O-GlcNAcylation is a post-translational protein modification of serine/threonine residues by N-acetyl-glucosamine (O-GlcNAc) that can negatively regulate the phosphorylation/activation of a variety of proteins, and thus alter important cellular processes.   For example, neurotransmitter release is thought to be regulated by O-GlcNAcylation of synaptic proteins. With diabetes, elevated glucose levels increase the extent of O-GlcNAcylation of key proteins in the insulin signaling pathway, thus impeding their functions; however, whether chronic hyperglycemia alters the O-GlcNAcylation of key proteins involved in bladder neurotransmission has not been previously investigated. Using an animal model of type 2 diabetes (T2D), this study examined the effect of hyperglycemia on the O-GlcNAcylation patterns of bladder proteins and investigated whether aberrant changes in O-GlcNAcylation of specific proteins involved in synaptic vesicle trafficking parallel the age-dependent changes in detrusor neurotransmission in T2D.

Urinary bladders were procured from 10, 20, 30, 40-week-old db/db mice, an animal model of T2D due to genetic deletion of leptin receptor. Bladders from age-matched C57BLKS/J were used as controls. Changes in protein O-GlcNAcylation were investigated by western blotting (WB) in db/db and control bladder tissues at each age. Protein co-localization and interaction in bladder tissue was investigated by multiplex WB and co-immunoprecipitation analysis respectively. For functional studies, longitudinal bladder smooth muscle tissue without mucosa was suspended in organ bath containing oxygenated Kreb’s at 37°C and stretched to 0.5 grams for isometric tension experiments. Detrusor responses to nerve or agonist induced stimulations were compared between diabetic and control animals at each age.

Blood glucose levels in db/db mice were significantly higher than those in control animals from 10 to 40 weeks of age. In db/db mice, proteins from whole bladder lysates were significantly more O-GlcNAcylated as early as 10 weeks of age compared to proteins from control bladders.  A particularly hyper O-GlcNAcylated protein band was colocalized with immunoreactivity for Akt, a protein kinase that is involved in the regulation of synaptic vesicle trafficking in neurons.   Bladder contractions induced by nerve stimulation were significantly lower in db/db mice than in control from 10 to 40 weeks of age. In contrast, no differences between db/db and control mice were detected in post-junctional bladder contractions elicited by agonists.  Immunoreactivity for myosin 5a (MYO5a), a motor protein involved in synaptic vesicles translocation within varicosities of peripheral nerves, was detected in Akt immuno-precipitates, as well as in immuno-precipitates obtained with an antibody that recognizes Akt-dependent phosphorylated substrates.

The increased levels of O-GlcNAcylated proteins found in diabetic bladders is consistent with other tissues in which chronic hyperglycemia causes elevated protein O-GlcNAcylation. The augmented O-GlcNAcylation of Akt in diabetic bladders together with the decreased neurogenic detrusor contractions in db/db mice suggest that altered regulation of Akt induced by hyperglycemia impairs nerve-mediated responses. Moreover, the molecular interaction between Akt and MYO5a suggests a role for Akt in phosphorylating/activating Myo5a in peripheral nerves to achieve neurotransmission.

Under conditions of chronic hyperglycemia, increased O-GlcNAcylation may cause impaired neurotransmission in diabetic bladders by altering the activation of Akt-dependent motor proteins involved in synaptic vesicle trafficking, and thus reducing neurotransmitter release.