Pregnane X Receptor and Yin Yang 1 Contribute to the Differential Tissue Expression and Induction of CYP3A5 and CYP3A4

The hepato-intestinal induction of the detoxifying enzymes CYP3A4 and CYP3A5 by the xenosensing pregnane X receptor (PXR) constitutes a key adaptive response to oral drugs and dietary xenobiotics. In contrast to CYP3A4, CYP3A5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. Using cell lines and mice transgenic for a CYP3A5 promoter we demonstrate that the CYP3A5 expression in these organs is non-inducible and independent from PXR. Instead, it is enabled by the loss of a suppressing yin yang 1 (YY1)-binding site from the CYP3A5 promoter which occurred in haplorrhine primates. This YY1 site is conserved in CYP3A4, but its inhibitory effect can be offset by PXR acting on response elements such as XREM. Taken together, the loss of YY1 binding site from promoters of the CYP3A5 gene lineage during primate evolution may have enabled the utilization of CYP3A5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. Our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. They also suggest an explanation for the considerable tissue expression differences between CYP3A5 and CYP3A4

Isolated hypercholesterolemia leads to steatosis in the liver without affecting the pancreas

Abstract Background Lipid accumulation in the liver and pancreas is primarily caused by combined hyperlipidemia. However, the effect of isolated hypercholesterolemia without hypertriglyceridemia is not fully described. Therefore, our aim was to investigate whether hypercholesterolemia alone leads to alterations both in hepatic and pancreatic lipid panel and histology in rats. Methods Male Wistar rats were fed with 2% cholesterol +0.25% cholate-supplemented diet or standard chow for 12 weeks. Blood was collected at weeks 0, 4, 8 and 12 to measure serum cholesterol and triglyceride levels. At week 12, both the pancreas and the liver were isolated for further histological and biochemical analysis. Hepatic and plasma fatty acid composition was assessed by gas chromatography. Expression of mRNA of major enzymes involved in saturated/unsaturated fatty acid synthesis was analyzed by qPCR. In separate experiments serum enzyme activities and insulin levels were measured at week 9. Results At week 12, rats fed with 2% cholesterol +0.25% cholate-supplemented diet were characterized by elevated serum cholesterol (4.09 ± 0.20 vs. 2.89 ± 0.22 mmol/L, *p < 0.05) while triglyceride (2.27 ± 0.05 vs. 2.03 ± 0.03 mmol/L) and glucose levels (5.32 ± 0.14 vs. 5.23 ± 0.10 mmol/L) remained unchanged. Isolated hypercholesterolemia increased hepatic lipid accumulation, hepatic cholesterol (5.86 ± 0.22 vs. 1.60 ± 0.15 ng/g tissue, *p < 0.05) and triglyceride contents (19.28 ± 1.42 vs. 6.78 ± 0.71 ng/g tissue, *p < 0.05), and hepatic nitrotyrosine level (4.07 ± 0.52 vs. 2.59 ± 0.31 ng/mg protein, *p < 0.05). The histology and tissue lipid content of the pancreas was not affected. Serum total protein level, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities remained unchanged in response to isolated hypercholesterolemia while serum alkaline phosphatase activity (ALP) significantly increased. Plasma insulin levels did not change in response to isolated hypercholesterolemia suggesting an intact endocrine function of the pancreas. Isolated hypercholesterolemia caused a significantly increased hepatic and serum fatty acid level associated with a marked alteration of fatty acid composition. Hepatic expression of Δ9-desaturase (SCD1) was increased 4.92×, while expression of Δ5-desaturase and Δ6-desaturase were decreased (0.447× and 0.577×, respectively) due to isolated hypercholesterolemia. Conclusions Isolated hypercholesterolemia leads to hepatic steatosis and marked alterations in the hepatic lipid profile without affecting the pancreas. Altered fatty acid profile might mediate harmful effects of cholesterol in the liver

Peripheral Effects of FAAH Deficiency on Fuel and Energy Homeostasis: Role of Dysregulated Lysine Acetylation

FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/-) mice.FAAH(-/-) mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH(-/-) mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/-) mice. Dysregulated hepatic FAAH(-/-) lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/-) acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH(-/-) mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/-) mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/-) aldolase B. Fed FAAH(-/-) alcohol dehydrogenase (ADH) acetylation was also decreased.Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/-) mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver's role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Plasma exosomes characterization reveals a perioperative protein signature in older patients undergoing different types of on-pump cardiac surgery

Although exosomes are extracellular nanovesicles mainly involved in cardioprotection, it is not known whether plasma exosomes of older patients undergoing different types of on-pump cardiac surgery protect cardiomyocytes from apoptosis. Since different exosomal proteins confer pro-survival effects, we have analyzed the protein cargo of exosomes circulating early after aortic unclamping. Plasma exosomes and serum cardiac troponin I levels were measured in older cardiac surgery patients (NYHA II-III) who underwent first-time on-pump coronary artery bypass graft (CABG; n = 15) or minimally invasive heart valve surgery (mitral valve repair, n = 15; aortic valve replacement, n = 15) at induction of anesthesia (T0, baseline), 3 h (T1) and 72 h (T2) after aortic unclamping. Anti-apoptotic role of exosomes was assessed in HL-1 cardiomyocytes exposed to hypoxia/re-oxygenation (H/R) by TUNEL assay. Protein exosomal cargo was characterized by mass spectrometry approach. Exosome levels increased at T1 (P &lt; 0.01) in accord with troponin values in all groups. In CABG group, plasma exosomes further increased at T2 (P &lt; 0.01) whereas troponin levels decreased. In vitro, all T1-exosomes prevented H/R-induced apoptosis. A total of 340 exosomal proteins were identified in all groups, yet 10% of those proteins were unique for each surgery type. In particular, 22 and 12 pro-survival proteins were detected in T1-exosomes of heart valve surgery and CABG patients, respectively. Our results suggest that endogenous intraoperative cardioprotection in older cardiac surgery patients is early mediated by distinct exosomal proteins regardless of surgery type

Comparison of immunophenotypes of primary breast carcinomas and multiple corresponding distant metastases: an autopsy study of 25 patients

Phenotypical change in metastatic breast carcinoma has widely been accepted as an inherent biological feature rather than technical fault. We analyzed the immunohistochemical phenotype and histopathological features of 25 primary breast carcinomas and 90 corresponding distant metastases in 23 organs retrospectively. Histological slides were reviewed for prognostic and predictive factors. Overall, metastases were more similar to each other and often differed from the primary tumor. We created a 3-step grouping system based on the localization of metastases. Regions: tumors metastasizing to the abdominal region were likely to lose ER (p = 0.002); we detected loss of PR in metastases to the thorax (p = 0.039) and abdomen (p < 0.001). Organ systems: loss of ER and PR was observed in metastases to the gastrointestinal system (p = 0.026 and p = 0.001, respectively), in the respiratory system only the loss of PR was significant (p = 0.05). Individual organs: the primaries were likely to lose the hormone receptors in liver metastases (ER p = 0.026; PR p = 0.004). In lung metastases only loss of PR was apparent (p = 0.049). We did not observe significant change in HER2 status, regarding Ki67 change occurred only in bone metastases compared to the primary (p = 0.048). 7/25 patients' distant metastases had heterogeneous immunoprofiles. The later the metastasis was discovered the more likely it had a differing IHC profile compared to the primary tumor, patients who had longer OS had a higher chance to develop a discordant metastasis. Immunoprofile of metastases may differ from primary breast cancer and from each other, probably resulting in different response to therapy