Michel Sternberg

Kidney sialidase and sialyltransferase activities in spontaneously and experimentally diabetic rats: Influence of insulin and sorbinil treatments

Kidney cortex sialic acid level, sialidase and sialyltransferase activities have been measured in spontaneously diabetic BB rats and in streptozotocin-diabetic rats (STZ). In untreated diabetic BB rats, at the onset of the disease, sialidase specific activity was found to be increased by 21% when compared with diabetes-resistant BB controls (P less than 0.05) whereas sialyltransferase activity was not significantly modified and bound sialic acid concentration was diminished (P less than 0.05). In diabetic BB rats submitted to a minimal insulin therapy, during 3 months of disease, sialidase activity and sialic acid concentration were similar to those of Wistar age-matched controls. In STZ-diabetic Wistar rats, sialidase specific activity was increased by 76% after 5 months of disease when compared to age-matched Wistar controls (P less than 0.01); in contrast, specific sialyltransferase activity was decreased by 21% (P less than 0.05); these enzymatic alterations were associated with a decrease in bound sialic acid concentration (P less than 0.01); 1 months insulin therapy, started 4 months after onset of the disease, normalized sialidase activity but had no effect on sialyltransferase activity and sialic acid concentration; treatment with sorbinil prevented cataract development but had no effect on sialidase activity whereas it emphasized the decrease in sialyltransferase activity and sialic acid concentration. The disturbances in the enzyme activities concerned with sialoglycoconjugate metabolism observed in experimental and spontaneous diabetes may be responsible for the decreased bound sialic acid content observed in the rat kidney cortex.

Studies on kidney sialidase in normal and diabetic rats

Rat kidney cortex sialidase was studied using alpha-sialyl-(2----3)-[3H]lactitol and alpha-sialyl-(2----6)-[3H]lactitol as substrates. The enzyme was found mainly in the lysosomal fraction. Only 23% of the sialidase activity of this fraction could be solubilized by a combination of freezing-thawing, sonication and Triton X-100 treatment. The optimal pH for the lysosomal enzyme activity was 4.2 and the enzymes Km values for alpha-sialyl-(2----3)-lactitol and alpha-sialyl-(2----6)-lactitol were 0.28 and 0.41 mM, respectively. The specific activity was twice as high with the former substrate than with the latter. Sialidase activities in dialyzed kidney cortex homogenates of streptozotocin-diabetic rats and of age-matched control rats were compared. The specific activity was found to be significantly increased in the diabetic animals when using both substrates 5950 +/- 720 (S.E.) dpm/h per mg protein (n = 7) vs. 3970 +/- 370 in the controls (n = 8) with alpha-sialyl-(2----3)-lactitol (P less than 0.025) and 2870 +/- 300 vs. 1820 +/- 170 with alpha-sialyl-(2----6)-lactitol (P less than 0.02). The activities were also found to be increased when expressed per whole kidney cortex (P less than 0.005 and P less than 0.001, respectively). The elevated sialidase activity in diabetic kidney cortex may be related to the reported decrease in sialic acid content of the glomerular basement membrane, which lowers its negative charges and which may contribute to an increased permeability to proteins.

Effect of an aldose reductase inhibitor on type IV collagen production by human endothelial cells cultured in high glucose

SummaryDiabetic microangiopathy is characterized by a thickening of capillary basement membranes associated with type IV collagen accumulation. An increase in type IV collagen content of the aortic wall is also observed in macroangiopathy. In order to analyse the importance of the polyol pathway in the development of the collagen metabolism alterations seen in diabetic angiopathy and their prevention by aldose reductase inhibitors, we have studied the effects of sorbinil on the high glucose-induced stimulation of type IV collagen biosynthesis in human umbilical vein endothelial cells. Primary cultures were exposed to high glucose (16.7 mmol/l), with and without 0.11 mmol/l sorbinil, for 3 or 6 days after beginning of confluence. We measured the soluble type IV collagen secreted into the culture medium and the insoluble type IV collagen accumulated in the extracellular matrix and cells, by ELISA. We also studied [14C]proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. High glucose decreased the number of cells and increased the amount of type IV collagen in the culture supernatant and in the extracellular matrix and cell fraction. It also increased proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. Sorbinil corrected all these high glucose-induced alterations. The corrective effects of sorbinil on the proliferation and on type IV collagen metabolism of endothelial cells cultured in high glucose may be attributed to prevention of polyol pathway dysregulation.

Relation between pancreatic islet cellular infiltration and plasma fibrinogen or alpha 1-acid glycoprotein levels in spontaneously and streptozotocin-diabetic rats: an increase in these protein levels is not necessary for inducing microcirculatory erythrocyte velocity alteration.

Plasma levels of fibrinogen, a,-acid glycoprotein (AG) and albumin, pancreatic insulitis quantitative scores, and erythrocyte velocity in the mesoappendix mi-crovessels were measured in BB diabetic (BBD) and streptozotocin-diabetic rats (WSTZ) in order to answer the following questions: (a) Does hyperfibrinogenemia or increase in AG plasma level occur in BBD and WSTZ rats, and if so, are these alterations secondary to the hy-perglycemia or to an inflammatory process such as insulitis? (b) Is there a decrease in microcirculatory flow in the BBD and WSTZ rats, and if so, is it secondary to the hyperfibrinogenemia and/or the hyperglycemia? Insulitis was present in the BBD rats after 5 weeks of disease (with a score of 2.9 ± 0.1 vs. 1.4 ± 0.6 in the normoglycemic controls), but absent in WSTZ rats after 5 months of disease (1.2 ± 0.06 vs. 1.1 ± 0.06). Increase in fibrinogen and AG plasma levels was observed in the BBD rats only and appears linked to the insulitis. The major acute phase protein AG level is increased in BBD rats already on the first day of appearance of glycosuria. In the WSTZ rats, without insulitis, chronic hyperglycemia alone did not induce an increase in fibrinogen and AG plasma levels. A decreased microcirculatory erythrocyte velocity has been found in both BBD and WSTZ rats. Thus an increase in fibrinogen or AG plasma levels is not necessary for inducing a decrease in erythrocyte velocity. Hyperglycemia is probably the main factor responsible for the decrease in microcirculatory flow in the BBD and WSTZ rats.

Cyclic guanosylmonophosphate urinary excretion in parasympathicomimetic or parasympatholytic syndromes induced by reserpine or diphemanil-methylsulfate

Parasympathetic hyperactivity is found in some infants presenting faint episodes and could be responsible of certain Sudden Infant Death Syndrome cases. Therefore it was interesting to look for a noninvasive biochemical indicator of parasympathetic activity. A parasympaticomimetic syndrome associated with muscarinic receptor stimulation, which has been followed during 48 h, was obtained in the awake rat by reserpine injection (6.25 mg/kg at T0 and T24h), and a model of prolonged parasympatholytic syndrome, by administration of diphemanil-methylsulfate (DPMS), a muscarinic receptor inhibitor, in drinking water (mean daily dosis: 150 mg/kg). Significant bradycardia and tachycardia were respectively observed. In the reserpine-treated rats we found significantly increased cyclic guanosylmonophosphate (cGMP) urinary excretion between T24h and T48h, when compared with vehicle-treated controls (+87% in one experiment, +135% in the other, when expressed in pmol/microg creatinine); norepinephrine urinary excretion between T24h and T48h was decreased (-44%); the increase in cGMP urinary excretion was not significantly modified by the NO-synthase inhibitor, L-nitroarginine-methyl-ester. In the DPMS-treated rats, we observed a significantly decreased cGMP (-20%) and increased norepinephrine urinary excretion (+61%). Thus cGMP excretion varied in opposite directions in the reserpine- and DPMS-treated rats. The link between these modifications in cGMP excretion and muscarinic receptor stimulation or blockade has still to be fully demonstrated. Urinary cGMP excretion could be tested as screening parameter in infants at risk of faint episodes associated with bradycardia.

Effect of Long-Term Treatment with a Purified Micronized Flavonoid Fraction on Pancreatic Mononuclear Cell Infiltration in Diabetic BB Rats

Bio Breeding (BB) rats develop a genetically determined insulin-dependent diabetes, because of the early destruction of pancreatic beta cells of Langerhans islets, massively infiltrated by inflammatory mononuclear cells. S 5682, registered as Daflon, 500 mg, is a purified micronized flavonoid fraction (90% diosmin, 10% hesperidin), which has been shown to possess antiinflammatory properties, including anti-free radical activity, effects on vascular permeability, venous tone, and perivenous inflammation. We studied the effect of S 5682 on the course of pancreatic insulitis in diabetic BB rats. All the diabetic BB rats were hyperglycemic, with an increase of plasma levels of fructosamine, α-1 acid glycoprotein, and fibrinogen, and a dramatic decrease of C-peptide level. These parameters were not modified by S 5682. Pancreas histologic studies showed that in S 5682-treated diabetic BB rats, lymphocytic infiltration of Langerhans islets was less important and frequent than in untreated diabetic BB rats. By quantitative analysis, a highly significant difference was observed for insulitis, as well as perivasculitis, between S 5682-treated and untreated diabetic BB rats. This inhibitory effect of S 5682 on pancreatic mononuclear cell infiltration may be useful for a complementary treatment to decrease the development of insulitis in human insulin-dependent diabetes mellitus.

A flavonoid fraction purified from Rutaceae aurantiae (DaflonR) inhibiting AGE formation, reduces urinary albumin clearance and corrects hypoalbuminemia in normotensive and hypertensive diabetic rats

AIM Advanced glycation endproducts (AGEs) have been shown to contribute to alteration of glomerular permselectivity to proteins in diabetes. Oxidative stress is required for AGE formation. Therefore we studied the effect of an antioxidant micronized purified flavonoid fraction (MPFF, Daflon(R) 500 mg), on urinary albumin clearance in diabetic rats. METHODS Hyperglycaemia was induced by streptozotocin 55 mg/kg IM at days 0 and 7 in normotensive Wistar rats (NWR, diabetes duration 5 months) or hypertensive Wistar Kyoto rats (SHR, diabetes duration 2 months). MPFF was administered at 300 mg/kg/day, from day -2 until sacrifice. RESULTS After 5 months of diabetes in NWR, MPFF reduced albumin clearance from 729±92 to 392±60 nl/min/kg, p<0.01, and restored albuminemia from 20.4±0.9 to 24.0±1 g/l, p<0.05; albumin fractional clearance was significantly diminished in the flavonoid-treated diabetic rats (0.360±0.037‰ versus 1.335±0.430‰ in the diabetic controls, p<0.001); MPFF did not significantly modify blood glucose and plasma fructosamine levels. After 2 months of diabetes in SHR, MPFF reduced albumin clearance from 243±121 to 101±47 nl/min/kg, p<0.05, and restored albuminemia from 21.1±1.6 to 26.7±2.2 g/l (p<0.05); MPFF also decreased plasma fluorescence characteristic of AGEs (p<0.02). Besides hesperetin, a main metabolite of MPFF recovered in plasma, inhibited in vitro the formation of the crosslinking AGE pentosidine in collagen incubated with high glucose (p<0.001). CONCLUSION Our results confirm the role of glycoxidative stress in diabetic nephropathy. MPFF might be useful as complementary treatment for preventing diabetic microangiopathy.

Renal and microvascular effects of an aldose reductase inhibitor in experimental diabetes: Biochemical, functional and ultrastructural studies

Aldose reductase inhibitors, and particularly sorbinil, have been reported to prevent glomerular basement membrane thickening (GBMT) and albuminuria development in diabetic rats, but contradictory observations have been published. The aim of this study was to answer the following questions (i) is the corrective effect of sorbinil on GBMT, if confirmed, associated with an effect on collagen metabolism alterations? (ii) Is it associated with an effect on microvascular functional alterations? We therefore studied the influence of sorbinil on glucosyl-galactosyl-hydroxylysyl-glucohydrolase activity (GGHG; EC 3.2.1.107 which is involved in the catabolism of collagen disaccharide units), 3- and 4-hydroxyproline content and GBMT by ultrastructural morphometry in the kidney cortex of streptozotocin-diabetic rats after 5 months of disease. In parallel, the effects on albumin renal clearance and another functional alteration, the microvascular response to norepinephrine, were evaluated. We confirmed a corrective effect of sorbinil on both renal albumin clearance and GBMT. In the diabetic rats, sorbinil diminished the 3-hydroxyproline (but not the 4-hydroxyproline) content, whether expressed per mg protein or per total kidney cortex relative to body weight. Sorbinil reduced GGHG activity measured in the dialysed 10,000 g supernatant whether expressed per mg protein or per total kidney cortex; this activity has been shown to be increased in diabetes. Sorbinil also corrected the microvascular response to norepinephrine which is altered in diabetes.

Inhibition of the ?-glucosidase specific for collagen disaccharide units in diabetic rat kidney by in vivo glucose levels: possible contribution to basement membrane thickening

SummaryThe activity of the α-glucosidase specific for collagen disaccharide units has been measured in kidney cortex homogenates of streptozotocin-diabetic rats under three different conditions: (1) in dialyzed homogenates; (2) in non-dialyzed homogenates; (3) in non-dialyzed homogenates to which glucose was added to compensate for dilution due to homogenization and to reach the glucose concentration determined in kidney cortex (37.5±2.8 mmol/kg diabetic cortex versus 6.8±0.3 mmol/kg normal cortex). Under the latter condition, the enzyme activity was markedly decreased in diabetic kidney cortex when compared with that of normal age-matched controls: 4.03±0.25 versus 6.82±0.29 units/mg protein (p < 0.001). Inhibition of enzyme activity was also significant in non-dialyzed diabetic homogenates without additional glucose. In the absence of glucose (in the dialyzed homogenates), it is confirmed that the enzyme activity is elevated in diabetic kidney. The glucose inhibition of the enzyme activity has been shown to be important under in vivo conditions. It may therefore contribute to kidney basement membrane thickening.

Aorta Collagen Metabolism in Spontaneously Hypertensive and Aortic-Constricted Rats: Variations in Enzyme Activities Concerned with Disaccharide Unit Synthesis and Degradation According to Blood Pressure and Age

Two enzyme activities concerned with collagen disaccharide unit metabolism (UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase) have been studied in the thoracic aortic wall together with 4-prolyl hydroxylase activity and 4-hydroxyproline content in spontaneously hypertensive rats (SHR) at the prehypertensive, hypertensive and sustained hypertensive stages (respectively 32 days, 12 weeks and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. Negative regressions of all three specific activities as function of age were observed. The most striking difference observed between the SHR and the WKY was the increase of glucosyltransferase specific activity, already found at the prehypertensive stage and continuing thereafter; the glucohydrolase specific activity was increased only during the establishment of hypertension whereas no modification was found with prolyl hydroxylase at any stage. However, a diminution of hydroxyproline concentration was seen at all ages while total hydroxyproline mass remained unaffected. The alterations of the aortic collagen metabolism observed in the ACR recall those seen in the SHR at the prehypertensive stage: the only significant modification was that of glucosyltransferase activity. Correlation was found between glucosyltransferase activity and blood pressure level in the two animal models.