Molecular targets for diabetes mellitus-associated erectile dysfunction

Abstract

Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCore suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-gamma, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-gamma, and Hsp47 confirmed increased p53 and 14-3-3-gamma and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a approximately 3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment.

Fig. 1.

Decreased erectile function in diabetic animals in response to cavernous nerve stimulation. The mean change in ICP/BP (*, p < 0.05 compared with non-diabetic) is shown. The error bars represent the standard deviation from the mean.

Fig. 2.

2D map of deep purple-labeled corpora tissue proteins indicating pick location of subset of proteins that changed in response to STZ-induced diabetes. Orientation of the pH gradients is indicated on the horizontal axes from 3 (left) to 10 pH unit (right), and approximate apparent molecular mass ranges are indicated along the vertical axes from 10 (bottom) to 200 kDa (top).

Fig. 3.

PCA of proteins mediated by STZ-induced diabetes. The protein expression profiles of experimental groups were visualized in two-dimensional Euclidian space by using the extended data analysis module of DeCyder software as described under “Materials and Methods.” The PCA distinctly clustered the 15 individual samples into four experimental groups (C-1W, 1-week control; D-1W, 1-week diabetes; C-2M, 2-month control; and D-2M, 2-month diabetes).

Fig. 4.

Protein networks associated with proteins differentially expressed in response to STZ-induced diabetes. The network was generated by the shortest paths algorithm of MetaCore (GeneGo) software using the list of differentially expressed proteins identified by 2D DIGE/MS analysis. Individual proteins are represented as nodes, and the different shapes of the nodes represent the functional class of the proteins. The edges define the relationships of the nodes: the arrowheads indicate the direction of the interaction. P indicates phosphorylation, T indicates transformation, B indicates binding, C indicates cleavage, and TR indicates transcriptional regulation. The color of the hexagons on edges between nodes describes activation (green), inhibition (red), and unspecified (black) interactions. PKC, protein kinase C; Apeh, acyl-peptide hydrolase; apoE, apolipoprotein E precursor; Apcs, serum amyloid P component; Pzp, pregnancy-zone protein; HSCO/ETHE1, ethylmalonic encephalopathy 1; LIPC, hepatic triglyceride lipase; c-Jun, jun oncogene; c-Fos, FBJ osteosarcoma oncogene; c-Myc, myelocytomatosis oncogene; Hadha, mitochondrial trifunctional protein, alpha subunit; Hbb, hemoglobin subunit beta-1; Hp, haptoglobin.

Fig. 5.

Confirmational immunoblots for selected proteins that were hypothetically identified by network analysis. Each lane was loaded with a sample from an independent biological replicate (n = 2/experimental group). The blots were probed with antibodies to the proteins indicated at the left. C-1W, 1-week control; D-1W, 1-week diabetes; C-2M, 2-month control; and D-2M, 2-month diabetes.

Fig. 6.

Confirmational immunoblots for selected proteins that were identified as differentially expressed by 2D DIGE. An equal amount of total protein was loaded in each lane, and the resulting blots were probed with anti-Hsp47, anti-14-3-3-γ, and anti-MPZ. Relative -fold changes for these proteins in STZ-induced diabetic rats compared with the age-matched controls are given in Table II. C-1W, 1-week control; D-1W, 1-week diabetes; C-2M, 2-month control; and D-2M, 2-month diabetes.