In this study, we investigated the functional role of the localization of human OTR in caveolin-1 enriched membrane domains. Biochemical fractionation of MDCK cells stably expressing the WT OTR-GFP indicated that only minor quantities of receptor are partitioned in caveolin-1 enriched domains. However, when fused to caveolin-2, the OTR protein proved to be exclusively localized in caveolin-1 enriched fractions, where it bound the agonist with increased anity and eciently coupled to Ga q/11 . Interestingly, the chimeric protein was unable to undergo agonist-induced internalization and remained con®ned to the plasma membrane even after prolonged agonist exposure (120 min). A striking dierence in receptor stimulation was observed when the OT-induced eect on cell proliferation was analysed: stimulation of the human WT OTR inhibited cell growth, whereas the chimeric protein had a proliferative eect. These data indicate that the localization of human OTR in caveolin-1 enriched microdomains radically alters its regulatory eects on cell growth; the fraction of OTR residing in caveolar structures may therefore play a crucial role in regulating cell proliferation.

Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11-), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic beta-cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic beta-cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN beta-cells. PDX-1 and HMGI(Y) transcription factors are down-regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor-associated PI 3-kinase/Akt/PHAS-I pathway in RIN beta-cells. Re-expression in RIN beta-cells chronically exposed to high glucose of the Ex11-, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining beta-cell dysfunction caused by chronic hyperglycemia.

BackgroundIn type 1 diabetes (T1D) vascular complications such as accelerated atherosclerosis and diffused macro-/microangiopathy are linked to chronic hyperglycemia with a mechanism that is not yet well understood. End-stage renal disease (ESRD) worsens most diabetic complications, particularly, the risk of morbidity and mortality from cardiovascular disease is increased several fold.Methods and FindingsWe evaluated protein regulation and expression in skin biopsies obtained from T1D patients with and without ESRD, to identify pathways of persistent cellular changes linked to diabetic vascular disease. We therefore examined pathways that may be normalized by restoration of normoglycemia with kidney-pancreas (KP) transplantation. Using proteomic and ultrastructural approaches, multiple alterations in the expression of proteins involved in oxidative stress (catalase, superoxide dismutase 1, Hsp27, Hsp60, ATP synthase δ chain, and flavin reductase), aerobic and anaerobic glycolysis (ACBP, pyruvate kinase muscle isozyme, and phosphoglycerate kinase 1), and intracellular signaling (stratifin-14-3-3, S100-calcyclin, cathepsin, and PPI rotamase) as well as endothelial vascular abnormalities were identified in T1D and T1D+ESRD patients. These abnormalities were reversed after KP transplant. Increased plasma levels of malondialdehyde were observed in T1D and T1D+ESRD patients, confirming increased oxidative stress which was normalized after KP transplant.ConclusionsOur data suggests persistent cellular changes of anti-oxidative machinery and of aerobic/anaerobic glycolysis are present in T1D and T1D+ESRD patients, and these abnormalities may play a key role in the pathogenesis of hyperglycemia-related vascular complications. Restoration of normoglycemia and removal of uremia with KP transplant can correct these abnormalities. Some of these identified pathways may become potential therapeutic targets for a new generation of drugs.

These data demonstrate that genetic factors, which play an important role in the regulation of body weight, may account for differences in the therapeutic response to LAGB.

To evaluate the effects of kidney-pancreas transplantation on hemostatic abnormalities in uremic type 1 diabetic patients, we conducted a cross-sectional study involving 12 type 1 diabetic patients, 30 uremic type 1 diabetic patients, 27 uremic type 1 diabetic patients who had a kidney-pancreas transplant, 12 uremic type 1 diabetic patients who had a kidney-alone transplant, and 13 healthy control subjects. We evaluated platelet and clotting system. Platelets in the group of uremic type 1 diabetic patients were significantly larger than platelets in the other groups. Resting calcium levels were significantly higher in the uremic type 1 diabetic patients and uremic type 1 diabetic patients who had a kidney-alone transplant than in the type 1 diabetic patients who had a kidney-pancreas transplant and control subjects. CD41 expression was significantly reduced in platelets from the uremic type 1 diabetic patients compared with the other groups. Levels of hypercoagulability markers in the type 1 diabetic patients who had a kidney-pancreas transplant and, to a lesser extent, the uremic type 1 diabetic patients who had a kidney-alone transplant but not the uremic type 1 diabetic patients were similar to those of the control subjects. A reduction in natural anticoagulants was evident in the uremic type 1 diabetic patients, whereas near-normal values were observed in the type 1 diabetic patients who had a kidney-pancreas transplant and uremic type 1 diabetic patients who had a kidney-alone transplant. Hemostatic abnormalities were not observed in type 1 diabetic patients who had a kidneypancreas transplant. This finding might explain the lower cardiovascular death rate observed in type 1 diabetic patients who had a kidney-pancreas transplant compared with uremic type 1 diabetic patients who had a kidney-alone transplant or uremic type 1 diabetic patients.