Study design, materials and methods
Three-week-old female Sprague-Dawley rats were randomly divided into two groups: a model group (fed a 60% fructose diet for 1 week) and a control group (fed a standard diet). Model establishment was confirmed by assessing body weight, blood biochemistry, urination behavior, and urodynamic function. AMPK antagonism experiments were performed via tail-vein injection of 10 μM Compound C, followed by urodynamic monitoring. In vitro, primary rat bladder smooth muscle cells (BSMCs) were cultured with fructose or glucose. Western blotting (WB) was used to quantify key proteins, including AMPK and HCN1, in tissues and cells.
Results
Compared to controls, fructose-fed rats showed OAB-like abnormal bladder function, including increased non-voiding contractions, shortened contraction intervals, elevated contraction amplitudes, reduced maximum bladder capacity, maximal contraction force and compliance; at the molecular level, their intracellular Ca²⁺, CaMKK2 levels and p-AMPK/AMPK ratio were significantly elevated while TRPV1 expression remained unchanged, and WB analysis revealed that AMPK (along with AMPKγ2 and CaMKK2) positively regulated HCN1 expression as Compound C administration reduced HCN1 levels, which was further confirmed by primary BSMCs experiments showing a direct correlation between AMPK phosphorylation and HCN1 expression in a time-dependent manner (with fructose levels, KHK activity and PFKP expression changing accordingly at 24 and 48 hours).
Interpretation of results
1.Short-term high-fructose diet activates AMPK phosphorylation via the Ca²⁺-CaMKK2 pathway, altering bladder contractility
Compared to controls, fructose-fed rats exhibited abnormal bladder pressure fluctuations, characterized by increased non-voiding contractions during the filling phase, shortened contraction intervals, and elevated contraction amplitudes, indicating heightened bladder sensitivity. The model group also showed reduced maximum bladder capacity, maximal contraction force, and compliance, consistent with OAB pathophysiology. At the molecular level, TRPV1 expression remained unchanged, but CaMKK2 levels and the p-AMPK/AMPK ratio were significantly elevated. Intracellular calcium assays confirmed higher Ca²⁺ levels in the fructose model group.
2.AMPK regulates HCN1 expression to influence bladder rhythmicity
WB analysis of AMPKγ2, AMPKα, p-AMPK, CaMKK2, and HCN1 in the fructose model, control, AMPK antagonist control, and AMPK antagonist model groups revealed elevated HCN1 expression in the model group. AMPKγ2, CaMKK2, and p-AMPK levels were also significantly higher. Compound C administration reduced HCN1 expression, indicating a close relationship between AMPK and HCN1, with synchronized changes in AMPKγ2, CaMKK2, and HCN1.
3.AMPK phosphorylation levels correlate directly with HCN1 expression in BSMCs
Primary BSMCs were cultured with 10 mM fructose or 5.5 mM glucose (control). Fructose levels in cells initially dropped at 24 hours and rebounded by 48 hours. At 24 hours, AMPK phosphorylation was low, and HCN1 expression decreased markedly. By 48 hours, reduced ketohexokinase (KHK) activity and increased phosphofructokinase platelet (PFKP) expression were observed, coinciding with restored AMPK phosphorylation and increased fructose and HCN1 levels.
Concluding message
This study demonstrates that fructose induces OAB in rats via the AMPK-HCN1 pathway. Glycolytic status, AMPK phosphorylation, intracellular fructose levels, and HCN1 expression are directly correlated, showing synchronized dynamics. AMPK phosphorylation likely regulates HCN1 expression. Smooth muscle cells may not directly internalize fructose to trigger symptoms, highlighting the need for further mechanistic exploration.