Original contribution
Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products

https://doi.org/10.1016/S0891-5849(00)00286-0Get rights and content

Abstract

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.

Introduction

Hyperglycemia is the major causal factor for the development of various diabetic complications, although how hyperglycemia provokes these complications has not been fully elucidated yet. Several biochemical mechanisms are postulated to explain the adverse effects of hyperglycemia and ensuing tissue damage. They are increased flux of glucose through the polyol pathway [1], enhanced nonenzymatic glycation [2], accelerated generation of reactive oxygen species [3], and activation of the diacylglycerol-protein kinase C pathway [4].

Aldose reductase (AR), the first and rate-limiting enzyme in the polyol pathway, is a member of the monomeric NADPH-dependent aldo-keto reductase family that catalyzes reduction of glucose to the corresponding sugar alcohol, sorbitol. It has been generally accepted that expression of AR gene is osmotically regulated: increased AR activity under hyperosmotic conditions supplies sorbitol as one of the physiological osmolytes in the inner medullary cells of the kidney [5]. In the lens, excessive accumulation of sorbitol by increased flux through this pathway was indicated to play a key role in the development of diabetic cataract [6]. In other target organs of diabetic complications, on the other hand, activation of polyol pathway alleviate glutathione reductase and nitric oxide synthase activities due to the depletion of the cofactor NADPH. This may result in the increased susceptibility to oxidative stress and vascular dysfunction, leading to the early stage of diabetic complications [1]. Under hyperglycemic conditions, elevated expression of AR was reported in a variety of tissues including the target organs of diabetic complications, and in cultured cells originated from humans and experimental animals [7], [8], [9]. Molecular mechanisms underlying such augmented expression of AR under hyperglycemia remain unresolved; however, a recent study indicated that oxidative stress may induce AR mRNA expression in a cell line derived from rat vascular smooth muscle cells [10].

Glucose-induced oxidative stress is one of the biochemical mechanisms postulated to precipitate the development of diabetic complications. Among multiple pathways by which hyperglycemia may accelerate the generation of free radicals, it has been demonstrated that advanced glycation itself participates in the production of oxygen free radicals [11]. Upon exposure to reducing sugars such as glucose, proteins undergo nonenzymatic glycation and oxidation. In the early stages of this reaction, reversible Schiff bases and Amadori products are formed within protein molecules. Amadori products undergo a series of oxidation, dehydration, and fragmentation reactions to irreversibly form yellow-brown and fluorescent adducts called advanced glycation end products (AGEs) [2]. Excessive formation and accumulation of AGEs in tissues, serum, and erythrocytes could lead to tissue damage through a variety of mechanisms: alteration of structure and function of tissue proteins [2]; stimulation of cellular responses by binding to specific cell surface receptors [12], [13], [14], [15], [16]; and generation of reactive oxygen intermediates [17]. Hence, we investigated the effect of AGEs on AR mRNA expression and the level of AR protein in human microvascular endothelial cells. To our knowledge, this is the first report indicating that AGEs may augment the expression of AR in the vascular tissue.

Section snippets

Preparation of AGEs-modified bovine serum albumin (BSA)

AGEs-modified BSA was prepared by incubating BSA (fraction V, very low endotoxin; Miles, Kankakee, IL, USA) in phosphate-buffered saline (PBS; 10 mM, pH 7.4) with 0.4 M glucose at 37°C for 6 weeks under sterile conditions. Unincorporated sugar was removed by dialysis against PBS. Unmodified BSA was prepared under the same conditions without glucose. The amount of AGEs present in AGEs-modified BSA was assessed by measuring crossline, an AGE that has cross-linking and fluorescence characteristics

Induction of AR by AGEs in endothelial cells

We first assessed the effect of AGEs on AR mRNA and AR protein levels in cultured endothelial cells. Cells were incubated for 16 h with 5 or 500 μg/ml AGEs-BSA; 50 μM H2O2; or in hyperosmotic medium (500 mOsm/kg). As shown in Fig. 1A and B , both AR mRNA and AR protein were markedly increased by addition of 500 μg/ml AGEs-BSA (mRNA 1.92-fold, protein 1.68-fold, respectively) compared with a control group incubated in serum-free medium. The extent of increased mRNA and protein levels in the

Discussion

Our present findings indicate for the first time that high concentrations of AGEs induce the expression of AR mRNA and protein in HDMVEC. Moreover, increased levels of AR protein were depicted in HDMVEC exposed to sera from diabetic patients with ESRD, even though the presence of unknown oxidant in the serum from the patients of chronic renal failure has been reported and it might contribute to increased AR expression [27]. Previously we found significant increments in AGEs in the membranes of

References (41)

  • S. Yamagishi et al.

    Advanced glycation end products-driven angiogenesis in vitro. Induction of the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor

    J. Biol. Chem.

    (1997)
  • C. Hirata et al.

    Advanced glycation end products induce expression of vascular endothelial growth factor by retinal Muller cells

    Biochem. Biophys. Res. Commun.

    (1997)
  • K. Arai et al.

    Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites

    J. Biol. Chem.

    (1987)
  • C.J. Mullarkey et al.

    Free radical generation by early glycation productsa mechanism for accelerated atherogenesis in diabetes

    Biochem. Biophys. Res. Commun.

    (1990)
  • Y. Hamada et al.

    Rapid formation of advanced glycation end products by intermediate metabolites of glycolytic pathway and polyol pathway

    Biochem. Biophys. Res. Commun.

    (1996)
  • Yabe-Nishimura, C. Aldose reductase in glucose toxicity: a potential target for the prevention of diabetic...
  • M. Brownlee et al.

    Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications

    N. Engl. J. Med.

    (1988)
  • J.V. Hunt et al.

    Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and aging

    Biochem. J.

    (1988)
  • T. Inoguchi et al.

    Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic ratsdifferential reversibility to glycemic control by islet cell transplantation

    Proc. Natl. Acad. Sci. USA

    (1992)
  • M.B. Burg

    Molecular basis of osmotic regulation

    Am. J. Physiol.

    (1995)
  • Cited by (0)

    View full text