Thickness of renal glomerular capillary basement membrane in the offspring of diabetic rats fed a regular or high-sucrose diet

Numerous animal model studies of diabetes mellitus have been reported. Diabetes-induced vascular damage is a common cause of systemic organ damage in humans and animals. Many investigations have been made of human and animal offspring of diabetic mothers. The present report documents the sequential glomerular basement membrane (GBM) thickness in fetuses and infants of diabetic rats. The postnatal increase in GBM thickness was similar in the offspring of control and diabetic rats, and was not related to the sucrose concentration in the diet.     

Increased nonenzymatically glycosylated proteins in the vitreous humor of diabetic animals

One of the earliest pathologic changes of diabetes mellitus is increased nonenzymatic glycosylation (i.e., glycation) of proteins, which results in abnormal aggregation of collagen fibrils and production of superoxide radicals. These abnormalities may be responsible for the precocious senescence of connective tissue associated with the disease. We sought to determine whether glycation is increased in the vitreous humor of short-term diabetic cats (6 months' duration) and rabbits (2 months' duration), using a nitroblue tetrazolium colorimetric assay for fructosamine. Vitreous protein fructosamine concentration was significantly higher in diabetic cats and rabbits, compared with that in control (nondiabetic) animals. These results indicate that glycation is increased in the vitreous humor of short-term diabetic animals, and therefore may be one of the initial triggers for clinically apparent diabetic retinopathy.     

Antioxidative enzyme and glutathione S-transferase activities in diabetic rats exposed to long-term ASA treatment

Low-dose acetylsalicylic acid (ASA) treatment is a standard therapeutic approach in diabetes mellitus for prevention of long-term vascular complications. The aim of the present work was to investigate the effect of long-term ASA administration in experimental diabetes on activities of some liver enzymes: glutathione peroxidase (GSHPx), catalase, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST). Blood glucose, glycated hemoglobin, as well as plasma ALT and AST activities increased in rats with streptozotocin-induced experimental diabetes. The long-term hyperglycemia resulted in decreased activities of GSHPx (by 26%), catalase (by 34%), GST (by 38%) and G6PDH (by 27%) in diabetic animals. We did not observe increased accumulation of membrane lipid peroxidation products or altered levels of reduced glutathione in livers. The linear correlation between blood glucose and glycated hemoglobin in diabetic animals was distorted upon ASA treatment, which was likely due to a chemical competition between nonenzymatic protein glycosylation and protein acetylation. The long-term ASA administration partially reversed the decrease in GSHPx activity, but did not influence the activities of catalase and GST in diabetic rats. Otherwise, some decrease in these parameters was noted in ASA-treated nondiabetic animals. Increased ASA-induced G6PDH activity was recorded in both diabetic and nondiabetic rats. While both glycation due to diabetic hyperglycemia and ASA-mediated acetylation had very similar effects on the activities of all studied enzymes but G6PDH, we conclude that non-enzymatic modification by either glucose or ASA may be a common mechanism of the observed convergence.     

Morphometric evaluation of capillary basement membrane thickness in the quadriceps muscle of diabetic and nondiabetic Chinese hamsters

Quadriceps muscle capillaries from 19-23 month old genetically diabetic (XA and AC) and nondiabetic (M) subline Chinese hamsters were morphometrically evaluated to determine if capillary basement membrane thickening (CBMT) is a quantifiable complication of diabetes. Significant CBMT was present in the diabetic XA Chinese hamsters (49.37 nm +/- 17.81, p less than 0.007) in comparison with the nondiabetic M hamsters (34.08 nm +/- 9.98). Although there was a trend towards expansion of the muscle capillary basement membranes in the diabetic AC Chinese hamsters, the value was not statistically significant. A nested analysis of variance showed that the greatest source of variation in basement membrane thickness occurred among capillaries within each animal. In addition, a positive correlation (r = 0.62; p less than 0.002) existed between blood glucose levels and CBMT in the XA subline. These data should serve as guidelines for evaluation of antimicrovascular disease compounds which will be tested to determine if they prevent or retard microangiopathy in the diabetic Chinese hamster.     

Diabetes mellitus in sand rats (Psammomys obesus): microangiopathy during development of the diabetic syndrome.

The purpose of the study was to investigate the development of microangiopathic complications in North African sand rats with diabetes induced by a long-term standard laboratory diet. Hyperinsulinaemic rats, whether non-diabetic obese or diabetic, developed capillary basement membrane (CBM) thickening in the skin; in insulin-dependent animals, this change was diffuse. Many PAS positive areas were demonstrated in skeletal muscle and myocardium, together with evidence of microangiopathy; the primary myocardial lesion in insulin-dependent disease was ischaemic fibrosis. The kidney was also affected with marked basement membrane thickening in Bowman's capsule and glomerular capillaries; glomerulosclerosis and tubular changes were found in insulin-dependent disease. No evidence of diabetic retinopathy was found, and there was a high incidence of cataract.     

The ultrastructure of the capillaries in the gingiva of alloxan-induced diabetic rats

The diabetic effects of alloxan (type I diabetes mellitus) were investigated in 40 Wistar albino rats (18 controls and 22 diabetics). Alloxan in sterile physiological saline was injected into animals intravenously. After the induction of diabetes with alloxan, the ultrastructure of the capillaries in the gingiva was examined by transmission electron microscopy. The thickness of the basement membranes was observed closely adherent to the endothelial cells of the capillary alloxan-diabetic rats. It was greatly thickened owing to the increase in its amorphous, granular and filamentous material with occasional scattered collagen fibres. In some sections, the capillary lumens of the diabetics were closed by epithelial cells. Loss of cytoplasmic material and hyalinization were seen in some smooth muscle cells. In addition, the mitochondrial cristae of smooth muscle cell and epithelial cells disappeared. There was endothelial integrity throughout the smooth muscle cells.     

Does enhanced expression of the Na+-CA2+ exchanger increase myocardial vulnerability to ischemia/reperfusion injury in rabbit hearts?

The present study focused on examining the efficacy of feeding a rutin-glucose derivative (G-rutin) to inhibit glycation reactions that can occur in muscle, kidney and plasma proteins of diabetic rats. Both thiobarbituric acid-reactive substance levels and protein carbonyl contents in muscle and kidney were significantly (p < 0.05) reduced in streptozotocin-induced diabetic rats fed G-rutin supplemented diet, compared to diabetic rats fed control diet. The N -fructoselysine content in muscle and kidney, a biomarker of early glycation reaction, was markedly (p < 0.05) increased by diabetes, but significantly (p < 0.05) reduced in diabetic rats fed G-rutin. Advanced glycation end-products (AGEs) in serum and kidney protein were measured by immunoblot using anti-AGE antibody, and were also reduced in diabetic rats fed dietary G-rutin. Feeding G-rutin also slightly inhibited aldose reductase activity in these animals. These results demonstrate for the first time that dietary G-rutin consumption can provide potential health benefits that are related to the inhibition of tissue glycation reactions common to diabetes.     

Hyperglycaemia alters the physico-chemical properties of proteins in erythrocyte membranes of diabetic patients.

1. The dynamic properties of erythrocyte membranes in diabetic children and of control erythrocyte membranes subjected to in vitro glycation have been investigated by means of fluorescence quenching of membrane tryptophan residues and ESR spectroscopy. 2. The apparent distance separating the membrane protein tryptophan and the bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased in erythrocyte membranes from children with diabetes. This resulted in a significant increase of the maximum energy transfer efficiency in diabetic membranes. 3. The relevant alterations occurred in the above parameters due to the in vitro nonenzymatic glycosylation of control membranes. 4. These changes were accompanied by the decreased hw/hs parameter of MSL and the increased relative rotational correlation time (tau c) of ISL in diabetic membranes and in the membranes subjected to in vitro glycation. 5. The results suggest that the conformational changes in membrane proteins may occur at both the intrinsic and exposed thiol groups. 6. Both the in vivo and the in vitro data indicate that nonenzymatic glycosylation of membrane proteins may be the major factor attributable to the alterations in the dynamic properties of erythrocyte membrane in diabetic state.     

Blood glucose control and glomerular capillary basement membrane thickening in experimental diabetes.

Glomerular capillary basement membrane thickness (BMT) was measured in 23 rats which had had streptozocin-induced diabetes for 14 months and in 12 age-matched controls. Diabetic rats were randomly allocated to different groups, either receiving no treatment or treated with a low carbohydrate diet or insulin, or both. Control rats were randomly allocated to a normal or low carbohydrate diet. Among the diabetic rats mean plasma glucose concentrations for the groups ranged from 27-4 mmol/l (494 mg/100 ml) in the untreated rats to 9-8 mmol/l (177 mg/100 ml) in those receiving both a low carbohydrate diet and insulin. A highly significant positive relation was found between BMT and plasma glucose concentration for individual rats. When BMT was corrected for body weight a similar relation was observed.     

Influence of maternal diabetes on basement membranes, type 2 cells, and capillaries in the developing rat lung

To determine the effect of maternal diabetes on rat lung development, we studied the ultrastructure of the alveolar wall from the ninteenth day of gestation (term = 22 days) through the eighth postnatal day in fetal and neonatal rats of mothers with streptozotocin-induced diabetes. In normal fetal lung development, epithelial basement membranes develop large discontinuities beneath type 2 cells, through which cytoplasmic foot processes extend into the interstitium. Maternal diabetes delays the appearances of these epithelial basement membrane discontinuities and reduces the number of type 2 cell processes that penetrate it. These alterations in epithelial basement membrane are reversed after birth. There is no ultrastructural evidence of a delay in type 2 cell maturation as assessed by lamellar body volume density morphometry. Endothelial basement membranes, which are not present around the growing pulmonary capillary bed in the pseudoglandular lung, are seen late in normal gestation, primarily around capillaries forming the mature air-blood barrier. This development of endothelial basement membrane may be delayed in the fetuses of diabetic mothers and reflects a significant delay in the expansion of the pulmonary capillary network in these animals as assessed by morphometric volume density measurements. This effect on capillary growth is not reversed in the newborn animals through 8 days after birth. The summation of these effects indicates a generalized slowing of fetal lung development by maternal diabetes, some of which effects persist after birth and may continue to influence lung development during the period of postnatal alveolar septal growth.     

AGE-breakers cleave model compounds,but do not break Maillard crosslinks in skin and tail collagen from diabetic rats

Advanced glycation end products (AGE), formed by nonenzymatic Maillard reactions between carbohydrate and protein, contribute to the increase in chemical modification and crosslinking of tissue proteins with age. Acceleration of AGE formation in collagen during hyperglycemia, with resultant effects on vascular elasticity and basement membrane permeability, is implicated in the pathogenesis of diabetic complications. AGE-breakers, such as N-phenacylthiazolium (PTB) and N-phenacyl-4,5-dimethylthiazolium (PMT) halides, have been proposed as therapeutic agents for reversing the increase in protein crosslinking in aging and diabetes. We have confirmed that these compounds, as well as the AGE-inhibitor pyridoxamine (PM), cleave the model AGE crosslink, phenylpropanedione, and have studied the effects of these compounds in reversing the increased crosslinking of skin and tail collagen isolated from diabetic rats. Crosslinking of skin collagen, measured as the half-time for solubilization of collagen by pepsin in 0.5M acetic acid, was increased approximately 5-fold in diabetic, compared to nondiabetic rats. Crosslinking of tail tendon collagen, measured as insolubility in 0.05 N acetic acid, was increased approximately 10-fold. Collagen preparations were incubated in the presence or absence of AGE-breakers or PM in phosphate buffer, pH 7.4, for 24h at 37 degrees C. These treatments did not decrease the half-time for solubilization of diabetic skin collagen by pepsin or increase the acid solubility of diabetic tail tendon collagen. We conclude that, although AGE-breakers and PM cleave model crosslinks, they do not significantly cleave AGE crosslinks formed in vivo in skin collagen of diabetic rats.     

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with³H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of³²P into purified neurofilament proteins when they were incubated with³²P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.     

Decreased interaction of fibronectin, type IV collagen, and heparin due to nonenzymatic glycation. Implications for diabetes mellitus

The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 degrees C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 X 10(-7) M while the KD for glycated fibronectin and type IV collagen was 2.9 X 10(-6) M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 X 10(-7) M for the interaction of control fibronectin with gelatin vs. KD of 1.2 X 10(-6) M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H]heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.     

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.     

Increased glycosylation of glomerular basement membrane collagen in diabetes

Basement membrane was purified from glomeruli isolated from normal and streptozotocin-diabetic rats. After extraction of non-collagen protein with 8M urea, the extent of glycosylation in glomerular basement membrane collagen was determined with a specific colorimetric reaction that detects carbohydrate in ketoamine linkage with proteins. The level of glycosylation of glomerular basement membrane collagen purified from diabetic rats was significantly greater than that in non-diabetic animals. Increased basement membrane glycosylation may alter structure-function relationships of the capillary filtration barrier.     

Delay of diabetic cataract in rats by the antiglycating potential of cumin through modulation of alpha-crystallin chaperone activity

alpha-Crystallin, a molecular chaperone of the eye lens, plays an important role in maintaining the transparency of the lens by preventing the aggregation/inactivation of several proteins and enzymes in addition to its structural role. alpha-Crystallin is a long-lived protein and is susceptible to several posttranslational modifications during aging, more so in certain clinical conditions such as diabetes. Nonenzymatic glycation of lens proteins and decline in the chaperone-like function of alpha-crystallin have been reported in diabetic conditions. Therefore, inhibitors of nonenzymatic protein glycation appear to be a potential target to preserve the chaperone activity of alpha-crystallin and to combat cataract under hyperglycemic conditions. In this study, we investigated the antiglycating potential of cumin in vitro and its ability to modulate the chaperone-like activity of alpha-crystallin vis-à-vis the progression of diabetic cataract in vivo. Aqueous extract of cumin was tested for its antiglycating ability against fructose-induced glycation of goat lens total soluble protein (TSP), alpha-crystallin from goat lens and a nonlenticular protein bovine serum albumin (BSA). The antiglycating potential of cumin was also investigated by feeding streptozotocin (STZ)-induced diabetic rats with diet containing 0.5% cumin powder. The aqueous extract of cumin prevented in vitro glycation of TSP, alpha-crystallin and BSA. Slit lamp examination revealed that supplementation of cumin delayed progression and maturation of STZ-induced cataract in rats. Cumin was effective in preventing glycation of TSP and alpha-crystallin in diabetic lens. Interestingly, feeding of cumin to diabetic rats not only prevented loss of chaperone activity but also attenuated the structural changes of alpha-crystallin in lens. These results indicated that cumin has antiglycating properties that may be attributed to the modulation of chaperone activity of alpha-crystallin, thus delaying cataract in STZ-induced diabetic rats.     

Chemical modification of muscle protein in diabetes

Levels of glycation (fructose-lysine, FL) and advanced glycoxidation and lipoxidation end-products (AGE/ALEs) were measured in total skeletal (gastrocnemius) muscle and myofibril protein and compared to levels of the same compounds in insoluble skin collagen of control and diabetic rats. Levels of FL in total muscle and myofibril protein were 3-5% the level of FL in skin collagen. The AGE/ALEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine, were also significantly lower in total muscle and myofibril protein, approximately 25% of levels in skin collagen. The newly described sulfhydryl AGE/ALE, S-(carboxymethyl)cysteine (CMC), was also measured in muscle; levels of CMC were comparable to those of CML and increased similarly in response to diabetes. Although FL and AGE/ALEs increased in muscle protein in diabetes, the relative increase was less than that seen in skin collagen. These data indicate that muscle protein is partially protected against the increase in both glycation and AGE/ALE formation in diabetes.     

Correlated endothelial caveolin overexpression and increased transcytosis in experimental diabetes.

We investigated the mechanism by which diabetes renders the capillary endothelium more permeable to macromolecules in the lungs of short-term diabetic rats. We used quantitative immunocytochemistry (ICC) to comparatively assess the permeability of alveolar capillaries to serum albumin in diabetic and normoglycemic animals. The effect of diabetes on the population of endothelial caveolae was evaluated by morphometry and by ICC and immunochemical quantification of the amount of caveolin in the whole cell or associated with the purified endothelial plasma membrane. A net increase in the amount of serum albumin taken up by the plasmalemmal vesicles of alveolar endothelial cells and transported to the interstitium was documented in diabetic animals. Interendothelial junctions were not permeated by albumin molecules. The alveolar endothelial cells of hyperglycemic rats contain more caveolae (1.3-fold), accounting for a larger (1.5-fold) fraction of the endothelial volume than those of normal animals. The hypertrophy of the caveolar compartment is accompanied by overexpression of endothelial caveolin 1. Although the aggregated thickness of the endothelial and alveolar epithelium basement membranes increases in diabetes (1.3-fold), the porosity of this structure appears to be unchanged. Capillary hyperpermeability to plasma macromolecules recorded in the early phase of diabetes is explained by an intensification of transendothelial vesicular transport and not by the destabilization of the interendothelial junctions.     

Evidence for a relationship between protein glycation and red blood cell membrane fluidity

This study examines the relationship between protein glycation and membrane fluidity in RBC membranes. Incubation of RBC membranes of healthy subjects with 25mM glucose or galactose at 37 degrees C induced a 38% (p less than 0.02) increase in protein glycation (using furosine determination by HPLC) and higher fluidity (p less than 0.05) in DPH polarization ratio). However, incubation of RBC membranes from diabetic subjects under the same conditions did not modify either membrane fluidity or protein glycation; protein glycation was above normal before incubation because of the high diabetic plasma glucose. There was no difference in the membrane fluidities of 21 healthy subjects and 32 diabetic subjects, despite a significantly elevated protein glycation in diabetics. Furthermore, there was no change with respect to age in either population. We conclude that other in vivo factors, such as membrane lipid changes (increase in CL/PL ratio) or formation of advanced Maillard products and peroxidation in the diabetic subjects, could be responsible for the difference between these in vitro results and the in vivo situation.