Role of Glyceraldehyde-Derived AGEs and Mitochondria in Superoxide Production in Femoral Artery of OLETF Rat and Effects of Pravastatin

Abstract

A complication of diabetes mellitus is the over-production of vascular superoxides, which contribute to the development of arteriosclerosis and peripheral arterial disease (PAD). Hyperglycemia induces the formation and accumulation of advanced glycation end-products (AGEs), which in turn stimulate vascular superoxide production. The mechanism underlying AGE-mediated vascular superoxide production remains to be clarified in lower limb complications associated with diabetes. In the present study, we investigated the role of AGEs and the mitochondrial respiratory complex in superoxide production in femoral arteries using the type 2 diabetes model Otsuka Long-Evans Tokushima Fatty (OLETF) rats [vs. non-diabetic Long-Evans Tokushima Otsuka (LETO) rats]. The effects of in vivo administration of pravastatin on superoxide production in femoral arteries were also examined. Using chemiluminescent assays, luminescence microscopy, and competitive enzyme-linked immunosorbent assay (ELISA), we determined that vascular superoxide production and serum glyceraldehyde-derived AGEs (Glycer-AGEs) increased in OLETF rats. Pravastatin inhibited these responses without changing serum total cholesterol concentrations. The mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA) also inhibited vascular superoxide production. Application of GlycerAGEs in situ increased superoxide production in the vascular wall of femoral arteries from pravastatintreated OLETF rats, which was then inhibited by TTFA. These results suggest that hyperglycemia increases serum Glycer-AGEs, which subsequently induce superoxide production in the femoral artery of OLETF rats in a mitochondrial complex II-dependent manner. Collectively, our results have partially elucidated the pathological mechanisms leading to diabetes-related PAD, and indicate dual beneficial actions of pravastatin for the prevention of oxidative damage to the vascular wall.

Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End-Products?

“…Furthermore, many commercial ELISA kits can be used to analyze major proteins. ELISA can be performed on various samples such as serum [5,[61][62][63][64][65][66][67], plasma [68], urine [69], saliva [5], supernatants [9,62], cell lysates [8,70], and tissue lysates [8]. The target in ELISA is generally one protein, such as interleukin (IL)-1β, IL-6, IL-17, IL-18, or tumor necrosis factor (TNF)-α [61,62,70].…”

“…Although AGEs have fluorescence, Indyk et al and Pinto et al especially focused on the fluorescence of pentosidine and its measurement in serum and plasma; the excitation/emission wavelengths were set at 335/385 nm (AU) [63,68]. In other studies, crude AGE proteins (e.g., various MGO-AGE-modified proteins) in serum [63,[65][66][67], plasma [68], saliva [5], cell lysates [9], and tissue lysates [8] were measured. As the fluorescence of AGEs can be measured at an excitation wavelength of 370 nm and an emission wavelength at 440 nm, Indyk et al and Pinto et al measured the total AGEs in serum and plasma (AU) [63].…”

“…As the fluorescence of AGEs can be measured at an excitation wavelength of 370 nm and an emission wavelength at 440 nm, Indyk et al and Pinto et al measured the total AGEs in serum and plasma (AU) [63]. However, some reports have quantified AGEs using standard AGE-modified proteins, e.g., low-molecular weight fraction of melibiose-derived glycation products [63], GA-AGE-BSA [65], TAGE-BSA [66], 1,5-AF-AGE-modified BSA [67], CML-modified BSA [8], and MGO-modified BSA [9]. Because researchers have quantified the mass of AGE-modified proteins as a standard, the amounts of crude AGEs have been calculated with their AGEmodified proteins.…”

“…Whether this novel slot blot method can measure other AGEs remains unclear. Although only the quantification of AGEs in cell lysates and tissue lysates has been reported for novel slot blot [15,[20][21][22][23][24][25][26][27][28][29][30], ELISA can measure AGEs in serum [5,[61][62][63][64][65][66][67], plasma [68], urine [69], saliva [5], supernatants [9,62], cell lysates [8,70], and tissue lysates [8]. It is not certain whether the AGEs in the serum, plasma, urine, and saliva can be measured using the novel slot blot.…”

Effect of Topiroxostat on Reducing Oxidative Stress in the Aorta of Streptozotocin-Induced Diabetic Rats

“…The effect of topiroxostat on intracellular XOR activity was investigated by oxidative fluorescence staining with dihydroethidium, as previously described. 24,25) Segments of aorta stored in Optimal Cutting Temperature (OCT) compound were used for these experiments. A frozen segment of embedded aorta was cut into 8-µm thicknesses under a cryostat and then mounted on MAS-coated glass slides.…”