ABSTRACT
Herein we have characterized CYPs and antioxidant enzymes in a new steatotic rat model induced with a high fat diet (HFD) combined with a low dose of streptozotocin (STZ). This model was recently put forward in order to better replicate the NAFLD human pathology. HFD/STZ rats developed hyperglycemia, hypercholesterolemia and overt steatosis. The treatment also caused liver damage, but not lipid peroxidation, suggesting this damage was due to hepatic fat deposition and excess formation of toxic free fatty acids, rather than to oxidative stress. In the HFD/STZ group, a significant rise in total CYP content was found, in conjunction with increased activity and protein levels of CYP2E1 and CYP4A, the latter also up-regulated at the transcriptional level. A significant decrease of CYP2C11 was observed at the transcriptional and protein level, whereas CYP3A2 did not change in response to HFD/STZ treatment. In our experimental conditions, the activity of the HO-1 and NQO1 enzymes, whose genes are regulated by Nrf2, were not affected, and nor were the antioxidant enzymes SOD and CAT, confirming the lack of oxidative stress. Our HFD/STZ treatment, which established overt steatosis and changes in CYPs expression, but not oxidative stress, likely reflects an early stage of NAFLD.
Subject(s)
Diet, High-Fat/adverse effects , Non-alcoholic Fatty Liver Disease/enzymology , Streptozocin/adverse effects , Animals , Blood Glucose/metabolism , Cholesterol/blood , Cytochrome P-450 CYP2E1/genetics , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 CYP4A/genetics , Cytochrome P-450 CYP4A/metabolism , Dose-Response Relationship, Drug , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase (Decyclizing)/metabolism , Lipid Peroxidation , Liver/drug effects , Liver/metabolism , Male , NAD(P)H Dehydrogenase (Quinone)/genetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Non-alcoholic Fatty Liver Disease/drug therapy , Oxidative Stress , Rats , Rats, Wistar , Streptozocin/administration & dosage , Triglycerides/blood , Up-RegulationABSTRACT
With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.
