Characterization of Prolylcarboxypeptidase Expression Pattern in Rat Cardiomyocytes in Nutrition Overload Conditions

Prolylcarboxypeptidase (PRCP) is a serine protease that cleaves the last amino acid at the carboxy-terminus of peptides with a penultimate proline such as angiotensin II (Ang II), angiotensin III (Ang III), prekallikrein (PK) and α-melanocyte-stimulating-hormone (1-13) (α-MSH1-13). By inactivating Ang II and Ang III, PRCP promotes vasodilation and insulin sensitivity. As well by activating PK, PRCP increases nitric oxide (NO) generation and vasorelaxation through bradykinin (BK) liberation from high molecular weight kininogen (HK). PK is activated to kallikrein by PRCP, implicating a role for PRCP in the coagulation pathway. The resultant kallikrein will activate FXII which in a reciprocal manner leads to the activation of FXI. Lastly, by metabolizing α-MSH1-13 to an inactive metabolite (α- MSH1-12), PRCP inhibits the anorexigenic response to the endogenous α-MSH1-13, leading to an increase in appetite. Thus, by means of vasodilation, insulin sensitization (via direct stabilization of IRS-1 and indirect reduction of Ang II), and suppressing reactive oxygen species (ROS) generation (via direct increase in NO generation and indirect reduction of Ang II) PRCP functions to protect cardiovascular system. Moreover, PRCP mutation is related to acute coronary syndrome in men, and individuals with obesity, diabetes and arteriosclerosis have elevated plasma PRCP concentration and activity. Since cardiovascular disease is the leading cause of mortality in diabetes and obesity, PRCP overexpression in nutrition excess conditions represents an important target for studying metabolic syndrome- related cardiac dysfunction. The overall goal of this study was to elucidate the hormetic effects of major nutrients on PRCP-dependent pathways. Therefore, the following methods were exploited to address our goals: cell culture of rat H9c2 cardiomyocytes, enzymatic assays for detecting plasma and cardiomyocyte PRCP activity, PRCP RNA and protein studies in high glucose and high fatty acid (saturated and unsaturated) conditions, enzymatic assays to detect PRCP-stimulated activation of PK and plasma protein studies of PK and its downstream target in the coagulation cascade, FXI. Using in vitro biochemical assays, plasma PRCP and kallikrein activity were significantly increased in uncontrolled diabetic patients; however metformin and insulin treated diabetic patients had reduced plasma PRCP and kallikrein activity. As well, uncontrolled diabetic patients had markedly elevated plasma prekallikrein (PK), PRCP and FXI protein which was reduced in insulin and metformin treated groups. In a rat cardiomyocyte model of nutrition overload, saturated fatty acid palmitate, unlike glucose, suppressed PRCP levels by 60% in a dose-dependent and time-dependent manner without affecting cell viability, while other tested saturated and unsaturated fatty acids did not alter the basal cardiomyocyte PRCP expression. Thyroxine and insulin, but not metformin, restored palmitate-induced cardiac PRCP depletion. Lastly, although fatty acid uptake inhibition by the CD36 antagonist increased palmitate- induced PRCP depletion by 2-fold, NFκB inhibition did not restore palmitate-stimulated PRCP suppression. Our results indicate that fatty acid-accelerated cardiomyocyte PRCP depletion along with diabetes-stimulated increase in plasma PK and FXI concentration, may contribute to nutrition overload-stimulated cardiovascular dysfunction. The details of fatty acid-induced PRCP down regulation in the heart could open new therapeutic avenues to protect against metabolic syndrome-related cardiovascular complications.

Name thesis:degree_name

Ph.D. in Pharmaceutical Sciences

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biomolecular Sciences

Year dc:date.available

2014

Author dc:creator

• Tabrizian, Tahmineh

Contributors dc:contributor

• Ziaeddin Shariat-Madar
• Kristine L. Willett
• Matthew Strum

• Molecular Biology

Repository record dc:identifier

https://egrove.olemiss.edu/etd/1476

OAI identifier oai:identifier

oai:egrove.olemiss.edu:etd-2475