Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: the response to glycated and nonglycated BSA is lost in metabolic syndrome

Abstract

The effects of nonglycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia, and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37 degrees C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six month old animals were used. Blood pressure, insulin, triglycerides, and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of nonglycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Nonglycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using nonglycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion, both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness.

Fig. 1

Fluorescence spectrum of AGE-BSA and nonglycated BSA. Both solutions were tested at 2 mg/mL of protein. The peak of fluorescence is not present in the nonglycated BSA. The interrupted line represents the saturation level of the fluorescence detection by the equipment employed.

Fig. 2

Purification of AGE-BSA on Affi-Gel-Blue. Three peaks were observed: the first one corresponds to highly glycated BSA which did not bind to the matrix, the second one corresponds to the less glycated BSA which bound moderately, and the third peak corresponds to the stronger bound BSA. The first peak was the only one to be further analyzed. The arrows indicate the buffer used at each stage.

Fig. 3

Evaluation of the specific activity (SA) of AGE-BSA. The increase of specific activity obtained by the purification process on Affi-Gel-Blue is observed. Bold circles represent the nonglycated BSA. Bold squares represent the unpurified AGE-BSA and bold triangles represent the purified AGE-BSA. Specific activity increased in approximately 33% by the purification process.

Fig. 4

Analysis of BSA and AGE-BSA by mass spectrometry. The mass increase of protein after glycation is observed. The mass of nonglycated BSA was 66,655.58 Da (A) and that of AGE-BSA was 74,461.15 Da (B). The net increase of mass was 7,805.57 Da, corresponding to the addition of 48 molecules of glucose to each BSA molecule. Inset: change of isoelectric point of BSA after glycation. Nonglycated BSA had a pI of 4.2 while AGE-BSA had a pI of 6.3. Line 2 is the broad range pI standard kit (pH 3–10).

Fig. 5

Effect of AGE-BSA on vascular contraction in aortic rings from control (solid bars) and MS rats (open bars). The contractions were induced by NE 1 μM and basal tension was normalized to 100% in control and MS rats. Tension values in grams are shown in Table III. Results are the means ± SEM of six independent experiments. ^*P < 0.05 between MS and control; ^**P < 0.001 between the Tyrode solution and L-NAME.

Fig. 6

Effect of nonglycated BSA and AGE-BSA at 40 AU/mL (3.8 mg/mL) on endothelium-dependent vasorelaxation in aortic rings from control (A) and MS rats (B). Results are expressed as relaxation percentage from the initial precontraction level with NE 1 μM. ^*P < 0.05 between the Tyrode solution and nonglycated BSA and AGE-BSA; ^#P < 0.05 between the Tyrode solution in MS and control rats.

Fig. 7

Immunohistochemistry for RAGE in aortas of control (Panel A) and MS (Panel B) rats. Negative control (Panel C). Magnification 40×. Asterisk indicates internal elastic lamina. There is an increased RAGE immunoreactivity in MS rats.