Arginine Reduces Glycation in γ2 Subunit of AMPK and Pathologies in Alzheimer's Disease Model Mice

Abstract

The metabolism disorders are a common convergence of Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). The characteristics of AD are senile plaques and neurofibrillary tangles (NFTs) composed by deposits of amyloid-β (Aβ) and phosphorylated tau, respectively. Advanced glycation end-products (AGEs) are a stable modification of proteins by non-enzymatic reactions, which could result in the protein dysfunction. AGEs are associated with some disease developments, such as diabetes mellitus and AD, but the effects of the glycated γ₂ subunit of AMPK on its activity and the roles in AD onset are unknown.

Methods: We studied the effect of glycated γ₂ subunit of AMPK on its activity in N2a cells. In 3 × Tg mice, we administrated L-arginine once every two days for 45 days and evaluated the glycation level of γ₂ subunit and function of AMPK and alternation of pathologies.

Results: The glycation level of γ₂ subunit was significantly elevated in 3 × Tg mice as compared with control mice, meanwhile, the level of pT172-AMPK was obviously lower in 3 × Tg mice than that in control mice. Moreover, we found that arginine protects the γ₂ subunit of AMPK from glycation, preserves AMPK function, and improves pathologies and cognitive deficits in 3 × Tg mice.

Conclusions: Arginine treatment decreases glycated γ₂ subunit of AMPK and increases p-AMPK levels in 3 × Tg mice, suggesting that reduced glycation of the γ₂ subunit could ameliorate AMPK function and become a new target for AD therapy in the future.

Keywords: AMPK; Alzheimer’s disease; L−arginine; advanced glycation end−products; glycation.

Figure 1

The level of glycation in γ₂ subunit of AMPK is increased and the level of pT172−AMPK is decreased in 3 × Tg mice. The hippocampal proteins are extracted from 7.5−month−old 3 × Tg mice and wild−type (WT) mice. (A,B) The expression and quantitative analysis of pT172−AMPK and AMPK were examined by western blot in the hippocampi of 3 × Tg and WT mice. (C,D) The level of advanced glycation end−products (AGEs) of brain proteins in 3 × Tg and WT mice were detected by dot blot. (E) Hippocampal extracts were immune−precipitated by antibody against AMPK γ₂ and then detected with antibody against AGEs. All data are presented as the means ± SD (n = 3), statistical analysis with Student’s unpaired t test; * p < 0.05 vs. WT mice.

Figure 2

Arginine decreases the glycation of proteins in a dose−dependent manner. (A,B) Bovine serum albumin (BSA, 30 mg/mL) and D−glucose (0.5 M) were incubated with L−arginine (0, 36, 180, 360 mM) at 37 °C for 3 months. Dot blot was used to measure the level of glycation with antibody against AGEs. (C,D) Mice brain proteins (13.5 mg/mL), methylglyoxal (MG, 250 mM) were reacted with L−arginine (0, 1.56, 6.25, 12.5, 50, 100, 200 mM) at 37 °C for a fortnight. Dot blot was performed. (E,F) Sequentially diluted samples from (C) (proteins + MG + Arg (6.25 mM)) were detected through dot blot with antibody against AGEs and assessed by correlation analyses. Significant correlation (r² = 0.9549, p = 0.0041). Data are exhibited as mean ± SD (n = 3). (B,D) statistical analysis with one−way ANOVA with a post hoc Turkey’s test. * p < 0.005, ** p < 0.001, **** p < 0.0001 vs. 0−Arg group.

Figure 3

Arginine mitigates glycation in γ₂ subunit of AMPK induced by MG and maintains the AMPK function in N2a cells. (A,B) The glycated proteins were detected and analyzed by dot blot with antibody against AGEs. (C) The N2a lysates were immune−precipitated with antibody against AMPK γ₂, followed by western blot with antibody against AGEs. (D–F) The level of pT172−AMPK, AMPK, pS79−ACC, and ACC were measured and analyzed by western blot in MG, MG + Arg groups. All data are presented as the means ± SD (n = 3), statistical analysis with one−way ANOVA with a post hoc Turkey’s test. * p < 0.05 vs. MG; # p < 0.05 vs. control.

Figure 4

Arginine defends γ₂ subunit of AMPK against its glycation and improves AMPK function in 3 × Tg mice. The hippocampi were extracted from 3 × Tg Saline group (Saline), 3 × Tg Arg−low group (Arg−low), 3 × Tg Arg−high group (Arg−high) at 7.5−month−old. (A,B) The levels of hippocampal AGEs were examined by dot blot with antibody against anti−AGEs. (C) Dissected hippocampi were immune−precipitated with antibody against AGEs and then examined by western blot with antibody against AMPK γ₂. (D–I) The expressions of AMPK, pT172−AMPK, pS79−ACC, ACC, Tau 5, pS396, and pS404 were examined by western blot. All data were exhibited as the means ± SD (n = 3), statistical analysis with one−way ANOVA with a post hoc Turkey’s test. * p < 0.05, ** p < 0.01 vs. Saline group, # p < 0.05 vs. Arg−low group.

Figure 5

Arginine attenuates the impairments of hippocampal−dependent spatial learning in memory in 3 × Tg mice. In the MWM test, mice (WT, n = 5; Saline group, n = 5; Arg−low, n = 8; Arg−high, n = 8) underwent 5 consecutive days for learning ability tests. (B) The latencies to reach the hidden platform were recorded each day. In the probe trial, the latency time of first reaching the platform (D), numbers of platform crossings (C), swimming speed (E), and representative swimming routes (A) were recorded. (F) The discrimination indexes were recorded. (G) Arg−high treatment increased the novel location recognition index in 3 × Tg mice. Data were expressed as mean ± SD (n = 3). (B) was analyzed by two−way repeated−measures ANOVA with a post hoc Bonferroni’s test. (C,D) were measured by one−way ANOVA with a post hoc Tukey’s test. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. 3 × Tg.