Einfluss von PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide) bzw. des PACAP Rezeptors PAC1 auf Metabolismus, Morphologie und Expression proinflammatorischer / oxidativen Stress -relevanter Proteine/Zytokine in der Leber bei 20 Wochen Cholesteringefütterten ApoE-/- Mäusen

Coronary heart disease is a major cause of death in industrialized countries. Vascular alteration and atherosclerosis are the main reasons for cardiac ischemia and heart damage. As the pathomechanism is still not fully understood, strategies for therapies and prevention remain a difficult subject for medical researchers. The lipid metabolism, PACAP and ApoE seem to play an important role for the development of atherosclerosis. The liver is a central organ for lipid metabolism and systemic inflammatory response so that it can be related to the formation of atherosclerotic plaques. Referring to this, it is unavoidable to consider the influence of PACAP, the PACAP-receptor PAC1 and ApoE on the liver metabolism. PACAP is an autonomic neurotransmitter and belongs to the glucagon, secretin, VIP and GHRH family. The immunomodulative, anti-apoptotic, circulatory, and metabolic effects of PACAP are mediated threw its receptors called PAC1-, VPAC1- und VPAC2-receptor. The present dissertation is an investigation on this unexplored field and creates a better understanding for the influence of PACAP and PAC1 on the liver metabolism. Therefore, we used four different genotypes: Wildtype-, ApoE-/--, PACAP-/-/ApoE-/-- and PAC1-/-/ApoE-/--mice. These were fed with a cholesterol-enriched diet for 20 weeks. With the help of applied immunohistochemical staining it was possible to quantify proinflammatory and metabolic tissue markers in the liver. Different biochemical procedures gave an overview of the metabolic status of the liver. Our results showed an 8,9 times higher plasma cholesterol level in ApoE-/--mice than in wildtype-mice and an 1,5 times higher plasma triglyceride level in PACAP-/-/ApoE-/--mice than in ApoE-/--mice. The concentration of hepatic cholesterol was 2,6 times higher in ApoE-/--mice than in wildtype-mice while PACAP-/-/ApoE-/-- and PAC1-/-/ApoE-/--mice had 54,6 % and 56,9 % lower hepatic cholesterol concentrations than ApoE-/--mice. PACAP-/-/ApoE-/--mice showed 73,4 % higher hepatic triglyceride concentrations than ApoE-/--mice. The concentration of proteinogenic and non-proteinogenic amino acids in the liver of ApoE-/--mice was 84,5 % and 83,8 % lower than in wildtype-mice. PAC1-/-/ApoE-/--mice had a 21,3 % lower hepatic concentration of non-proteinogenic amino acids than ApoE-/--mice. In the liver of ApoE-/--mice we found a significant concentration of hydroxyproline while there was no detectable hepatic hydroxyproline concentration in wildtype-mice. PACAP-/-/ApoE-/--mice had a 24,4 % lower hepatic concentration of aspartic acid than ApoE-/--mice and a 37,5 % higher hepatic concentration of tyrosine than ApoE-/--mice. PAC1-/-/ApoE-/--mice showed a 25,8 % lower hepatic urea concentration, a 26,3 % lower threonine concentration, a 23,8 % lower asparagine concentration and a 15,1 % lower ornithine concentration than ApoE-/--mice. The hepatic concentrations of GSH and rGSH were 30,5 % and 32,8 % higher in ApoE-/--mice than in wildtype-mice while the rGSH2/GSSG-ratio was 95,4 % higher than in wildtype-mice. PAC1-/-/ApoE-/--mice showed an 18,6 % lower hepatic GSH concentration and a 19,0 % lower hepatic rGSH concentration than ApoE-/--mice. Immunohistochemical staining showed a 2,1 times higher density of CD68-positive macrophages around the hepatic glisson trias of ApoE-/--mice than in wildtype mice. The density of MOMA-2-macrophages around the glisson trias of ApoE-/--mice was 3,5 times higher than in wildtype-mice. In ApoE-/--mice we found a 1,5 times higher concentration of COX-2-positive cells around the hepatic glisson trias than in wildtype-mice. PACAP-/-/ApoE-/--mice had a non-significant 1,7 times higher density of IL-1β-positive cells around the glisson trias than ApoE-/--mice. The results suggest that PACAP-deficiency might cause hypertriglyceridemia and elevated hepatic triglyceride levels. Whether PACAP could possibly decrease the plasmatic and hepatic triglyceride concentration remains unclear. These observations reveal new possibilities for pharmacological targets for hypertriglyceridemia, a disease that is found in patients with specific ApoE-subtypes. Considering the results, there is strong evidence that PACAP and its receptor PAC1 mediate the uptake of cholesterol in the liver. Regarding the pharmacological aspect, it would be possible to study PACAP- or PAC1-antagonists as a treatment for fatty liver disease caused by cholesterol. Possible side effects of this treatment could be elevated plasmatic and hepatic triglyceride concentrations. The present results show a decreased concentration of proteinogenic and nonproteinogenic amino acids in the liver of ApoE-/--mice that is probably associated with a fibrotic change in the liver tissue. The additional absence of PAC1 in ApoE-deficient mice causes increased levels of substrates and decreased levels of products of the urea metabolism as well as decreased levels of the hepatoprotective amino acids aspartate and ornithine. PACAP-deficiency seems to lead to higher concentrations of inflammatory amino acids like tyrosine. It can be suspected that ApoE-deficiency is associated with elevated levels of GSH, rGSH and a higher GSH2/GSSG-ratio in liver tissue. In contrast, PAC1-deficiency seems to provide decreased levels of total and reduced glutathione and by that cause a decreased tolerance to hepatic cells against oxidative stress. These observations are comparable to earlier studies concerning PACAP and its cytoprotective effects on ischemic induced liver injury. According to the immunohistochemical analyses, ApoE-deficiency seems to cause an increased influx of macrophages and a higher density of COX-2-positive cells in the liver, while PACAP-deficiency could lead to a higher density of IL-1β-positive cells. These results confirm the anti-inflammatory properties of ApoE and PACAP that have been explored in various studies. As autoimmune diseases make up a big part of morbidity of the younger generation, our results give a new perspective for PACAP based pharmacological treatments and possible alternatives to IL-1β-inhibitors. Our findings are in accordance with a big part of previous studies on PACAP and PAC1, especially considering its effect on inflammatory processes in the body. Apart from that, there are some new findings about the influence of PACAP and PAC1 on the hepatic metabolism. To achieve therapeutic approaches with PACAP-/PAC1-agonists or -antagonists, it is necessary to start further investigations with a larger number of cases.