Divergent AKT Signalling Mechanisms Regulate GLUT4 Translocation and Glucose Uptake in Skeletal Muscle and Adipose Tissue

Background The serine/threonine kinase AKT is a key regulator of glucose and energy metabolism. Prevailing dogma suggests that AKT is an obligate intermediate for glucose uptake in all metabolic tissues and that impaired AKT signalling is a major molecular driver of insulin resistance in obesity. However, whether AKT is universally required for insulin‐stimulated glucose uptake across tissues in vivo has remained unresolved. Method Several mouse models of adipose‐specific AKT2 deletion (F‐AKT2KO) and skeletal muscle‐specific AKT1, AKT2 and combined AKT1/AKT2 knockout mice (M‐AKT1KO, M‐AKT2KO and M‐AKTDKO) were generated. Skeletal muscle and adipose tissues were analysed following in vivo administration of insulin (2 U/kg), using Western blotting, phosphoproteomics, PI(3,4,5)P3 ELISA and mitochondrial respiration assays. Glucose metabolism was assessed using [ 3 H]‐2‐deoxyglucose uptake, hyperinsulinemic‐euglycemic clamps, glucose and insulin tolerance tests. Global phosphoproteomics was performed in insulin‐stimulated skeletal muscle lacking AKT isoforms. Results Loss of AKT2 in adipose tissue impaired insulin signalling, including reduced pAS160 Thr649 , and markedly decreased insulin‐stimulated glucose uptake (~2–3 fold reduction in F‐AKT2KO vs F‐Control, p < 0.001, n = 7–11), resulting in systemic insulin resistance. In contrast, M‐AKTDKO mice exhibited a robust increase in insulin‐stimulated glucose uptake (~3–4 fold increase) despite complete loss of AKT signalling, including pAS160 Thr649 . Phosphoproteomic analysis of M‐AKTDKO ( n = 3–4) identified ~7088 phosphosites, with 795 uniquely upregulated in insulin‐stimulated M‐AKTDKO muscle (fold change > 2, p < 0.05), enriched in PI3K and AMPK pathways. Consistently, ~8‐fold ( p < 0.05) increase in PIP3 levels was observed in M‐AKTDKO muscle in response to insulin. Additionally, AKT deficiency was associated with reduced complex I–dependent mitochondrial respiration (~37% decrease in state 3 respiration), consistent with altered energetic status and AMPK activation. Genetic epistasis experiments demonstrated that both AKT and AMPK activity are required for insulin‐stimulated glucose uptake, systemic glucose homeostasis and whole body insulin sensitivity. Conclusion These findings challenge the long‐standing assumption that AKT is universally required for insulin‐stimulated glucose uptake in vivo. The study demonstrates that while AKT is essential in adipose tissue, it is dispensable for insulin‐stimulated glucose uptake in skeletal muscle. AKT exerts negative feedback on PI3K signalling in both tissues; however, only skeletal muscle engages AMPK in the abscence of AKT to preserve glucose uptake. These findings redefine tissue‐specific insulin signalling mechanisms and identify AMPK as a critical downstream target of PI3K that coordinates with AKT to regulate glucose uptake.