Effect of glucose on stress-activated protein kinase activity in mesangial cells and diabetic glomeruli.
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BACKGROUND: We have reported that hyperglycemia increases c-jun mRNA levels in isolated glomeruli of diabetic rats. The transcriptional activity of c-jun can be modified by phosphorylation of serine residues in the regulatory domain of the protein by stress-activated protein kinases (SAPKs), but the effect of high glucose concentrations on SAPK expression and activity is unknown. Accordingly, we studied p42/44 MAPK, p38 MAPK, and SAPK expression and activity in primary mesangial cells exposed to high glucose concentrations, as well as SAPK expression and activity in glomeruli of normal and streptozotocin-induced diabetic rats. METHODS: Mesangial cells were incubated in 40 mM glucose for 30 and 60 minutes and 6, 12, 24, and 48 hours, whereas glomeruli of streptozotocin-induced diabetic rats were isolated one day and one and two weeks after the onset of hyperglycemia (blood glucose levels more than 15 mmol/liter), and were compared with age-matched normal rats. Cell lysates were subjected to Western blot analysis of SAPK and phosphorylated SAPK and an in vitro SAPK assay using recombinant c-jun. RESULTS: Western blot analysis revealed that SAPK was expressed, but unphosphorylated, in unstimulated mesangial cells and whole glomerular lysates from normal rats. In accord with these observations, no SAPK activity was detected in lysates from mesangial cells or whole glomeruli from normal rats, although mesangial cell SAPK activity was readily induced in vitro by sorbitol. High glucose concentrations did not increase SAPK activity or lead to detectable phosphorylated SAPK either in vitro or in vivo. In contrast, short-term exposure to 40 mM of glucose activated both p42/44 MAPK and p38 MAPK. CONCLUSIONS: We conclude that high glucose concentrations do not activate SAPK in primary cultured mesangial cells or in diabetic glomeruli during the early phase of diabetic renal hypertrophy. (+info)
A model for evaluation of the peroral insulin therapy: short-term treatment of alloxan diabetic rats with oral water-in-oil-in-water insulin emulsions.
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Alloxan diabetic rats with fasting blood glucose levels above 300 mg/100 ml were treated with oral administration of water-in-oil-in-water (W/O/W) insulin emulsions at a dose of 50 U/100 g body weight, three times daily for 10 to 14 days. The course of diabetes was followed by determinations of glucose levels in blood and urine. During treatment with oral W/O/W insulin emulsions, daily excretion of urinary glucose decreased by about 30 to 40% (2 to 3 g/day) in all of the five rats studied, and returned to the pre-treatment levels after the treatment being discontinued. During treatment, a significant reduction in fasting blood glucose levels was observed in 4 out of 5 rats, giving the decrease by 18 to 44%. Quantitative estimates suggested that the effectiveness of 50 U/100 g of oral W/O/W insulin emulsions was comparable to that after intramuscular regular insulin at a dose of 0.5 U/100 g. Although oral W/O/W insulin emulsions are still of low efficiency, these results would indicate that diabetes can be controlled by effective oral insulin preparations. (+info)
Localization of lipoprotein lipase in the diabetic heart: regulation by acute changes in insulin.
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Vascular endothelium-bound lipoprotein lipase (LPL) is rate limiting for free fatty acid (FFA) transport into tissues. In streptozotocin (STZ)-diabetic rats, we have previously demonstrated an increased heparin-releasable LPL activity from perfused hearts. Because heparin can traverse the endothelial barrier, conventional Langendorff retrograde perfusion of the heart with heparin could release LPL from both the capillary luminal and abluminal surfaces. To determine the precise location of the augmented LPL, a modified Langendorff retrograde perfusion was used to isolate the enzyme at the coronary lumen from that in the interstitial effluent. In response to heparin, a 4-fold increase in LPL activity and protein mass was observed in the coronary perfusate after 2 weeks of STZ diabetes. Release of LPL activity into the interstitial fluid of control hearts was slow but progressive, whereas in diabetic hearts, peak enzyme activity was observed within 1 to 2 minutes after heparin, followed by a gradual decline. Immunohistochemical studies of myocardial sections confirmed that the augmented LPL in diabetic hearts was mainly localized at the capillary endothelium. To study the acute effects of insulin on endothelial LPL activity, we examined rat hearts at various times after the onset of hyperglycemia. An increased heparin-releasable LPL activity in diabetic rats was demonstrated shortly (6 to 24 hours) after STZ injection or after withdrawal from exogenous insulin. Heparin-releasable coronary LPL activity was also increased after an overnight fast. These studies indicate that the intravascular heparin-releasable fraction of cardiac LPL activity is acutely regulated by short-term changes in insulin rather than glucose. Thus, during short periods (hours) of hypoinsulinemia, increased LPL activity at the capillary endothelium can increase the delivery of FFAs to the heart. The resultant metabolic changes could induce the subsequent cardiomyopathy that is observed in the chronic diabetic rat. (+info)
Microencapsulation of rat islets prolongs xenograft survival in diabetic mice.
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OBJECTIVE: To protect the transplanted islets from the host's immune system by means of immunoexclusion membranes. METHODS: Rat islets were isolated from Wistar rat pancreas by ductal collagenase distention, stationary digestion, and finally with the aid of dextran gradient separation. Then the islets were encapsulated in alginate-polylysine-alginate (APA) semipermeable membranes. RESULTS: In vitro studies demonstrated that encapsulated islets secreted insulin in response to glucose challenge for at least 8 weeks, which was similar to free islets. In vivo studies showed that 15 streptozotocin (STZ)-induced diabetic mice were transplanted intraperitoneally with 1000 encapsulated islets without immunosuppression. Diabetes was reversed within 3 days, and the mice remained normoglycemic for up to 160 days, with a mean xenograft survival time of 126 days. The encapsulated islets had a significantly greater effect than unencapsulated islets, which functioned for less than 8 days. CONCLUSIONS: Encapsulation of pancreatic islets in semipermeable membranes can effectively prolong xenograft survival without immunosuppression in an animal model. (+info)
Protective effect of the angiotensin-converting enzyme inhibitor perindopril on diabetic glomerulopathy in streptozotocin-induced diabetic rats.
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OBJECTIVES: To evaluate the protective effect of the angiotensin-converting enzyme inhibitor perindopril on diabetic glomerulopathy in rats with experimentally induced diabetes and explore its possible mechanisms. METHODS: Ninety-two adult male Wistar rats were randomly allocated into diabetes mellitus (DM), diabetes mellitus + perindopril (DMP) and control (C) groups. According to the duration of diabetes or observation (1, 3, 6 months), each group was randomly subdivided into DM1, DM3, DM6; DMP1, DMP3, DMP6; and C1, C3, C6 groups. Diabetes was induced by intraperitoneal injection of streptozotocin. The rats in the DMP groups received perindopril 1 mg.kg-1.d-1, through gastric intubation. Urinary protein excretion rate was determined by the method of Coomassie brilliant blue. Plasma renin activity, renal tissue renin activity, and plasma and renal tissue angiotensin II concentration were assayed by radioimmunoassay (RIA). Renal tissue total RNA was extracted by the Chomezymskis AGPC method. Renal angiotensinogen mRNA expression level was assessed by slot blot hybridization using a full length rat angiotensinogen cDNA probe labelled with 32P-dCTP and a random primer. RESULTS: There was increased activity of the renin angiotensin system in diabetic rats. Perindopril decreased proteinuria and delayed the progression of glomerular basement membrane thickening. However, it did not reduce the expansion of the mesangial matrix (P < 0.05). Renin activity increased and angiotensin II concentration decreased significantly in both plasma and renal tissue in diabetes + perindopril groups (P < 0.05). CONCLUSIONS: Perindopril may help prevent the progression of diabetic glomerulopathy, and the inhibition of renin angiotensin system activity may be a mechanism for this action. (+info)
Neogenesis vs. apoptosis As main components of pancreatic beta cell ass changes in glucose-infused normal and mildly diabetic adult rats.
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We have investigated in adult rats made mildly diabetic by a low dose of streptozotocin (35 mg/kg; STZ rats) and in nondiabetic rats (ND rats) the mechanisms leading to adaptive changes in the beta cell mass, during glucose infusion and several days after stopping infusion. As early as 24 h of glucose infusion, the beta cell mass was maximally increased in ND and STZ rats. In both groups, this increase was due mainly to a rapid activation of neogenesis of new endocrine cells rather than to an increase in beta cell proliferation. Seven days after stopping glucose infusion, the beta cell mass returned to basal values in both groups as a result of stimulation of beta cell apoptosis and a decrease in beta cell replication rate. In glucose-infused ND rats, changes in the beta cell mass were correlated to insulin secretion, whereas in STZ rats, insulin secretion in response to glucose was still impaired whatever the beta cell mass. In conclusion, the data stress the impressive plasticity of the endocrine pancreas of adult rats. They also show that changes in beta cell mass in ND and STZ rats resulted from a disruption in the balance between neogenesis and apoptosis. (+info)
No correlation of plasma cell 1 overexpression with insulin resistance in diabetic rats and 3T3-L1 adipocytes.
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Membrane glycoprotein plasma cell 1 (PC-1) has been shown to be increased in type 2 diabetes and involved in insulin resistance through inhibiting the insulin receptor tyrosine kinase, which was demonstrated using cultured breast cancer cells. However, other reports have shown contradictory results in Chinese hamster ovary cells and in vitro kinase assay. Thus, we considered it necessary to investigate the effect of PC-1 using highly insulin-sensitive cells. Here, we used two of the following approaches: 1) investigating PC-1 expression levels in insulin-responsive tissues in rat models of diabetes and 2) overexpressing PC-1 in 3T3-L1 adipocytes. We found that PC-1 was highly expressed in insulin-responsive tissues, such as liver and adipose tissue, in normal rats. However, high-fat feeding or streptozotocin-induced diabetes did not change its expression levels in liver, adipose tissue, and skeletal muscle. Thus, PC-1 expression levels were not associated with high-fat-diet-induced insulin resistance or hyperglycemia. Although PC-1 was increased in adipose tissue in Zucker fatty rats (protein level, by 50%; mRNA level, by 90%), its expression levels in liver and skeletal muscle, tissues that are more responsible for whole body glucose metabolism than adipose tissue, did not significantly differ from those in normal rats. Next, we overexpressed PC-1 in 3T3-L1 adipocytes using an adenovirus transfection system. PC-1 expression was markedly increased to a level 16-fold greater than that in normal human adipose tissue, which is higher than the previously reported levels in diabetic patients. However, insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1, activation of phosphatidylinositol 3-kinase, and glucose uptake were not affected by PC-1 overexpression. These results strongly suggest that increased PC-1 expression is not causally related to insulin resistance. (+info)
Why do microencapsulated islet grafts fail in the absence of fibrotic overgrowth?
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The survival of microencapsulated islet grafts is limited, even if capsular overgrowth is restricted to a small percentage of the capsules. In search of processes other than overgrowth contributing to graft failure, we have studied the islets in non-overgrown capsules at several time points after allotransplantation in the rat. All recipients of islet allografts became normoglycemic. Grafts were retrieved at 4 and 8 weeks after implantation and at 15.3 +/- 2.3 weeks postimplant, 2 weeks after the mean time period at which graft failure occurred. Overgrowth of capsules was complete within 4 weeks postimplant, and it was usually restricted to <10% of the capsules. During the first 4 weeks of implantation, 40% of the initial number of islets was lost. Thereafter, we observed a decrease in function rather than in numbers of islets, as illustrated by a decline in the ex vivo glucose-induced insulin response. At 4 and 8 weeks postimplant, beta-cell replication was 10-fold higher in encapsulated islets than in islets in the normal pancreas, but these high replication rates were insufficient to prevent a progressive increase in the percentage of nonviable tissue in the islets. Necrosis and not apoptosis proved to be the major cause of cell death in the islets. The necrosis mainly occurred in the center of the islets, which indicates insufficient nutrition as a major causative factor. Our study demonstrates that not only capsular overgrowth but also an imbalance between beta-cell birth and beta-cell death contributes to the failure of encapsulated islet grafts. Our observations indicate that we should focus on finding or creating a transplantation site that, more than the unmodified peritoneal cavity, permits for close contact between the blood and the encapsulated islet tissue. (+info)