Antidiabetic drug metformin (GlucophageR) increases biogenesis of Alzheimer's amyloid peptides via up-regulating BACE1 transcription

Abstract

Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of beta-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of beta-amyloid (Abeta) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage(R)), affects APP metabolism and Abeta generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular Abeta species. Furthermore, the effect of metformin on Abeta generation is mediated by transcriptional up-regulation of beta-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on Abeta degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on Abeta. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on Abeta generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on Abeta generation, in combined use, metformin enhances insulin's effect in reducing Abeta levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients.

Fig. 1.

Effects of metformin on APP/Aβ metabolism. (A) Dose-dependent effects on extracellular Aβ40/42 in N2a695 cells as measured by ELISAs. Data represented as means from 6 independent experiments. (B) Effects on extracellular Aβ40/42 in primary neurons. Data collected from 3 independent experiments. (C) Western analysis of APP metabolites, including intracellular Aβ, sAPPα, and CTFs upon metformin treatment. (D) Alterations of surface and total protein levels of APP, LRP and BACE1 upon metformin treatment. (E) Immunocytochemistry showing increased Aβ40 species in the trans-Golgi network colocalized with the TGN marker (TGN-38) in metformin-treated cells compared with the untreated control. Fluorescent images (100× magnification): green, TGN-38; red, anti-Aβ40.

Fig. 2.

Effects of metformin on BACE 1 expression and activity. Data presented as mean values from at least 3 independent experiments. (A) Metformin's effect on BACE 1 enzymatic activity as measured by Sigma's activity assay kit. Data therefore represent the relative total BACE1 activity per cell. (B) Effect of metformin on BACE1 mRNA level. The BACE1 transcript levels were determined by semiquantitative RT-PCR in both N2a695 and primary neurons. (C) Metformin up-regulates BACE1 promoter activity. Data represents the luciferase activity of a 1.5-kb BACE1 promoter-luciferase construct after transient transfection into N2a695 cells with or without treatment with metformin (10 mM) or insulin (1 μM) for 24 h. The BACE1 promoter activity is presented with respect to the activity of a control plasmid. The gray bars represent the relative luciferase activity with full-length BACE1 promoter (BACE_F1), whereas the black bars represent the activity of the truncated promoter (BACE1_F2) lacking the first 3 PPAR/RXR binding elements as illustrated in the scheme above the bar graph. In both settings, metformin up-regulates the promoter activity of BACE1_F1 and BACE1_F2 to a similar degree, suggesting a PPARγ-independent mechanism. n = 5.

Fig. 3.

Metformin's effect is independent of glucose levels and insulin signaling. (A) For low glucose conditions, cells were cultured in low-glucose DMEM (5 mM) overnight and then metformin was added for an additional 24 h in this condition compared with normal glucose (25 mM). For serum-free conditions, cells were cultured in DMEM/Opti-MEM for 24 h. Intracellular Aβ production was measured by ELISA of lysates (diluted 50-fold) collected from cells after the last 4 h incubation in serum-free media. (B) Effects of various tyrphostins on metformin's modulation of Aβ levels. Cells were pretreated with metformin for 24 h and inhibitors were added at 10 μM concentrations the second day after switching to serum-free media. Aβ ELISAs were performed using cell lysates collected 4 h after incubation with various inhibitors. n = 4.

Fig. 4.

Metformin's effect depends on AMPK activation. (A) Metformin activates AMPK in N2a695 cells. Western blot analysis shows a marked elevation of the Thr-172 phosphorylated AMPK. (B) Dose-dependent effect of metformin on activating ACC, the AMPK downstream substrate, as measured by Western blot analysis of the phosphorylated ACC (Ser-79). (C) Effect of the AMPK inhibitor Compound C (Comp. C) on abolishing metformin's effect on Aβ levels. Comp. C completely abolishes the effect of metformin on intracellular Aβ production as measured by ELISA of cell lysates (diluted 50-fold). (D) Combinatory effects of metformin and Comp. C on BACE1 transcription as determined by semiquantitative RT-PCR.

Fig. 5.

Metformin activates AMPK/BACE1 in WT C57B6 mice. (A) Western analysis of phosphorylated AMPK, phosphorylated ACC and BACE1 protein levels in mouse brain lysates (frontal region) after receiving metformin in drinking water for 6 days. n = 4 animals in each groups. (B) The bar graph shows quantitative data of A.

Fig. 6.

Combination of insulin and metformin reduces Aβ generation. (A) N2a695 cells were pretreated with 10 mM metformin for 24 h, and exogenous insulin was added at the 2 concentrations (0.25 μM or 1 μM) in serum-free media for an additional 4 h in combination with metformin. Intracellular Aβ levels were measured by IP-Western analysis. To measure insulin's effect, it was added directly to the cultures in serum-free media for 4 h before the Aβ assays. (B) The bar graph shows quantitative data of the representative IP-Western blot of A. n = 3.