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PGC1α-Related Coactivator (PRC) is a transcriptional coactivator promoting cytokine expression in vitro in response to mitochondrial injury and oxidative stress, however, its physiological role has remained elusive. Herein we investigate aspects of the immune response function of PRC, first in an in vivo thioacetamide (TAA)-induced mouse model of drug-induced liver injury (DILI), and subsequently in vitro in human monocytes, HepG2, and dendritic (DC) cells. TAA treatment resulted in the dose-dependent induction of PRC mRNA and protein, both of which were shown to correlate with liver injury markers. Conversely, an adenovirus-mediated knockdown of PRC attenuated this response, thereby reducing hepatic cytokine mRNA expression and monocyte infiltration. Subsequent in vitro studies with conditioned media from HepG2 cells overexpressing PRC, activated human monocytes and monocyte-derived DC, demonstrated up to 20% elevated expression of CD86, CD40, and HLA-DR. Similarly, siRNA-mediated knockdown of PRC abolished this response in oligomycin stressed HepG2 cells. A putative mechanism was suggested by the co-immunoprecipitation of Signal Transducer and Activator of Transcription 1 (STAT1) with PRC, and induction of a STAT-dependent reporter. Furthermore, PRC co-activated an NF-κB-dependent reporter, indicating interaction with known major inflammatory factors. In summary, our study indicates PRC as a novel factor modulating inflammation in DILI., (© 2020 The Authors. FASEB BioAdvances published by the Federation of American Societies for Experimental Biology.)

Context: There is an ongoing search for specific and translational biomarkers of drug-induced liver injury (DILI). MicroRNA-122 (miR-122) has previously shown potential as a sensitive, specific, and translational biomarker of DILI in both rodent, and human studies., Objective: To build on previous work within the field, we examined biomarker kinetics in a rat model of acetaminophen (APAP)-induced liver injury to confirm the sensitivity, and specificity of miR-122 and glutamate dehydrogenase (GLDH)., Materials and Methods: qRT-PCR and a standard enzymatic assay were used for biomarker analysis., Results: Both miR-122 and GLDH were demonstrated to be more readily-detectable biomarkers of APAP-DILI than alanine aminotransferase (ALT). Peak levels for all biomarkers were detected at 2 days after APAP. At day 3, miR-122 had returned to baseline; however, other biomarkers remained elevated between 3 and 4 days. We were also able to demonstrate that, although miR-122 is present in greater quantities in exosome-free form, both exosome-bound and non-vesicle bound miR-122 are released in a similar profile throughout the course of DILI., Discussion and Conclusions: Together, this study demonstrates that both GLDH and miR-122 could be used during preclinical drug-development as complementary biomarkers to ALT to increase the chance of early detection of hepatotoxicity.

Alanine aminotransferase (ALT) in serum is the standard biomarker for liver injury. We have previously described a clinical trial with a novel selective peroxisome proliferator-activated receptor α (PPARα) agonist (AZD4619), which unexpectedly caused increased serum levels of ALT in treated individuals without any other evidence of liver injury. We pinpointed a plausible mechanism through which AZD4619 could increase serum ALT levels; namely through the PPARα-specific activation of the human ALT1 gene at the transcriptional level. In the present study, we present data from the preceding rat toxicity study, demonstrating that AZD4619 had no effect on rat serum ALT activity levels, and further experiments were performed to elucidate the mechanisms responsible for this species-related difference. Our results revealed that AZD4619 increased ALT1 protein expression in a dose-dependent manner in human, but not in rat primary hepatocytes. Cloning of the human and rat ALT1 promoters into luciferase vectors confirmed that AZD4619 induced only the human, but not the rat ALT1 gene promoter in a dose-dependent manner. In PPARα-GAL4 reporter gene assays, AZD4619 was >100-fold more potent on the human vs. rat PPARα levels, explaining the differences in induction of the ALT1 gene between the species at the concentration range tested. These data demonstrate the usefulness of the human and rat ALT1 reporter gene assays for testing future drug candidates at the preclinical stage. In drug discovery projects, these assays elucidate whether elevations in ALT levels observed in vivo or in the clinic are due to metabolic effects rather than a toxic event in the liver.

Background & Aims: There is a demand for more sensitive, specific and predictive biomarkers for drug-induced liver injury (DILI) than the gold standard used today, alanine aminotransferase (ALT). The aim of this study was to qualify novel DILI biomarkers (keratin-18 markers M65/M30, microRNA-122, glutamate dehydrogenase and alpha-foetoprotein) in human DILI., Methods: Levels of the novel biomarkers were measured by enzyme-linked immunosorbent assay or real-time quantitative reverse-transcription PCR (qRT-PCR) in two human DILI cohorts: a human volunteer study with acetaminophen and a human immunodeficiency virus (HIV)/tuberculosis (TB) study., Results: In the acetaminophen study, serum M65 and microRNA-122 levels were significantly increased at an earlier time point than ALT. Furthermore, the maximal elevation of M65 and microRNA-122 exceeded the increase in ALT. In the HIV/TB study, all the analysed novel biomarkers increased after 1 week of treatment. In contrast to ALT, the novel biomarkers remained stable in a human cohort with exercise-induced muscular injury., Conclusions: M65 and microRNA-122 are potential biomarkers of DILI superior to ALT with respect to sensitivity and specificity., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

PPARδ is involved in the inflammatory response and its expression is induced by cytokines, however, limited knowledge has been produced regarding its regulation. Since recent findings have shown that microRNAs, which are small non-coding RNAs that regulate gene expression, are involved in the immune response, we set out to investigate whether PPARδ can be regulated by microRNAs expressed in monocytes. Bioinformatic analysis identified a putative miR-9 target site within the 3'-UTR of PPARδ that was subsequently verified to be functional using reporter constructs. Primary human monocytes stimulated with LPS showed a downregulation of PPARδ and its target genes after 4 h while the expression of miR-9 was induced. Analysis of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages showed that human PPARδ mRNA as well as miR-9 expression was higher in M1 compared to M2 macrophages. Furthermore, treatment with the PPARδ agonist, GW501516, induced the expression of PPARδ target genes in the pro-inflammatory M1 macrophages while no change was observed in the anti-inflammatory M2 macrophages. Taken together, these data suggest that PPARδ is regulated by miR-9 in monocytes and that activation of PPARδ may be of importance in M1 pro-inflammatory but not in M2 anti-inflammatory macrophages in humans.

Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Promoter polymorphisms in microsomal triglyceride transfer protein (MTTP) have been associated with decreased plasma lipids but an increased risk for ischemic heart disease (IHD), indicating that MTTP influences the susceptibility for IHD independent of plasma lipids. The objective of this study was to characterize the functional promoter polymorphism in MTTP predisposing to IHD and its underlying mechanism. Use of pyrosequencing technology revealed that presence of the minor alleles of the promoter polymorphisms -493G>T and -164T>C result in lower transcription of MTTP in vivo in the heart, liver, and macrophages. In vitro experiments indicated that the minor -164C allele mediates the lower gene expression and that C/EBP binds to the polymorphic region in an allele-specific manner. Furthermore, homozygous carriers of the -164C were found to have increased risk for IHD as shown in a case-control study including a total of 544 IHD patients and 544 healthy control subjects. We concluded that carriers of the minor -164C allele have lower expression of MTTP in the heart, mediated at least partly by the transcription factor CCAAT/enhancer binding protein, and that reduced concentration of MTTP in the myocardium may contribute to IHD upon ischemic damage.

In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) alpha agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPARalpha agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at -574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum.

The peroxisome proliferator-activated receptor delta (PPARdelta) is a transcription factor that regulates genes of importance in lipid and glucose metabolism. ApoA-II is one of the major proteins of the HDL-particle. The aim of this study was to investigate the regulation of apoA-II gene expression by PPARdelta. Treatment of HepG2 cells with the PPARdelta specific agonist GW501516 increased apoA-II mRNA expression. Likewise, reporter gene assays using a construct containing 2.7 kb of the proximal apoA-II promoter showed increased activity after treatment with GW501516, both in HepG2 and in HuH-7 cells. Mutation of two putative PPAR response elements (PPREs) in this region showed that the PPRE at position -737/-717 is the functional site. Binding of PPARdelta to this site was confirmed by chromatin immunoprecipitation and gel retardation analyses. In conclusion, PPARdelta increases the expression of the human apoA-II gene in liver cells via a PPRE in the proximal promoter.

Background: Peroxisome proliferator-activated receptor delta (PPAR delta) is a member of the nuclear receptor superfamily. Numerous studies have aimed at unravelling the physiological role of PPAR delta as a transcriptional regulator whereas the regulation of PPAR delta gene expression has been less studied., Results: The principal transcription start site in the human PPAR delta gene identified here is positioned upstream of exon 1, although four alternative 5'-ends related to downstream exons were identified. The demonstration of multiple 5'-UTR splice variants of PPAR delta mRNA, with an impact on translation efficiency, suggests a translational regulation of human PPAR delta expression. Five untranslated exons identified in this study contribute to the variability among the 5'-UTRs of human PPAR delta mRNAs. Moreover, in vitro studies of a 3'-splice transcript encoding a truncated variant of PPAR delta (designated PPAR delta 2) show that this isoform constitutes a potential dominant negative form of the receptor., Conclusion: We propose that alternative splicing of human PPAR delta constitutes an intrinsic role for the regulation of PPAR delta expression and thus activity, and highlight the significance of alternative splicing of this nuclear receptor in physiology and disease.