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. 2018 Sep 13;19(9):2751.
doi: 10.3390/ijms19092751.

Protective Actions of Anserine Under Diabetic Conditions

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Free PMC article

Protective Actions of Anserine Under Diabetic Conditions

Verena Peters et al. Int J Mol Sci. .
Free PMC article

Abstract

Background/aims: In rodents, carnosine treatment improves diabetic nephropathy, whereas little is known about the role and function of anserine, the methylated form of carnosine.

Methods: Antioxidant activity was measured by oxygen radical absorbance capacity and oxygen stress response in human renal tubular cells (HK-2) by RT-PCR and Western-Immunoblotting. In wildtype (WT) and diabetic mice (db/db), the effect of short-term anserine treatment on blood glucose, proteinuria and vascular permeability was measured.

Results: Anserine has a higher antioxidant capacity compared to carnosine (p < 0.001). In tubular cells (HK-2) stressed with 25 mM glucose or 20⁻100 µM hydrogen peroxide, anserine but not carnosine, increased intracellular heat shock protein (Hsp70) mRNA and protein levels. In HK-2 cells stressed with glucose, co-incubation with anserine also increased hemeoxygenase (HO-1) protein and reduced total protein carbonylation, but had no effect on cellular sirtuin-1 and thioredoxin protein concentrations. Three intravenous anserine injections every 48 h in 12-week-old db/db mice, improved blood glucose by one fifth, vascular permeability by one third, and halved proteinuria (all p < 0.05).

Conclusion: Anserine is a potent antioxidant and activates the intracellular Hsp70/HO-1 defense system under oxidative and glycative stress. Short-term anserine treatment in diabetic mice improves glucose homeostasis and nephropathy.

Keywords: Hsp70; anserine; carnosine; diabetes; diabetic nephropathy; proteinuria; vascular permeability.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effect of co-incubation with high glucose and anserine in human tubular cells (HK-2) on cellular heat shock protein 70 (Hsp70), hemeoxygenase (HO-1), Sirtuin-1 (Sirt-1) and Thioredoxin (Trx). Hsp70 (A), HO-1 (B), Sirt-1 (C) and Trx (D) cellular protein concentrations significantly increased in HK-2 cells with glucose stress (25 mM for 24 h), determined by Western blotting, compared to cells incubated with medium containing normal glucose concentration (11 mM). Densitometric units (D.U.) after normalization against β-actin are given (n = 3). Co-incubation with anserine (0.1 and 1 mM) further increased Hsp70 and HO-1 protein but had no additional effect on Sirt-1 and Trx. Anserine alone does not alter tubular cell defense systems. p < 0.05 (*); p < 0.01 (**); n.s. = not significantly.
Figure 2
Figure 2
Effect of co-incubation with glucose and anserine in human tubular cells (HK-2) on total protein carbonylation. Glucose stress (25 mM) increased total protein carbonylation in HK-2 cells, compared to cells incubated under normal glucose concentration (11 mM; n = 3). Co-incubation with anserine (0.1 and 1 mM) reduced protein carbonylation. In unstressed cells, anserine had no effect on overall protein carbonylation. Protein carbonylation was visualized by derivatization with 2,4-dinitrophenolhydrazine (DNPH) and quantified immunochemically.
Figure 3
Figure 3
Effect of anserine and carnosine in human tubular cells exposed to oxidative and glycative stress. Human tubular cells (HK-2) were stressed by H2O2 (60 µM) and glucose (25 mM) and co-incubated with 1 mM anserine (red bars) and carnosine (blue bars), respectively, compared to control (grey bars). Cellular heat shock protein 70 (Hsp70) mRNA was measured by RT-PCR and normalized to expression of β-actin. Hsp70 expression significantly increased with co-incubation of anserine but not with carnosine. Since a nitrated form of anserine was applied, an independent effect of nitrate (green bars) on Hsp70 was ruled out. p < 0.01 (**); p < 0.001 (***).
Figure 4
Figure 4
Antioxidant capacity of carnosine and anserine. (A) Antioxidant capacity of anserine and carnosine were assessed by a standardized oxygen radical absorbance capacity (ORAC) assay. Antioxidant capacity, given as relative fluorescence units (RFU), of anserine (red line) is significantly higher as compared to carnosine (black line). (B) Molecular structure of carnosine and its methylated derivate anserine.
Figure 5
Figure 5
Effects of anserine on blood glucose, proteinuria and renal vascular permeability in diabetic mice (db/db). Twelve-week-old db/db were treated with three intravenous anserine injections every other day and scarified at week 14. Anserine treatment lowered blood glucose (A), proteinuria (B) and vascular permeability (C). p < 0.05 (*); p < 0.001 (***).

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