Dysfunctional endothelium is associated with and, likely, predates clinical complications of diabetes mellitus, by promoting increased vascular permeability and thrombogenicity. Irreversible advanced glycation end products (AGEs), resulting from nonenzymatic glycation and oxidation of proteins or lipids, are found in plasma, vessel wall, and tissues and have been linked to the development of diabetic complications. The principal means through which AGEs exert their cellular effects is via specific cellular receptors, one of which, receptor for AGE (RAGE), is expressed by endothelium. We report that blockade of RAGE inhibits AGE-induced impairment of endothelial barrier function, and reverses, in large part, the early vascular hyperpermeability observed in diabetic rats. Inhibition of AGE-and dia- IntroductionExposure of proteins or lipids to reducing sugars results in nonenzymatic glycation and oxidation. Initially, reversible early glycation adducts, Schiff bases and Amadori products, form on free amino groups (1). Further complex molecular rearrangements produce irreversible advanced glycation end products (AGEs):1 heterogeneous structures of yellow-brown color, characteristic fluorescence, and a propensity to form cross-links, which generate reactive oxygen intermediates and interact with specific cellular receptors (1-4). The presence of AGEs in tissue has been linked to development of vasculopathy, especially in the setting of diabetes (1, 3). AGE-modified adducts on long-lived proteins in extracellular matrix alter basement membrane structure by trapping plasma macromolecules and by increasing vessel wall rigidity through formation of cross-links (3). The principal means through which AGEs influence cellular properties is by binding to specific receptors (4-6), the best characterized of which is the receptor for AGEs (RAGE), a member of the immunoglobulin superfamily expressed by endothelial cells (ECs), smooth muscle cells, and mononuclear phagocytes (7), cells central to both vascular homeostasis and the pathogenesis of vascular lesions. A potential role for RAGE in vascular dysfunction is suggested by two lines of evidence: ( a ) engagement of AGEs by cellular RAGE affects critical properties of these cells in a manner contributory to vascular dysfunction (4); and ( b ) there is enhanced expression of RAGE in diabetic vasculopathy and in arteriosclerotic and other vascular lesions, such as inflammatory vasculitides (8).Increased vascular permeability is characteristic of diabetic vasculopathy (9), even at the earliest stages in which microalbuminuria may be the only harbinger of vascular complications yet to come (10). As ECs are the critical guardians of vascular barrier function, we postulated that AGEs in plasma or the subendothelium would promote vascular hyperpermeability by interacting with RAGE. We demonstrate that when diabetic rat red cells bearing AGEs are infused into normal animals, increased vascular permeability results, an effect which is prevented by blockade of RAGE. Hyperperme...

OBJECTIVEHeterozygous mutations in the human preproinsulin (INS) gene are a cause of nonsyndromic neonatal or early-infancy diabetes. Here, we sought to identify INS mutations associated with maturity-onset diabetes of the young (MODY) or nonautoimmune diabetes in mid-adult life, and to explore the molecular mechanisms involved.RESEARCH DESIGN AND METHODSThe INS gene was sequenced in 16 French probands with unexplained MODY, 95 patients with nonautoimmune early-onset diabetes (diagnosed at <35 years) and 292 normoglycemic control subjects of French origin. Three identified insulin mutants were generated by site-directed mutagenesis of cDNA encoding a preproinsulin–green fluorescent protein (GFP) (C-peptide) chimera. Intracellular targeting was assessed in clonal β-cells by immunocytochemistry and proinsulin secretion, by radioimmunoassay. Spliced XBP1 and C/EBP homologous protein were quantitated by real-time PCR.RESULTSA novel coding mutation, L30M, potentially affecting insulin multimerization, was identified in five diabetic individuals (diabetes onset 17–36 years) in a single family. L30M preproinsulin-GFP fluorescence largely associated with the endoplasmic reticulum (ER) in MIN6 β-cells, and ER exit was inhibited by ∼50%. Two additional mutants, R55C (at the B/C junction) and R6H (in the signal peptide), were normally targeted to secretory granules, but nonetheless caused substantial ER stress.CONCLUSIONSWe describe three INS mutations cosegregating with early-onset diabetes whose clinical presentation is compatible with MODY. These led to the production of (pre)proinsulin molecules with markedly different trafficking properties and effects on ER stress, demonstrating a range of molecular defects in the β-cell.

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