AimsThe purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD), regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting).Methods and ResultsMale C57BL/6 (WT) and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase) and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36) and Fatty acid transport protein (FATP) 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar) α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK) phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters — CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle.ConclusionEnhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.

The potential for drug-drug interactions (DDIs) arising from transcriptional regulation of drug-disposition genes via activation of nuclear receptors (NRs), such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR), remains largely unexplored, as highlighted in a recent guidance document from the European Medicines Agency. The goal of this research was to establish PXR-/CAR-/AhR-specific drug-metabolizing enzyme (DME) and transporter gene expression signatures in sandwich-cultured cryopreserved human hepatocytes using selective activators of PXR (rifampin), CAR (CITCO), and AhR (omeprazole). Dose response for ligand-induced changes to 38 major human DMEs and critical hepatobiliary transporters were assessed using a custom gene expression array card. We identified novel differentially expressed drug-disposition genes for PXR (↑ABCB1/MDR1, CYP2C9, CYP2C19, and EPHX1, ↓ABCB11), CAR [↑sulfotransferase (SULT) 1E1, uridine glucuronosyl transferase (UGT) 2B4], and AhR (↑SLC10A1/NTCP, SLCO1B1/OATP1B1], and coregulated genes (CYP1A1, CYP2B6, CYP2C8, CYP3A4, UGT1A1, UGT1A4). Subsequently, DME gene expression signatures were generated for known CYP3A4 inducers PF-06282999 and pazopanib. The former produced an induction signature almost identical to that of rifampin, suggesting activation of the PXR pathway, whereas the latter produced an expression signature distinct from those of PXR, CAR, or AhR, suggesting involvement of an alternate pathway(s). These results demonstrate that involvement of PXR/CAR/AhR can be identified via expression changes of signature DME/transporter genes. Inclusion of such signature genes could serve to simultaneously identify potential inducers and inhibitors, and the NRs involved in the transcriptional regulation, thus providing a more holistic and mechanism-based assessment of DDI risk for DMEs and transporters beyond conventional cytochrome P450 isoforms.

Number of references: 40Number of words in Abstract: 250 Number of words in Introduction: 640Number of words in Discussion: 1214 Abbreviations (alphabetical order):5' UTR, 5' untranslated region; ABCG2, ATP-binding cassette subfamily G member 2; AF, allele frequency; ARE, antioxidant response element; AHR, aryl hydrocarbon receptor; BCRP, breast cancer resistance protein; CYP, cytochrome P450; DRE, dioxin response element; EGFR, epidermal growth factor receptor; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; ERE, estrogen response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF1α, hypoxia inducible factor 1 alpha; HRE, hypoxia response element; NRF2, nuclear factor erythroid 2-related factor 2; PPARγ, peroxisome proliferator-activated receptor gamma; PPRE, peroxisome proliferator response element; PBS/T, PBS with 0.5% Tween-20; PR, progesterone receptor; RPL13A, ribosomal protein L13A; SNP, single nucleotide polymorphism; SP1, SP1 transcription factor; SP3, SP3 transcription factor; TSS, transcriptional start site contribute to our understanding of the interindividual regulation of BCRP expression in term placentas and may help to identify infants at risk for increased fetal exposure to chemicals due to low expression of this efflux protein.