-products" (AGEs), which alter protein structure and functions, and participate in diabetic long-term complications. Glycation and oxidative stress are closely linked, and are often referred to as "glycoxidation" processes. Experimental data support these interactions. a) All glycation steps generate oxygen free radicals, some of these steps being common with these of lipid peroxidation. b) AGEs bind to membrane receptors such as RAGE, and induce an oxidative stress and a pro-inflammatory status. c) Glycated proteins modulate cellular oxidative functions: glycated collagens induce an inappropriate oxidative response in PMNs. d) Products of lipid peroxidation (MDA) bind to proteins and amplify glycoxidation-induced damages. Glycoxydation intensity increases in diabetes mellitus, ageing, renal failure and other pathological states with oxidative stress. Therapies aiming at limiting glycoxidation take into account its oxidative part.     

Overexpression of glyoxalase-I reduces hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ～50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.     

The Effects of Iron-Mediated Oxidative Stress in Isolated Renal Cortical Brush Border Membrane Vesicles at Normothermic and Hypothermic Temperatures

Experiments on renal cortical brush border membrane vesicles have been undertaken in order to assess the involvement of iron in oxidative stress at physiological temperatures and under conditions of hypothermia. A decrease in temperature stimulated iron-induced lipid peroxidation. The results are discussed in relation to the role of the oxidation state of the iron and iron(II)/iron(III) ratios in the initiation of peroxidative events.     

The Effects of Iron-Mediated Oxidative Stress in Isolated Renal Cortical Brush Border Membrane Vesicles at Normothermic and Hypothermic Temperatures

Experiments on renal cortical brush border membrane vesicles have been undertaken in order to assess the involvement of iron in oxidative stress at physiological temperatures and under conditions of hypothermia. A decrease in temperature stimulated iron-induced lipid peroxidation. The results are discussed in relation to the role of the oxidation state of the iron and iron(II)/iron(III) ratios in the initiation of peroxidative events.     

Distribution of parathyroid hormone-stimulated adenylate cyclase in plasma membranes of cells of the kidney cortex.

Free flow electrophoresis was employed to separate renal cortical plasma membranes into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. During the separation adenylate cyclase activity was found to parallel the activity of Na+-K+-activated ATPase, an enzyme which is present in contraluminal but not in luminal membranes. In the basal-lateral membrane fraction the specific activities of adenylate cyclase and Na+-K+-activated ATPase were 4.4 and 4.6 times greater, respectively, than in the brush border fraction. The adenylate cyclase of the basal-lateral membrane fraction was specifically stimulated by parathyroid hormone which maximally increased enzyme activity eightfold. The biologically active (1-34) peptide fragment of paratyhroid hormone produced a 350% increase in adenylate cyclase activity. In contrast, calcitonin, epinephrine and vasopressin maximally stimulated the enzyme by only 55, 35 and 30%, respectively. These results indicate that adenylate cyclase, specifically stimulated by parathyroid hormone, is distributed preferentially in the contraluminal region of the plasma membrane of renal cortical epithelial cells.     

Use of phospholipid transfer protein as a probe to study the lipid dynamics and alkaline phosphatase activity in the brush border membrane of human term placenta

Incubation of placental brush border membrane (BBM) along with sonicated vesicles of exogenous lipids (egg yolk PC) in the presence of phospholipid-transfer protein (PL-TP) showed a decrease in the alkaline phosphatase activity due to the change in the membrane micro-environment, such as fluidity. Effect of substrate concentration was tested by Lineweaver-Burk plot, which showed decreased V(max) and K(M). The effect of temperature was probed by the Arrhenius plot, which showed no change in transition temperature, but a decline in the energy of activation both below and above the transition temperature. The protein-catalyzed transfer of phospholipid from the donor unilamellar vesicles resulted in a substantial increase in the BBM phospholipid and a net decrease in cholesterol/phospholipid molar ratio. The change in membrane fluidity was assessed by translational as well as rotational diffusion of membrane extrinsic fluorescent probes, pyrene and diphenyl-hexatriene. An increased lateral mobility was recorded by the increased pyrene excimer formation. A decrease in fluorescent polarization of diphenyl-hexatriene was observed, which led to the decrease in fluorescence anisotropy and order parameter, and therefore, an increase in membrane fluidity (rotational diffusion). Mean anisotropy parameter was also decreased in the presence of PL-TP. Thus, the placental BBM alkaline phosphatase activity showed a distinct lipid dependence which may have important physiological consequences.     

Oxidative and drug-induced alterations in brush border membrane hemileaflet fluidity, functional consequences for glucose transport

Oxidation of biological membranes has been suggested as a major pathological process in a variety of disease states including intestinal ischemia and inflammatory bowel disease. Previous studies on the small intestinal brush border membrane have shown that part of the decrease in the activity of the Na(+)-dependent glucose transporter (SGLT1) observed after oxidation could be secondary to the derangement in membrane fluidity that accompanied oxidative damage. The present study examined the relationship between oxidative-induced hemileaflet fluidity alterations and the resultant change in Na(+)-dependent glucose transport activity. To address this issue, in vitro oxidation of guinea pig brush border membrane vesicles was induced by incubation of the vesicles with ferrous sulfate and ascorbate. We found that oxidation decreased the fluidity of both the outer and inner hemileaflets, the decrease being greater in the outer leaflet. Moreover, the preferential alteration in hemileaflet fluidity was accompanied by a decrease in glucose transport. However, when membrane perturbing agents such as hexanol and A(2)C were used to restore membrane fluidity to levels comparable to controls, rates of glucose transport could not be interpreted in terms of variation of bulk membrane fluidity or variation in fluidity of any specific membrane leaflet. On the basis of these experiments, we propose that previous studies that reported coincidental alteration in membrane fluidity and glucose transport cannot be interpreted on the basis of bulk fluidity or hemileaflet fluidity.     

Effects of piperine on antioxidant pathways in tissues from normal and streptozotocin-induced diabetic rats

Using diabetes mellitus as a model of oxidative damage, this study investigated whether subacute treatment (10 mg/kg/day, intraperitoneally for 14 days) with the compound piperine would protect against diabetes-induced oxidative stress in 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively) content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Piperine treatment of normal rats enhanced hepatic GSSG concentration by 100% and decreased renal GSH concentration by 35% and renal glutathione reductase activity by 25% when compared to normal controls. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with piperine reversed the diabetic effects on GSSG concentration in brain, on renal glutathione peroxidase and superoxide dismutase activities, and on cardiac glutathione reductase activity and lipid peroxidation. Piperine treatment did not reverse the effects of diabetes on hepatic GSH concentrations, lipid peroxidation, or glutathione peroxidase or catalase activities; on renal superoxide dismutase activity; or on cardiac glutathione peroxidase or catalase activities. These data indicate that subacute treatment with piperine for 14 days is only partially effective as an antioxidant therapy in diabetes.     

Antioxidant effect of carnosine treatment on renal oxidative stress in streptozotocin-induced diabetic rats

Nitric oxide (NO) plays a significant role in the development of diabetic nephropathy. We investigated the effects of an antioxidant, carnosine, on streptozotocin (STZ)-induced renal injury in diabetic rats. We used four groups of eight rats: group 1, control; group 2, carnosine treated; group 3, untreated diabetic; group 4, carnosine treated diabetic. Kidneys were removed and processed, and sections were stained with periodic acid-Schiff (PAS) and subjected to eNOS immunohistochemistry. Examination by light microscopy revealed degenerated glomeruli, thickened basement membrane and glycogen accumulation in the tubules of diabetic kidneys. Carnosine treatment prevented the renal morphological damage caused by diabetes. Moreover, administration of carnosine decreased somewhat the oxidative damage of diabetic nephropathy. Appropriate doses of carnosine might be a useful therapeutic option to reduce oxidative stress and associated renal injury in diabetes mellitus.     

Effects of lindane on fluidity and lipid composition in rat renal cortex membranes.

The influence of lindane upon dynamic properties of plasma membranes from rat renal cortex has been investigated using a fluorescence polarization technique. Preincubation with lindane increased membrane fluidity in a manner that is dose-dependent. This increase was higher in brush border membranes than in basolateral membranes. However, a significant decrease of the membrane fluidity was found in brush border membranes when rats were injected with lindane for 12 days. A possible solution to this difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the amount of cholesterol and phospholipid classes in the renal cortex membranes of lindane-injected rats.     

Modulation of intestinal brush border membrane chemical composition during postnatal development in rats: effect of gestational diabetes

There was a significant increase in fucose (52%), total hexoses (16%) and hexosamine (56%) except sialic acid, which was reduced (77%) in the microvillus membrane of infants born to rat mothers made diabetic by injecting alloxan on day 3 of gestation. Expressed on the protein basis there were a significant increase in membrane, triglyceride, total cholesterol, and phospholipids content of brush border in pups from diabetic group between 5-45 days of postnatal age. Intestinal morphology in diabetic group showed, regression of tubular glands, distorted cellular organization of mucosal cells, reduction in the mucosal cell height and number of secretory goblet cells. These findings suggest that the gestational diabetes affects the sugar and lipid composition of the intestinal brush border membrane in rats during early stages of the postnatal development, which may be associated with compromised tissue functions later in life.     

Green tea impedes dyslipidemia, lipid peroxidation, protein glycation and ameliorates Ca2+ -ATPase and Na+/K+ -ATPase activity in the heart of streptozotocin-diabetic rats

Diabetes-induced hyperlipidemia, oxidative stress and protein glycation impair cellular calcium and sodium homeostasis associated with abnormal membrane-bound enzyme activities resulting in cardiac dysfunction in diabetes. To explore the cardioprotective mechanism of green tea in diabetes, we measured the changes in the levels of calcium, sodium, potassium and the activities of Na+/K+ -ATPase and Ca2+ -ATPase in green tea treated diabetic rat hearts. The effect of green tea on triglycerides, lipid peroxidation and protein glycation in diabetic heart were also measured to elucidate the underlying mechanisms. Diabetes was induced by streptozotocin (STZ, 60 mg/kg i.p.). Six weeks after the induction of diabetes, some of the diabetic rats were treated orally with green tea extract (GTE) (300 mg/kg/day) for 4 weeks. GTE produced reduction in blood glucose and lowered the levels of lipid peroxides, triglycerides and extent of protein glycation in the heart of diabetic rats. GTE blunted the rise in cardiac [Ca2+] and [Na+] whereas increased the activities of Ca2+ -ATPase and Na+/K+ -ATPase in diabetic rats. In conclusion, the data provide support to the therapeutic effect of GTE and suggest that a possible mechanism of action may be associated with the attenuation of the rise in [Ca2+] and [Na+] by ameliorating Ca2+ -ATPase and Na+/K+ -ATPase activities.     

Correction of abnormal lipid fluidity and composition of rat ileal microvillus membranes in chronic streptozotocin-induced diabetes by insulin therapy

Diabetes was induced in rats by administration of streptozotocin. After 90-120 days, one group of chronic diabetic animals was treated with insulin for chronic diabetic animals was treated with insulin for 10 days. The lipid fluidity and composition of microvillus membranes prepared from ileal enterocytes of control, diabetic, and insulin-treated diabetic animals were determined. Lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the probes 1,6-diphenyl-1,3,5-hexatriene, DL-2-(9-anthroyl)stearic acid and DL-12-(9-anthroyl)stearic acid, was decreased in membranes of diabetic animals compared to membranes of control and insulin-treated diabetic membranes. The differences in fluidity resulted from an increased cholesterol content and cholesterol/phospholipid molar ratio in membranes of diabetic animals. The activities of sucrase and alkaline phosphatase were also found to be higher in membranes of diabetic animals. Insulin treatment, however, failed to significantly influence the enzymatic activities of these membranes. These studies, therefore, demonstrate that alterations in the lipid fluidity, lipid composition, and certain enzymatic activities exist in microvillus membranes of enterocytes prepared from chronic streptozotocin-induced diabetic rats. Administration of insulin for 10 days to these animals restored membrane fluidity and lipid composition but not enzymatic activities to control membrane levels.