Absence of endogenous carnosine synthesis does not increase protein carbonylation and advanced lipoxidation end products in brain, kidney or muscle

Abstract

Carnosine and other histidine-containing dipeptides are expected to be important anti-oxidants in vertebrates based on various in vitro and in vivo studies with exogenously administered carnosine or its precursor β-alanine. To examine a possible anti-oxidant role of endogenous carnosine, mice lacking carnosine synthase (Carns1^-/-) had been generated and were examined further in the present study. Protein carbonylation increased significantly between old (18 months) and aged (24 months) mice in brain and kidney but this was independent of the Carns1 genotype. Lipoxidation end products were not increased in 18-month-old Carns1^-/- mice compared to controls. We also found no evidence for compensatory increase of anti-oxidant enzymes in Carns1^-/- mice. To explore the effect of carnosine deficiency in a mouse model known to suffer from increased oxidative stress, Carns1 also was deleted in the type II diabetes model Lepr^db/db mouse. In line with previous studies, malondialdehyde adducts were elevated in Lepr^db/db mouse kidney, but there was no further increase by additional deficiency in Carns1. Furthermore, Lepr^db/db mice lacking Carns1 were indistinguishable from conventional Lepr^db/db mice with respect to fasting blood glucose and insulin levels. Taken together, Carns1 deficiency appears not to reinforce oxidative stress in old mice and there was no evidence for a compensatory upregulation of anti-oxidant enzymes. We conclude that the significance of the anti-oxidant activity of endogenously synthesized HCDs is limited in mice, suggesting that other functions of HCDs play a more important role.

Keywords: Advanced lipoxidation end products; Carnosine; Diabetes; Protein carbonylation.

Fig. 1

Carns1^−/− mice. A Schematic presentation of the wild type Carns1 gene and the Carns1 knock-out allele in Carns1^−/− mice analysed in this study. P/F indicate position of loxP and FRT sites that remain after cre mediated recombination in the knockout allele. All coding exons are deleted. B Western blot analysis (10% SDS-PAGE; 20 µg protein/lane) of skeletal muscle confirmed absence of Carns1 protein in Carns1^−/− mice. C Kaplan–Meier curve showed no significant difference between genotypes in survival rate

Fig. 2

Protein carbonylation in brain, skeletal muscle, kidney and heart from Carns1^+/+ and Carns1^−/− mice. A Tissue homogenates were treated with DNPH and analysed by 8% SDS-PAGE (10 µg protein/lane) and Western blotting using DNP specific antiserum. Age of mice was 18 months. Ponceau S staining served as loading control. In total, samples from at least 4 mice per genotype were examined. Representative experiments are shown. B Densitometric analysis showed that protein carbonyl bands at 50 kDa in kidney and 53 kDa in heart (labeled by arrow heads in A) had higher intensity in Carns1^−/− mice. Data shown are mean ± SD (n = 4 mice per genotype). Quantification data for other protein bands are given in supplementary Fig. S1

Fig. 3

Western blot analysis of MDA and HNE adducts in brain, kidney, heart and skeletal muscle from 18-month-old mice. Proteins (20 µg/lane) were separated by 10% SDS-PAGE. A MDA Western blots. B Quantification of MDA Western blots. C HNE Western blots. D Quantification of HNE Western blots. Shown are the mean ± SD; mean of Carns1^+/+ set to 1 (n = 4 mice per genotype). Quantification data for individual protein bands are shown in supplementary Fig. S2 and S3

Fig. 4

Western blot analysis of anti-oxidant enzymes. Proteins (10 µg/lane) were separated by 12.5% SDS-PAGE. A Representative Western blots of brain, heart, kidney and skeletal muscle from 18-month-old Carns1^+/+ and Carns1^−/− mice are shown. Blots were stained with antibodies against GSR, SOD1, SOD2 and tubulin. B Densitometric quantification of Western blots (normalized to tubulin). No significant differences were observed. Shown are the mean ± SD (n = 4 mice per genotype)

Fig. 5

Analysis of MDA-adducts and anti-oxidant enzymes in Lepr^db/db/Carns1^−/− mice. A Western blot analysis (10% SDS-PAGE, 20 µg protein/lane) of MDA adducts in kidneys from Lepr^db/db/Carns1^−/− mice. B Densitometric quantification of MDA Western blots (mean ± SD, n = 4 mice per genotype). *p < 0.05; ns, not significant. C Western blot analysis (12.5% SDS-PAGE; 20 µg/lane) of anti-oxidant enzymes GSR, SOD1 and SOD2 in kidneys from 5-month-old mice (genotypes as indicated). D Densitometric quantification of GSR, SOD1 and SOD2 (mean ± SD, n = 4 mice per genotype). No significant differences were observed

Fig. 6

FBG, insulin and Hb1Ac levels in Lepr^db/db/Carns1^−/− mice. A FBG was measured in male and female Carns1^−/−, Carns1^± and Carns1^+/+ mice at 2, 3 and 18 months of age (n = number of animals examined). No significant differences were observed. B Hb1Ac levels in blood from 18-month-old female Carns1^+/+ and Carns1^−/− mice (n = 5 per genotype). No significant difference was found. C Hb1Ac level in blood from 5-month-old Carns1^+/+ and Carns1^−/− mice (n = 4 per genotype). D FBG level in 2-month- and 5-month-old mice of the genotypes indicated. For the non-diabetic mice data from Carns1^−/−/Lepr^db/+ and Carns1^−/−/Lepr^+/+ (2 months: n = 11; 5 months: n = 8), as well as Carns1^+/+/Lepr^db/+ and Carns1^+/+/Lepr^+/+ (2 months: n = 24; 5 months: n = 14) were combined (n = 6 for the other genotypes). E Insulin concentration in 5-month-old mice of the genotypes indicated (n = 8 for Carns1^−/−/Lepr^db/+ and Carns1^+/+/Lepr^db/+; n = 6 for Carns1^−/−/Lepr^db/+ and Carns1^+/+/Lepr^db/+. F Body weights at 5 months of age (n = 6 per genotype). G Relative kidney weights at 5 months of age (n = 6 per genotype). All data shown are the mean ± SD. Data were analysed by one-way ANOVA (except B: t test): *p < 0.05, **p < 0.001 (one-way ANOVA with post hoc Tukey HSD test)