The physiological changes that occur in Type 1 Diabetes involve multiple molecular alterations. The ability to monitor simultaneously the expression of thousands of genes originally made possible by microarray technology is now being joined by evolving mass spectrometry-based proteomics approaches where the more complex protein universe is being dissected.
In Aim 1 we will utilize two dimensional, liquid chromatography coupled mass spectrometry (2D-LC/MS and 2D-LC/MS/MS) to study the effects of streptozotocin (STZ) induced diabetes on protein expression and posttranslational modifications in the bladder and corporal myocytes of rats that have already been characterized with respect to the degree of bladder and erectile dysfunction in vivo. This will include examination of control, early stage diabetic (1-wk STZ), intermediate stage diabetic (8-wk STZ), and late stage diabetic (16-wk STZ) animals. Our working hypothesis in Aim 1 is that we will identify multiple surrogate protein markers (as well as potential causative changes in protein functionality) for the progression of Type 1 Diabetes and the development of complications in an animal model that recapitulates relevant aspects of the corresponding human condition.
In Aim 2, using immuno-precipitation of the Maxi-K channel (Slo gene product) followed by protease digestion and LC-mass spectrometry, we will determine which Slo gene splice variants are expressed as protein isoforms and determine changes in their possible phosphorylation states during diabetes progression in the STZdiabetes model. Preliminary data show changes in Slo gene splice variant gene expression during diabetic progression; including data from human patients. In addition, expression of splice variants in exogenous expression systems (HEK293 cells and Xenopus oocytes) induces electrophysiological changes in Maxi-K channel function consistent with the known complications of the disease. Our working hypothesis in Aim 2 is that we will confirm these predictions of splice variants through identification of the appropriate protein isoforms, first expressed in HEK cells, then in diabetic bladder and corpora. This research, if successful, will lead to an improved understanding of the molecular alterations in myocytes that attend the end-organ complications of diabetes mellitus. The long term goal of these studies is to identify surrogate markers of disease progression as well as shed light on novel therapeutic targets.
