RESUMO
Human serum albumin nonenzymatically condenses with glucose to form stable Amadori adducts that are increased with the hyperglycemia of diabetes. The present study evaluated the influence of fatty acids, which are major endogenous ligands, on albumin glycation and of glycation on albumin conformation and exogenous ligand binding. Physiologic concentrations of palmitate, oleate, and linoleate reduced the ability of albumin to form glucose adducts, whereas glycation decreased intrinsic fluorescence, lowered the affinity for dansylsarcosine, and diminished the fatty acid-induced increase in limiting fluorescence of protein-bound warfarin that was observed with nonglycated albumin. The findings indicate that fatty acids impede the ability of albumin to undergo Amadori glucose modification and induce conformational changes affecting exogenous ligand binding, and that nonenzymatic glycation of albumin induces alterations in structural and functional properties that may have import in lipid transport and atherogenesis.
Assuntos
Compostos de Dansil/metabolismo , Ácidos Graxos Insaturados/metabolismo , Glicosilação , Sarcosina/análogos & derivados , Albumina Sérica/metabolismo , Varfarina/metabolismo , Fluorescência , Glicosilação/efeitos dos fármacos , Humanos , Ácido Linoleico/metabolismo , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Sarcosina/metabolismoRESUMO
Glomerular cells in culture respond to albumin containing Amadori glucose adducts (the principal serum glycated protein), with activation of protein kinase C-beta(1), increased expression of transforming growth factor (TGF)-beta1, the TGF-beta type II signaling receptor, and the extracellular matrix proteins alpha(1)(IV) collagen and fibronectin and with decreased production of the podocyte protein nephrin. Decreasing the burden of glycated albumin in diabetic db/db mice significantly reduces glomerular overexpression of TGF-beta1 mRNA, restores glomerular nephrin immunofluorescence, and lessens proteinuria, mesangial expansion, renal extracellular matrix protein production, and increased glomerular vascular endothelial growth factor (VEGF) immunostaining. In the present study, db/db mice were treated with a small molecule, designated 23CPPA, that inhibits the nonenzymatic condensation of glucose with the albumin protein to evaluate whether increased glycated albumin influences the production of VEGF receptors (VEGFRs) and type IV collagen subchains and ameliorates the development of renal insufficiency. Renal levels of VEGF and VEGFR-1 proteins and serum creatinine concentrations were significantly higher and renal levels of alpha(3)(IV) collagen and nephrin proteins and endogenous creatinine clearance values were significantly lower in control diabetic than in age-matched nondiabetic (db/m) mice. These changes were significantly attenuated in db/db littermate mice treated from 9 to 18 wk of age with 23CPPA. The findings indicate that inhibiting excess nonenzymatic glycation of serum albumin improves renal molecular biology abnormalities and protects against the development of renal insufficiency in the db/db mouse.
Assuntos
Colágeno Tipo IV/biossíntese , Diclofenaco/análogos & derivados , Glicosilação/efeitos dos fármacos , Rim/efeitos dos fármacos , Insuficiência Renal/prevenção & controle , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Albuminas/química , Animais , Creatinina/sangue , Diabetes Mellitus Experimental/fisiopatologia , Diclofenaco/farmacologia , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos ObesosRESUMO
BACKGROUND: Albumin modified by Amadori-glucose adducts has been linked to the development of diabetic nephropathy through its ability, independent of hyperglycemia, to activate protein kinase C-beta (PKC-beta), up-regulate the transforming growth factor-beta (TGF-beta) system, and stimulate expression of extracellular matrix proteins in glomerular cells, and by the demonstration that reducing the burden of glycated albumin ameliorates renal structural and functional abnormalities in the db/db mouse. METHODS: To probe whether the salutary effects consequent to lowering glycated albumin, which include reduction of albuminuria, relate to an influence of the Amadori-modified protein on nephrin, the podocyte protein critical to regulation of protein excretion, and on the angiogenic vascular endothelial growth factor (VEGF), which induces microvascular permeability, diabetic db/db mice were treated with a small molecule that inhibits the nonenzymatic glycation of albumin. RESULTS: Compared to nondiabetic db/m mice, diabetic controls exhibited increased urinary excretion of albumin and type IV collagen, elevated renal TGF-beta1 protein levels, reduced glomerular nephrin immunofluorescence and nephrin protein by immunoblotting, and increased glomerular VEGF immunostaining and renal VEGF protein content. Diabetic animals receiving test compound showed significant lowering of proteinuria, normalization of renal TGF-beta1 protein, and significant restoration of altered glomerular nephrin and VEGF expression. CONCLUSION: The findings causally implicate the increased glycated albumin associated with the diabetic state in the abnormal renal nephrin and VEGF expression found in diabetes, thereby promoting proteinuria and glomerulosclerosis.
Assuntos
Nefropatias Diabéticas/metabolismo , Glomérulos Renais/metabolismo , Proteínas de Membrana/metabolismo , Proteinúria/metabolismo , Albumina Sérica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Albuminúria/metabolismo , Animais , Colágeno Tipo IV/urina , Imunofluorescência , Produtos Finais de Glicação Avançada , Masculino , Camundongos , Camundongos Mutantes , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Albumina Sérica GlicadaRESUMO
Nonenzymatic glycation of apolipoprotein B in the low-density lipoprotein (LDL) complex has been considered a proatherogenic modification contributory to the increased susceptibility of patients with diabetes to atherosclerosis. We postulated that glycated LDL concentrations might be associated with other markers of cardiovascular disease. To explore this hypothesis, we measured glycated LDL concentrations by a monospecific immunoassay in 50 patients with type 1 and 100 patients with type 2 diabetes and examined relationships with the amount of albumin excretion and the serum cholesterol and triglyercide concentrations. Plasma glycated LDL showed a significant positive correlation (r = 0.325; P < 0.001) with urinary albumin excretion that was higher in type 1 (r = 0.463) than in type 2 (r = 0.245) patients. The mean glycated LDL concentration progressively increased with increasing albumin excretion when patients were subcategorized into groups of normoalbuminuria, low (=100 microg/mg of creatinine), and high (101-300 microg/mg) microalbuminuria, and proteinuria. Glycated LDL also correlated positively and significantly with cholesterol (r = 0.578) and triglyceride (r = 0.350) concentrations. The significant correlations in this cross-sectional analysis between glycated LDL and urinary albumin excretion, an index of cardiovascular mortality, and cholesterol and triglyceride concentrations, traditional markers of risk for cardiovascular disease, support the hypothesis that an elevated level of glycated LDL represents an atherogenic risk factor in patients with diabetes.
Assuntos
Arteriosclerose/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Lipoproteínas LDL/sangue , Adulto , Idoso , Albuminúria , Biomarcadores/sangue , Feminino , Hemoglobinas Glicadas/análise , Produtos Finais de Glicação Avançada , Humanos , Masculino , Pessoa de Meia-Idade , Análise de RegressãoRESUMO
BACKGROUND: Glycated albumin has been causally linked to the pathobiology of diabetic renal disease through its ability to stimulate the expression of transforming growth factor-beta1 (TGF-beta1), activate protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), and promote production of extracellular matrix proteins in cultured glomerular cells. Whether glycated albumin modulates glomerular TGF-beta1 expression in vivo is not known. To address this issue, we assessed glomerular TGF-beta1 expression and pathology in response to reducing the burden of glycated albumin in vivo. METHODS: We measured serum glycated albumin, urine protein, glomerular TGF-beta1 expression and morphometry, and collagen IV and fibronectin mRNA in db/m and db/db controls and in db/db mice treated for eight weeks with a synthetic compound that inhibits the condensation of glucose with albumin. RESULTS: In situ hybridization studies showed markedly increased glomerular TGF-beta1 mRNA in control db/db mice, which was significantly reduced in db/db mice treated for eight weeks with test compound. The treatment protocol, which normalized serum glycated albumin, concomitantly reduced the elevated protein excretion and the renal overexpression of mRNAs encoding fibronectin and collagen IV, and significantly decreased the mesangial matrix expansion, observed in db/db control animals. CONCLUSIONS: These findings, to our knowledge, provide the first evidence that glomerular overexpression of TGF-beta1 in diabetes derives at least in part from elevated glycated albumin concentrations, and can be partially suppressed by inhibiting the formation of this glycated protein. The results further suggest that glycated albumin has an important nephropathogenic role in diabetes that is operative, and can be therapeutically addressed, independent of glycemic status.