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. 2025 Jul;603(14):3961-3971.
doi: 10.1113/JP288854. Epub 2025 Jun 30.

Muscle-specific AXIN1 and AXIN2 double knockout does not alter AMPK/mTORC1 signalling or glucose metabolism

Kaspar W Persson  1 , Roberto Meneses-Valdés  1 , Nicoline R Andersen  1 , Frederik S Pedersen  1 , Samantha Gallero  1 , Sofie A Hesselager  1 , Carlos Henriquez-Olguin  1   2 , Thomas E Jensen  1
Affiliations

Muscle-specific AXIN1 and AXIN2 double knockout does not alter AMPK/mTORC1 signalling or glucose metabolism

Kaspar W Persson et al. J Physiol. 2025 Jul.

Abstract

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are crucial kinase signalling hubs that regulate the balance between catabolism and anabolism in skeletal muscle. The scaffold protein AXIN1 has been proposed to regulate the switch between these pathways and be required for GLUT4 translocation in skeletal muscle and adipocyte cell lines. Muscle-specific AXIN1 knockout (KO) mice exhibit no discernable phenotype, possibly due to compensation by AXIN2 upon AXIN1 loss. Thus we generated and characterized muscle-specific inducible AXIN1 and AXIN2 double knockout (dKO) mice. Surprisingly AXIN1/2 dKO mice displayed normal AMPK and mTORC1 signalling and glucose uptake in response to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), insulin and in situ muscle contraction. These findings suggest that AXIN proteins are not essential for the regulation of AMPK and mTORC1 signalling or glucose uptake in skeletal muscle. This study challenges the previously indicated critical roles of AXIN1 in exercise-stimulated AMPK activation and GLUT4-mediated glucose uptake in skeletal muscle. KEY POINTS: Phenotyping of tamoxifen-inducible muscle-specific AXIN1/2 double knockout (dKO) mice. We find no evidence for AXIN-dependent AMPK or mTORC1 regulation in skeletal muscle by insulin, AMPK activation or contraction. Glucose uptake regulation by insulin and AMPK activation is normal in AXIN1/2 dKO mice.

Keywords: AMPK; exercise; glucose transport; mTOR; skeletal muscle.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Verification of skeletal muscle AXIN1/2 double knockout
A, schematic overview of the tamoxifen induction protocol. B, C, quantification of the Axin1 and Axin2 flox allele excision in quadriceps muscle. Data are presented as means ± standard deviation with individual data points. Statistical analysis was performed using Student's t test. n = 5–8. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2. AXIN1 and AXIN2 knockout does not affect ex vivo 5‐aminoimidazole‐4‐carboxamide ribonucleotide (AICAR)‐stimulated 2‐deoxyglucose (2‐DG) uptake and downstream AMPK and mechanistic target of rapamycin complex 1 (mTORC1) signalling
A, experimental overview of ex vivo AICAR‐stimulated 2‐DG uptake in incubated extensor digitorum longus (EDL) and soleus (SOL) muscle. B, ex vivo AICAR (4 mm)‐stimulated 2‐DG uptake. C–G, levels of AMPK‐ and mTORC1‐related phosphorylated protein after AICAR stimulation. H, representative blots of EDL and SOL muscle. Data are presented as means with sex‐colour‐coded, paired individual values (pink: female; blue: male). Sex‐specific (pink/blue) and pooled data (black) P‐values were derived from two‐way ANOVA with Sidak's multiple comparisons test in case of a significant ANOVA main effect (ME). n = 18–20 (males: 8–10; females: 10). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3. AXIN1 and AXIN2 knockout does not affect ex vivo insulin‐stimulated 2‐deoxyglucose (2‐DG) uptake and downstream AMPK and mechanistic target of rapamycin complex 1 (mTORC1) signalling
A, experimental overview of ex vivo insulin‐stimulated 2‐DG uptake in incubated extensor digitorum longus (EDL) and soleus (SOL) muscle. B, ex vivo insulin (60 nm)‐stimulated 2‐DG uptake. C–G, levels of mTORC1‐related phosphorylated protein after insulin stimulation. H, representative blots of EDL and SOL muscle. Data are presented as means with paired individual values and analysed using two‐way ANOVA with Sidak's multiple comparisons test in case of ANOVA main effect (ME). n = 4 (females). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4. Contraction‐induced AMPK and mechanistic target of rapamycin complex 1 (mTORC1) signalling is not affected by AXIN1 and AXIN2 muscle knockout
A, experimental overview of unilateral, percutaneous in situ contraction (CTXN) in quadriceps (QUAD) muscle. B–F, levels of AMPK and mTORC1‐related phosphorylated protein acutely after in situ CTXN. G, representative blots of QUAD muscle. Data are presented as means with sex colour‐coded, paired individual values (pink: female; blue: male). Sex‐specific (pink/blue) and pooled data (black) P‐values were derived from two‐way ANOVA with Sidak's multiple comparisons test in case of ANOVA main effect (ME). n = 16–18 (males: 7–10; females: 7–9). [Colour figure can be viewed at wileyonlinelibrary.com]
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