Butyrate acts as a G-protein-coupled receptor ligand that prevents high glucose-induced amyloidogenesis in N2a cells through the protein kinase B/glycogen synthase kinase-3β pathway.

Abstract

To the Editor: Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are major causes of morbidity and mortality in older individuals, and their epidemiological link generates substantial challenges for the management of these devastating diseases. Abnormal glucose metabolism, accompanied by inhibition of the protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) signaling pathway, is a key factor in amyloid precursor protein (APP) accumulation and amyloid β (Aβ) formation in neuronal cells.[1-3] Therefore, activation of the Akt/GSK-3β pathway may be a promising target for AD treatment. Several lines of evidence indicated that a major microbial metabolite, butyrate (NaB) downregulated the amyloidogenic pathway and improved learning or memory ability in vivo or in vitro models of AD. NaB improved glucose metabolism in T2DM models through the Akt/GSK-3β pathway, which suggests its beneficial effect in the management of T2DM and AD.[4] However, there are few studies evaluating the mechanism of NaB on T2DM-related AD. Therefore, we used mouse neuroblastoma-derived N2a cells (serial TCM29, obtained from National Collection of Authenticated Cell Cultures of China), a cell model extensively used for studying AD-related signal transduction, to investigate the potential impact and mechanism of NaB on high glucose-induced amyloidogenesis. We hypothesized that high glucose exposure would inhibit Akt and GSK-3β phosphorylation and induce amyloidogenesis in N2a cells, and NaB would downregulate pathological changes via activation of the Akt/GSK-3β pathway. To establish a cell model of abnormal glucose metabolism, N2a cells were exposed to different concentrations of glucose (25, 50, 75, 100, 125, 150 mmol/L) for 24, 48, or 72 h, and mannitol was used to exclude the effect of high osmolarity [Supplementary Figure 1A–1E, https://links.lww.com/CM9/B383]. Compared to 5.5 mmol/L glucose (control), cell viability within 24 h decreased significantly from 90.15 ± 2.21% to 55.87 ± 1.22%. We detected markers of glucose uptake and metabolism, that is, glucose transporter 3 (GLUT3), glucose transporter 4 (GLUT4), and phosphorylated insulin receptor substrate 1 (p-IRS1). After 24 h of high glucose treatment, we found that the expression levels of GLUT3, GLUT4, and p-IRS1 were statistically significantly reduced in the 75 mmol/L glucose group. Furthermore, the transcription and protein levels of APP and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) were examined to upregulate in the 75 and 100 mmol/L glucose groups [Supplementary Figure 1F–1H, https://links.lww.com/CM9/B383]. These findings suggest that 75 mmol/L glucose treatment for 24 h induces amyloidogenesis, and the cell model was used in the subsequent investigations. Next, we investigated the effect of NaB on high glucose-induced amyloidogenesis and compared to acetate (NaA) and propionate (NaP), the other two major short-chain fatty acids (SCFAs). Different concentrations ranging from 0.5 to 8.0 mmol/L NaA, NaB, and NaP did not statistically significantly decrease cell activity. To further determine the effects of SCFAs on amyloidogenesis under high glucose conditions, related mRNA and protein levels in N2a cells under pretreatment with 0.5, 1.0, or 2.0 mmol/L NaA, NaB, or NaP for 2 h before 75 mmol/L glucose stimulation for 24 h were assessed. As shown in Supplementary Figure 2, https://links.lww.com/CM9/B383, NaB and NaP, but not NaA decreased APP and BACE1 mRNA levels, and APP and BACE1 protein concentrations were decreased by 1 mmol/L and 500 μmol/L NaB treatment, and 500 μmol/L NaB showed the most significant inhibitory effect. Similar results were obtained from immunostaining: cells in the hyperglycemia model exhibited a higher level of Aβ than control cells, and pretreatment with 500 μmol/L NaB decreased the high glucose-induced accumulation of Aβ [Supplementary Figure 3A, https://links.lww.com/CM9/B383]. These results indicated that the inhibitory effects of NaA, NaB, and NaP on AD-related pathology were different despite their similar structure, which was documented in other studies and partially related to different water solubilities and affinities for cell receptors. However, the exact underlying mechanisms require further investigation. Moreover, Akt and GSK-3β phosphorylation levels were assessed in N2a cells treated with various glucose concentrations and SCFAs. High glucose (50, 75, and 100 mmol/L) lowered the relative p-Akt expression in cells in a concentration-dependent manner [Supplementary Figure 4A, https://links.lww.com/CM9/B383]. Only NaB treatment increased the level of p-Akt by a small margin in the 2 mmol/L and 1 mmol/L NaB-treated groups and significantly reversed the glucose-stimulated reduction of p-Akt to 96.39 ± 7.11% in the 500 μmol/L NaB-treated group [Supplementary Figure 4B–4D, https://links.lww.com/CM9/B383]. Meanwhile, the p-GSK-3β level shown in immunofluorescence, a downstream signal of Akt, was lowest in the glucose-treated group and was higher in the NaB-treated group than the glucose-treated group [Supplementary Figure 3B, https://links.lww.com/CM9/B383]. In agreement, the p-GSK-3β protein level in the high glucose group showed a significant reduction to 56.45 ± 6.62% compared to the control, and pretreatment with 500 μmol/L NaB significantly prevented their reduction in the glucose-induced group [Figure 1A]. These results showed that high glucose diminished Akt-mediated GSK-3β deactivation and enhanced amyloidogenesis in N2a cells.Figure 1: Inhibitory effect of NaB on high glucose-induced amyloidogenesis through inhibition of GSK-3β pathway. N2a cells were pretreated with NaB and ARA (5 μmol/L) for 2 h prior to treatment of high glucose for 24 h. The expression of p-GSK-3β (A), APP (B), BACE1 (C) were measured by Western blotting. GAPDH was used as a loading control. n = 6. Data are represented as individual points, ∗ P < 0.05, † P < 0.01 compared to the control group, ‡ P < 0.05 compared to the treatment of high glucose and NaB group. APP: amyloid precursor protein; BACE1: Beta-site amyloid precursor protein cleaving enzyme 1; GAPDH: Glyceraldehyde-3phosphate dehydrogenase; GSK-3β: glycogen synthase kinase-3β.To further determine the internal signaling pathway, we used the GSK-3β inhibitor N-(4-methoxybenyl)-N′-(5-nitro-1,3-thiazol-2-yl) urea (Sigma-Aldrich; AR-A014418) to investigate whether the Akt/GSK-3β pathway interfered with this progress. Compared to the glucose-treated group, N2a cells exposed to 5 μmol/L GSK-3b inhibitor or NaB before glucose treatment had significantly reduced APP and BACE1 levels [Figure 1B and 1C], which showed that high glucose-induced upregulation of APP and BACE1 was partially dependent on the inhibition of GSK-3β. Pretreatment with 500 μmol/L NaB in the presence of 5 μmol/L GSK-3b inhibitor significantly decreased the levels of APP and BACE1 compared to NaB pretreatment, indicating that the effect of NaB on high glucose-induced amyloidogenesis occurred through activation of the Akt/GSK-3β pathway. NaB affects cell signaling pathways through two different approaches, inhibition of class I and most class II histone deacetylase (HDAC) or interaction with G-protein-coupled receptor (GPCR), especially the Gαi family of G proteins. In this study, we examined the activated way of NaB by using pertussis toxin (PTX) to inhibit Gαi and ibuprofen to inhibit sodium-coupled monocarboxylate transporter 1, which is the co-transporter for the entry of HDAC inhibitors. As shown in Supplementary Figure 5, https://links.lww.com/CM9/B383, cells in the presence of NaB and PTX prevented the ability of NaB to affect high glucose-induced alterations in APP and BACE1 expression; in contrast, when cells were pretreated with NaB and ibuprofen, NaB decreased high glucose-induced APP and BACE1 upregulation. These findings suggest that NaB was absorbed into N2a cells via GPCRs, which activated Gαi to attenuate high glucose-induced amyloidogenesis. The present study provides several important findings on the effects of NaB on high glucose-induced amyloidogenesis. First, N2a cells treated with a high concentration of glucose (75 mmol/L, more than threefold the normal concentration) for 24 h exhibited significant insulin resistance and induced amyloidogenesis. Similar conclusions were reported in the correlation between long-term hyperglycemia and insulin resistance of T2DM and AD-related pathology and cognitive impairment. Second, our study found that high glucose treatment decreased activated Akt, which activated GSK-3β and enhanced APP processing, suggesting that high glucose induces amyloidogenesis via GSK-3β signaling. Akt is a serine/threonine-protein kinase that is an important upstream regulator of the inhibitory phosphorylation of GSK-3β involved in insulin receptor signaling. Several studies evaluated the underlying mechanisms of AD and T2DM and suggested that impaired insulin signaling played a critical role in the pathogenesis of both disorders. Third, we found the involvement of the Akt/GSK-3β pathway in NaB-mediated anti-amyloidogenesis effect in neuronal cells under high glucose conditions. Increasing evidence suggests that Akt/GSK-3β signaling is linked to age-related structural and functional changes in humans and is crucial to protect against several stress stimuli in neuronal cells. GSK-3β activity may play a central role in the development of AD, and GSK-3β activity was implicated in APP processing and Aβ production.[5] Overexpression of GSK-3β resulted in amyloidogenesis and neuronal death, and inhibition of GSK-3β activity decreased Aβ production and plaque accumulation in transgenic models of AD.[6] Other biological substances or drugs such as neuroglobin, ethosuximide, and ghrelin, were also found to alleviate amyloidogenesis by activating the Akt/GSK-3β pathway in cell and animal models of AD. Among them, NaB may be the most promising compound to target high glucose-induced amyloidogenesis. Firstly, humans could obtain NaB supplementation by consuming dietary carbohydrates, and diets rich in fiber were reported to decrease brain Aβ and improve memory in AD models and older adults.[7] Besides, gut microbiota generates NaB, and prebiotics and probiotics to increase NaB production would be a rather innovative approach for AD treatment. Taken together, we revealed that NaB inhibited high glucose-induced amyloidogenesis in N2a cells by acting as a GPCR ligand and mediating the Akt/GSK-3β pathway. Our findings provide new insights into the prevention and treatment of T2DM-associated AD pathogenesis. Funding This study was supported by grants from the Active Health and Aging Technologic Solutions Major Project of the National Key R&D Program (No. 2020YFC2006300), the Department of Science and Technology of Sichuan Province (No. 2021YJ0156), and the West China Nursing Discipline Development Special Fund Project of Sichuan University (No. HXHL20006). Conflicts of interest None.