# What mechanisms, beyond canonical pathways, are indicated by GLP-1R?

## GLP-1R employs various alternative signaling mechanisms, including PLC/PKC and GTP-binding protein pathways, alongside organelle interactions and concentration-dependent pathway switching.

# Abstract

GLP‑1 receptor signaling involves several distinct, noncanonical mechanisms alongside its classic Gαs/cAMP/PKA pathway. Studies report that picomolar GLP‑1 concentrations trigger insulin release via a phospholipase C/protein kinase C (PLC/PKC) pathway rather than through PKA activation. In addition, the GLP‑1(9‑36) metabolite reduces glucagon secretion by engaging an inhibitory GTP‐binding protein independently of PKA. Other reports describe a cAMP/Epac/PI3K/Akt cascade mediating cardioprotective effects and a mode of Akt activation that bypasses traditional PI3K‐dependent phosphorylation in β‑cells.

Additional findings indicate that signaling bias plays a role in GLP‑1R function. Differential β‑arrestin recruitment is linked to receptor internalization and altered downstream responses, while concentration‐dependent shifts—such as a switch from PKC‐ to PKA‐dependent pathways—modulate physiologic outcomes. Furthermore, evidence from in vitro studies shows that GLP‑1R associates with endoplasmic reticulum and mitochondrial proteins (for example, VAP‑B and SPHKAP), suggesting a role in inter‑organelle communication and mitochondrial remodeling. These convergent observations demonstrate that GLP‑1R orchestrates a multifaceted network of PKA‑independent and context‑dependent signaling events.

## Methods

We analyzed 40 sources from an initial pool of 497, using 7 screening criteria. Each paper was reviewed for 5 key aspects that mattered most to the research question. More on methods

### Papers identified with Elicit search

n = 497

### Papers screened using: 
- **GLP-1R Signaling Focus**: Does the study investigate GLP-1R signaling pathways (either canonical or non-canonical mechanisms)?
- **Molecular Methods**: Does the study employ molecular biology techniques (such as receptor binding assays or signal transduction studies) to investigate mechanistic aspects?
- **Study Design**: Is this an in vitro and/or in vivo experimental study?
- **Receptor Interactions**: Does the study examine GLP-1R interactions with cellular components or signaling molecules?
- **Beyond Canonical Pathway**: Does the study investigate pathways beyond or in addition to the canonical cAMP/PKA pathway?
- **Receptor Involvement**: Does the study directly investigate GLP-1R (rather than only GLP-1 effects without receptor involvement)?
- **Mechanistic Investigation**: Does the study include mechanistic investigation (rather than only clinical efficacy outcomes)?

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

## Results

### Characteristics of Included Studies

Study | Study Type | Cell/Tissue Type | Key Mechanisms Investigated | Primary Findings | Full text retrieved
--- | --- | --- | --- | --- | ---
Athauda et al., 2024 | In vitro cellular studies, Clinical trial | Human iPSC models of synucleinopathy | Glucagon-like peptide-1 receptor (GLP-1R) agonism effects on neuronal insulin signaling and glial suppression | GLP-1R agonism restores insulin signaling, suppresses MAPK pathways, reduces oxidative stress, improves mitochondrial and lysosomal function, and reduces α-synuclein aggregation | No
Austin et al., 2024 | In vitro cellular studies | Pancreatic β-cells | Inter-organelle contact between GLP-1R, endoplasmic reticulum (ER), and mitochondria | GLP-1R interacts with VAP-B and SPHKAP to trigger mitochondrial Protein kinase A (PKA) signaling and MIC19 phosphorylation | No
Bitsi et al., 2023 | Animal studies | Adult β cell–specific β-arrestin 2 knockout mice | β-arrestin-mediated GLP-1R signaling | β-arrestin 2 regulates GLP-1R desensitization, recycling, and signaling in a sex-dimorphic manner | No
Buteau et al., “GLP-1 Induces Proliferation” | In vitro cellular studies | INS(832/13) cells, Isolated rat islets, LNCaP cells | GLP-1R-mediated transactivation of EGFR | GLP-1 activates PI 3-kinase and promotes β-cell proliferation through EGFR transactivation via c-Src | No
Capozzi et al., 2020 | Animal studies, In vitro cellular studies | Isolated mouse islets, Gnasβcell-/- mice | Gαs-dependent and independent mechanisms of incretin-stimulated insulin secretion | GLP-1R can signal through both Gαs and non-Gαs mechanisms, including Gq | No
...

### Key Findings from GLP-1R Signaling Mechanism Studies

#### Study Types

- In vitro cellular experiments: 35/40 studies
- Animal studies: 14/40 studies
- Clinical trials: 1 study
- Computational/modeling studies: 2 studies
- Design not clearly specified: 3 studies

#### Cellular/Biological Model

- Mouse islets: 12 studies
- INS cells: 7 studies
- MIN6 cells: 6 studies
- Human islets: 5 studies
- Knockout mice: 6 studies

#### Key Mechanisms

- General GLP-1R signaling: 4 studies
- β-arrestin-mediated signaling: 2 studies
- Biased agonism: 2 studies
- Non-canonical Akt activation: 2 studies
- PLC/PKC-dependent pathways: 2 studies

#### Other Mechanisms

- EGFR transactivation
- Gαs-dependent and independent mechanisms
- ER stress/UPR effects
- Various kinase cascades (e.g., MAPK, PI3K/Akt)

The included studies predominantly used in vitro and animal models to investigate various signaling pathways and mechanisms related to GLP-1R, with many focusing on β-cell function and insulin secretion.

## Non-Canonical Signaling Mechanisms

### PKA-Independent Signaling Pathways

Several studies identified PKA-independent signaling pathways activated by GLP-1R:

- PLC/PKC-dependent pathway:
  - Shigeto et al. (2017a, 2017b) reported GLP-1 stimulates insulin secretion at physiological (picomolar) concentrations through this pathway
- Inhibitory GTP-binding protein mechanism:
  - Guida et al. (2019) found GLP-1(9-36) metabolite inhibits glucagon secretion through this PKA-independent mechanism
- GLP-1R/cAMP/Epac/PI3K/Akt signaling pathway:
  - Nuamnaichati et al. (2020) identified this novel pathway mediating cardioprotective effects
  - Epac acts as a key regulator instead of PKA
- Non-canonical Akt activation:
  - Widenmaier et al. (2009) and Widenmaier “Non-canonical Activation” discovered GLP-1 can activate Akt in β-cells without requiring PI3K or typical Akt activation site phosphorylation

These studies report that GLP-1R can engage multiple signaling pathways independently of PKA. The authors suggest this may allow for fine-tuned cellular responses depending on the cellular context, GLP-1 concentration, and the specific GLP-1R agonist used.

### Concentration-Dependent Alternative Pathways

Several studies reported concentration-dependent activation of alternative signaling pathways by GLP-1R:

- PKC vs PKA-dependent pathways:
  - Shigeto and Kaku (2014) found:
    - Picomolar GLP-1: PKC-dependent pathway for insulin secretion
    - Nanomolar GLP-1: Canonical PKA-dependent pathway
- Receptor affinity and internalization:
  - Jones et al. (2018) observed:
    - Low-affinity agonists: Less receptor internalization, greater insulin secretion
    - High-affinity agonists: More receptor internalization, less insulin secretion
- β-arrestin 2 roles:
  - Zaïmia et al. (2023) demonstrated:
    - Physiological doses: β-arrestin 2 dampens insulin secretion by partially uncoupling cAMP/PKA signaling
    - Pharmacological doses: β-arrestin 2 required for ERK/CREB pathway activation

These studies report concentration-dependent effects, suggesting that the dose of GLP-1 or GLP-1R agonists may be an important factor in experimental design and interpretation.

## Physiological Implications

### Cell-Type Specific Effects

Studies reported diverse effects of GLP-1R signaling in different cell types:

- Pancreatic β-cells:
  - Enhanced insulin secretion through canonical and non-canonical pathways
  - Mechanisms vary with GLP-1 concentration and signaling molecules involved
  - Studies: Shigeto et al., 2017a; Shigeto et al., 2017b; Zaïmia et al., 2023
- Cardiomyoblasts:
  - Novel GLP-1R/cAMP/Epac/PI3K/Akt signaling pathway
  - Mediates cardioprotective effects
  - Study: Nuamnaichati et al. (2020)
- Gut intraepithelial lymphocytes (IELs):
  - Modulates inflammation and affects gut microbiota composition
  - Expands known functions of GLP-1 to include immunomodulation
  - Study: Wong et al. (2022)
- Neurons:
  - Neuroprotective effects in Parkinson’s disease models
  - Involves improvements in mitochondrial and lysosomal function
  - Study: Athauda et al. (2024)

These studies report cell-type specific effects, suggesting that cellular context may be an important factor in GLP-1R signaling research and potential therapeutic applications.

### Integration with Known Pathways

Studies revealed complex interactions between GLP-1R signaling and other established signaling pathways:

- EGFR transactivation:
  - Buteau et al. (2003) and Buteau et al. “GLP-1 Induces Proliferation” showed GLP-1R can transactivate EGFR
  - Leads to activation of PI3-kinase and promotion of β-cell proliferation
- mTORC1 signaling:
  - Le et al. (2021) and Le et al. “GLP-1 Receptor Activation” demonstrated GLP-1R activation can stimulate mTORC1 signaling
  - Involves a novel PKA-dependent mechanism and phosphorylation of Raptor
- RhoA-ROCK signaling:
  - Kong et al. (2014) revealed GLP-1 can enhance insulin secretion by inhibiting the RhoA-ROCK pathway
  - Involves PKA-mediated phosphorylation of RhoA
- Kinase networks:
  - Xiao et al. (2023) used proteomic profiling to identify a wide range of kinases affected by GLP-1R activation
  - Includes MAPKs, PKCs, and LKB1

These studies report that GLP-1R signaling interacts with numerous other cellular signaling networks, potentially allowing for regulation of diverse physiological processes.