The effect of high-fat diet on diabetic bladder dysfunction in type 2 diabetes
Lower urinary tract dysfunction is one of the major but often unrecognized complications associated with DM2. Patients present with detrusor overactivity or underactivity. Although diabetic bladder dysfunction affects up to 80% of patients with DM2, its underlying pathophysiology is poorly understood. We used a hepatic-specific conditional knockout of insulin receptor substrates 1 & 2 (Irs1, Irs2) genetic mouse model which develops DM2, and demonstrate that the Irs1/Irs2 double knockout (DKO) mice mimic many of the signs and symptoms seen in human patients with diabetic lower urinary tract dysfunction. We demonstrate that the bladders of the DKO mice are overactive (a compensated state of bladder) in early/mid life of the mice, and underactive (a decompensated state of bladder) in later adult life. We show that TNF-a, a molecule that is significantly upregulated in the bladder of diabetic animals, directly stimulates bladder smooth muscle contraction and activates Rho A signalling. Systemic inhibition of TNF-a in DKO animals prevents upregulation of Rho A signaling and reverses the bladder dysfunction. When inhibition of TNF-a is combined with metformin, bladder dysfunction is improved beyond that achieved with metformin alone. We hypothesize that TNF-a, a major inflammatory mediator, is a key effector of diabetic cystopathy. We postulate that obesity, in combination with DM2 will further increase TNF-a levels, leading to worsening of bladder dysfunction. In this project, we propose to test this hypothesis in murine models of DM2.

Genetic Model for Bladder Dysfunction in Type 2 Diabetes
Type 2 diabetes mellitus (DM2) stems from insulin resistance and affects 7% of the US population. Lower urinary tract dysfunction is one of the major urologic complications associated with DM2, and the patients present with detrusor overactivity or underactivity. Although diabetic bladder dysfunction affects up to 80% of patients with DM2 its underlying pathophysiology is poorly understood. Here, we demonstrate a genetic mouse model with DM2 with conditional knockout of insulin receptor substrates 1 & 2 (Irs1, Irs2), which mimic the diabetic lower urinary tract dysfunction in humans. The DKO (Irs1, Irs2) organ-specific (hepatocyte) conditional knockout animal system is a powerful model for DM2 which demonstrates the secondary bladder cystopathy observed in humans. As opposed to many previous animal models that have utilized the streptozotocin system, which more closely resembles type 1 diabetes, the DKO model is associated with insulin resistance which resembles DM2. In addition, the DKO mice are not obese, therefore, uncoupling DM2 from obesity. The DM2 in the DKO model is reversed in the TKO model (Irs1, Irs2 & Foxo1 knockout), enabling us to examine fundamental questions associated with correction of DM2 and bladder dysfunction and the molecular mechanisms that may be responsible for the compensated and decompensated states of diabetic cystopathy. We hypothesize that the phosphorylated myosin light chain (pMLC) is a key regulator of the compensated and decompensated states of diabetic cystopathy. Specific Aim 1: To determine if correction of DM2 in a genetic animal model will restore the bladder function. The DKO mice develop hyperglycemia and dyslipidemia the fifth week of life and the metabolic abnormalities last beyond 30 weeks of life. The metabolic dysregulation of DM2 in DKO mice is reversed by inhibition of the FOXO1 gene, therefore, we will determine whether the bladder dysfunction and altered expression of pMLC that is observed in the DKO is reversible and age-dependent. Specific Aim 2: To determine the effect of high-glucose concentration on inflammatory mediators in DKO mice. Hyperglycemia secondary to insulin resistance is one of the hallmarks of DM2. One of the key associated findings with hyperglycemia is hyper-glucosuria which can significantly affect a DM2 patient’s lower urinary tract symptoms. However, the molecular alterations associated with hyper-glucosuria are not well understood. Here we will investigate the effect of high glucose concentration on inflammatory modulators, TNFa and IL-6, and examine how they may regulate the phosphorylated myosin light chain (pMLC) in the bladder smooth muscle of DKO mice.

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