Activation of AMPK by GLP-1R Agonists Mitigates Alzheimer-Related Phenotypes in Transgenic Mice

I. Research Background and Scientific Question

Alzheimer’s disease (AD) is the most common neurodegenerative disease worldwide, with pathological features mainly including neuronal loss, neurofibrillary tangles, and senile plaques (mainly formed by amyloid-β [Aβ] deposition). According to statistics, the number of AD patients continues to rise each year, posing a serious threat to the quality of life of the elderly population and placing a huge burden on society and healthcare systems.

Meanwhile, epidemiological studies have found that patients with type 2 diabetes mellitus (T2DM) are at a significantly increased risk of developing AD. Disruption of glucose homeostasis and insulin resistance in the brain are closely related to the pathogenesis of AD. Increasing evidence indicates that abnormal glucose metabolism, impaired energy metabolism, and damage to insulin signaling may serve as important biomarkers for the early diagnosis of AD, with the potential to play a role in early intervention and prevention of the disease.

Driven by the development of diabetes management drugs, glucagon-like peptide-1 receptor agonists (GLP-1RAs) have been widely used for glycemic control in T2DM. Furthermore, a growing amount of clinical cohort and basic research indicates that GLP-1RAs possess neuroprotective properties and show broad therapeutic prospects in AD and other neurodegenerative diseases. However, the mechanisms by which GLP-1RAs affect AD pathology are not yet fully clarified, especially their impact mechanisms in cerebral energy metabolism, Aβ generation, and neuroinflammation, which urgently require systematic investigation.

Against this background, the present paper systematically explores the role of GLP-1R agonists in the pathogenesis of AD, seeking to elucidate their molecular mechanisms and propose new ideas for the prevention and treatment of AD.

II. Paper Source and Basic Information

This paper, titled “activation of ampk by glp-1r agonists mitigates alzheimer-related phenotypes in transgenic mice,” was jointly completed by Yun Zhang, Huaqiu Chen, Yijia Feng, and other scholars, with the main authors coming from Wenzhou Medical University, Dalian Medical University, the University of Hong Kong, and other well-known domestic and international universities and medical research institutions. The article was published in June 2025 in the internationally renowned journal “Nature Aging” (Volume 5, pages 1097-1113).

III. Research Design and Specific Workflow

This research is an original basic medical study combining human data, animal models, cell experiments, and molecular mechanisms in a multi-dimensional approach to systematically elucidate how GLP-1R agonists regulate multiple AD pathological phenotypes via the AMPK pathway. The specific workflow is as follows:

1. Correlation Analysis of GLP-1 Levels and AD Pathology in Human Populations and Model Animals

• Subjects and Methods: Peripheral plasma samples were collected from AD model mice (app23/ps45 double transgenic mice) and wild-type (WT) control mice (12 in each group, with equal numbers of males and females) to measure GLP-1 levels. Meanwhile, plasma was collected from 12 strictly selected (excluding diabetes and other confounding factors) AD patients, and 18F-AV45 PET imaging was used to assess brain Aβ deposition load, with correlation analysis between GLP-1 levels and brain Aβ pathology.
• Experimental Techniques: Enzyme-linked immunosorbent assay (ELISA) to quantify GLP-1 levels; PET/MR with quantitative analysis of regional brain Aβ plaque burden.

2. Effects of GLP-1RA on Energy Metabolism of Neurons and Astrocytes in AD Model Mice

• Subjects and Methods: Embryonic day 17 (E17) primary neurons and astrocytes were isolated from AD model and WT mice to study the regulatory effects of GLP-1RA (Exendin-4) on glucose uptake, ATP generation, fatty acid oxidation (FAO), oxidative phosphorylation (Oxphos), and other metabolic activities.
• Technical Highlights: Seahorse XF96 cell mitochondrial stress test was used to assess cellular oxygen consumption rate (OCR) and ATP synthesis capacity. Immunofluorescence was used to detect neuronal membrane glucose transporter GLUT3 distribution, with analysis of cellular metabolic status via metabolic products (such as pyruvate and reactive oxygen species [ROS]).

3. Molecular Mechanisms of the AMPK Signaling Pathway

• Subjects and Methods: Focusing on AMP-activated protein kinase (AMPK) signaling, the study used pharmacological activators (AICAR), inhibitors (Compound C), and gene editing (shRNA interference of GLP-1R and AMPK) to verify the dependence and activation pathway of AMPK under GLP-1RA treatment.
• Mechanistic Exploration: The roles of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and liver kinase B1 (LKB1) in AMPK phosphorylation were investigated, with cellular Ca2+ levels measured by Fluo-4 AM calcium fluorescence probe, and STO609 inhibitor blockade to validate the mechanism.

4. Study of the Mechanism by which GLP-1RA Influences APP Processing and Aβ Generation

• Models and Operations: In primary neurons and the APP Swedish mutant stable expression cell line (20E2), GLP-1RA treatment was used to monitor β-site APP cleaving enzyme 1 (BACE1) expression, APP cleavage products (C99, C89), and Aβ40/Aβ42 secretion levels, with AMPK inhibitors and gene knockdown confirming the molecular dependency of drug effects.

5. Regulation of BACE1 Transcription by AMPK/NF-κB Mechanism

• Technical Workflow: Construction of a human BACE1 promoter (-1942 to +292 bp) fused to a luciferase (Luc) reporter gene, transfected into N2A cells with pharmacological intervention to quantify promoter activity changes. Combined with qPCR and western blot to detect endogenous BACE1 expression changes.
• Molecular Mechanism: Subcellular fractionation and chromatin immunoprecipitation (ChIP) experiments determined the binding of NF-κB p65 to the BACE1 promoter under AMPK inhibition or activation, fully connecting the AMPK→NF-κB→BACE1 transcriptional regulation pathway.

6. Effects of GLP-1RA on Microglial Phagocytosis and Inflammation

• Cell Experiments: Mouse BV2 microglia were treated with GLP-1RA, with Aβ oligomer stimulation, to assess AMPK phosphorylation and differential gene expression (DEGs) via RNA sequencing.
• Functional Detection: RNA-Seq results were summarized, GO/KEGG enrichment analyses compared changes in signaling pathways regulated by GLP-1RA. Immunofluorescence and western blot were used to detect the expression of CD68 and LAMP1 (phagocytosis and lysosome markers), fluorescently labeled Aβ42 phagocytosis function was evaluated, and levels of inflammatory factor products (ELISA) were used to measure drug effects.

7. In Vivo Intervention with GLP-1RA in AD Mouse Models

• Animal Administration and Detection: Starting at 6 weeks old, app23/ps45 AD model mice were given daily intraperitoneal injections of Exendin-4 (25 nmol/kg) for 8 weeks, with control group mice receiving vehicle. After the experiment, tissue samples were taken and analyzed for brain AMPK activation, APP processing, Aβ production, BACE1 expression, and neuroinflammation.
• Pathological and Behavioral Detection: 4G8 antibody and Thioflavin S staining were used to quantify neuronal amyloid plaques in the brain, with Morris water maze testing used to evaluate cognitive function recovery.

IV. Key Research Findings and Result Chain

1. GLP-1 Level is Closely Linked to AD Pathology

Plasma GLP-1 levels were significantly decreased in AD model mice, and low GLP-1 was highly negatively correlated with hippocampal Aβ load in patients (r = -0.825), suggesting that decreased GLP-1 is a key feature of disturbed energy metabolism and amyloid burden in AD. AD model neurons also showed impaired glucose uptake and ATP synthesis, indicating a close relationship between brain energy metabolism disorders and GLP-1.

2. GLP-1RA Restores Energy Metabolism and Modulates Cellular Metabolic Activities

GLP-1RA (Exendin-4) treatment significantly increased glucose uptake and ATP generation in AD neurons and enhanced FAO and Oxphos functions in astrocytes. Immunodetection further showed that GLUT3 expression and membrane translocation increased, PGC-1α (a key mitochondrial biogenesis regulator) was upregulated, enhancing neuronal energy supply. Analysis of metabolic products indicated that the drug significantly inhibited the AD-related rise in ROS and suppressed oxidative stress injury.

3. CaMKK2-AMPK Signaling Pathway as the Core of Drug Effect

In vitro cell experiments demonstrated that Exendin-4 and Tirzepatide (a dual GIP/GLP-1RA) dose-dependently increased phosphorylation activities of AMPK and downstream ACC, and this effect was dependent on GLP-1R expression. The increase in phosphorylation was primarily mediated by CaMKK2 through Ca2+ signaling, with no significant change in the LKB1 pathway. STO609 blockade trials confirmed the pivotal role of CaMKK2 in this signaling axis.

4. GLP-1RA Suppresses BACE1 Expression via AMPK and Reduces Aβ Generation

Under drug treatment, BACE1 protein and activity were significantly downregulated (to 53.6% in neurons and 39.8% in the cell line), and the downstream APP cleavage products and Aβ40/42 levels were also markedly reduced. AMPK inhibitors and gene knockdown both weakened or eliminated the drug effect, confirming AMPK as the core signaling hub.

5. NF-κB Mediates AMPK Regulation of BACE1 Gene Transcription

Luciferase reporter and ChIP experiments confirmed that AMPK activity directly affects BACE1 promoter activity: under AMPK activation, BACE1 transcription was downregulated; the activator AICAR significantly reduced nuclear and cytoplasmic NF-κB p65 levels, while AMPK inhibition increased NF-κB binding to the BACE1 promoter and strengthened its expression. This revealed that the AMPK/NF-κB/BACE1 pathway forms the molecular mechanism by which GLP-1RA modulate AD occurrence.

6. GLP-1RA-Driven AMPK Activation Promotes Microglial Phagocytosis of Aβ and Alleviates Neuroinflammation

RNA-seq combined with GO/KEGG analyses revealed that GLP-1RA improved the expression patterns of metabolism-related genes, strengthened pathways of phagocytosis, glucose transport, and energy metabolism, and inhibited inflammation-related signaling. Immunodetection confirmed the upregulation of CD68 and LAMP1, enhanced phagocytosis of fluorescent Aβ, downregulation of neuroinflammatory factors (IL-1β, IL-6, TNF), and increased anti-inflammatory factor (TGF-β). Blocking AMPK signaling reversed these drug effects.

7. In Vivo Intervention in Animal Models Verified the Multidimensional Efficacy of GLP-1RA

After long-term intervention with Exendin-4, AD model mice had increased brain AMPK phosphorylation, significant reductions in BACE1 and Aβ production, with no change in total APP protein level. Pathological assessment showed approximately half the number of plaques, significantly reduced inflammation (decreased IL-1β, IL-6, TNF), and substantial improvement in cognitive behavior (Morris water maze escape latency and spatial memory indicators).

V. Conclusions and Significance

This study systematically demonstrated that GLP-1RA, via activation of the CaMKK2-AMPK signaling axis, restores brain cell energy metabolism, suppresses Aβ production and deposition, improves neuroinflammation, and promotes Aβ clearance, thereby reversing key pathological and behavioral abnormalities in AD model animals. The AMPK signaling pathway is the central target integrating the multifaceted effects of GLP-1RA. The research not only further enriches the molecular mechanism map of AD pathogenesis and progression, but also, for the first time, proposes a novel GLP-1RA/AMPK/NF-κB/BACE1 signaling axis, offering a promising new direction for future “metabolic activation”-based interventions in AD.

This study has dual scientific and clinical application value: On one hand, it enriches the interactive regulatory network of brain energy metabolism and AD pathology, providing a theoretical basis for drug screening and target development; on the other hand, as a mainstream clinical medication for T2DM and obesity management, the safety and accessibility of GLP-1RA lay a practical foundation for its transformation into AD prevention and treatment drugs, and related clinical trials are worth accelerating.

VI. Research Highlights and Innovations

• For the first time, it clearly identifies that peripheral GLP-1 reduction is an important marker of disturbed brain energy metabolism and Aβ deposition in AD.
• It reveals that the action of GLP-1RA regulates multiple links, including glucose and fatty acid metabolism, APP processing, and neuroinflammation, via CaMKK2-AMPK activation.
• The molecular mechanism innovatively elucidates the AMPK-to-NF-κB transcriptional regulation of BACE1, achieving direct inhibition of Aβ production.
• The study is designed with multiple models, multiple scales (human-animal-cell), and multiple technology platforms, featuring rigorous logic and complete data chains.
• It provides a new paradigm and empirical basis for AD drug development based on the “metabolic optimization–neuroprotection” concept.

VII. Other Noteworthy Points

• The study emphasizes sex as a biological variable, obtaining consistent results in both male and female animals and patients, demonstrating scientific rigor.
• It provides detailed descriptions of the sources of cells and animal models used, drug dosages, and data statistical methods, laying a foundation for subsequent replication and in-depth mechanistic studies by peers.
• Multiple sets of experiments are accompanied by multiple repetitions and quantitative statistics to ensure the reliability and scientific validity of the results.

VIII. Summary and Outlook

This is a foundational study that combines theoretical innovation with translational prospects, systematically demonstrating how GLP-1R agonists improve AD-associated energy metabolism disorders, pathological protein deposition, inflammatory responses, and cognitive impairments through the AMPK pathway. It provides a solid foundation for exploring and developing novel metabolic regulation therapeutic strategies for AD and other neurodegenerative diseases. With further elucidation of these mechanisms and intensified clinical research, GLP-1RA or AMPK-centered metabolic regulatory drug strategies are expected to become important new breakthroughs in the field of AD treatment.