Pharmacological activation of NRF2 (nuclear factor erythroid 2-related factor 2) arises from blocking the interaction of NRF2 with its negative regulator, KEAP1 (Kelch-like ECH-associated protein 1). We previously reported an isoquinoline-based NRF2 activator, but this compound showed negative logD 7.4 and a −2 charge at physiological pH, which may have limited its membrane permeability. In this work, we report potent, metabolically stable analogs that result from replacing a carboxymethyl group at the 4-position with a fluoroalkyl group. Article pubs.acs.org/jmc

Nrf2 and HIF1αtranscription factors protect against ischemicacute kidney injury (AKI) by upregulating metabolic and cytoprotective gene expression. In this study, we tested the hypothesis that Nrf2 is required for HIF1α-mediated hypoxic responses using Nrf2-sufficient (WT) and Nrf2-deficient (Nrf2-/-) primary murine kidney tubular epithelial cells (RTECs) and human immortalized tubular epithelial cells (HK2 cells) with HIF1 inhibition and activation. HIF1 pathway inhibitor digoxin blocked hypoxia-stimulated HIF1α activation and heme oxygenase (HMOX1) expression in HK2 cells. Hypoxia mimicking CoCl2 stimulated HMOX1 expression was significantly lower in Nrf2-/- RTECs than in WT counterparts. Similarly, hypoxia-stimulated HIF1α-dependent metabolic gene expression was markedly impaired in Nrf2-/- RTECs. Nrf2 deficiency impaired hypoxia-induced HIF1α stabilization independent of increased prolyl 4-hydroxylase gene expression. We found decreased HIF1α mRNA levels in Nrf2-/- RTECs both in normoxia and hypoxia-reoxygenation conditions. In silico analysis and chromatin immunoprecipitation assays demonstrated Nrf2 binding to the HIF1α promoter in normoxia, but its binding decreased in hypoxia exposed HK2 cells. However, Nrf2 binding at the HIF1α promoter was enriched following reoxygenation demonstrating that Nrf2 maintains constitutive HIF1α expression. Consistent with this result, we found decreased levels of Nrf2 in hypoxia and that were restored following reoxygenation. Inhibition of mitochondrial complex I prevented hypoxia-induced Nrf2 downregulation and also increased basal Nrf2 levels. These results demonstrate a crucial role for Nrf2 in optimal HIF1α activation in hypoxia, that mitochondrial signaling downregulates Nrf2 levels in hypoxia whereas reoxygenation restores it. Nrf2 and HIF1α interact toprovide optimal metabolic and cytoprotective responses in ischemic AKI.

Bronchopulmonary dysplasia (BPD) is a chronic disease of preterm babies with poor clinical outcomes. Nrf2 transcription factor is crucial for cytoprotective response, whereas Keap1—an endogenous inhibitor of Nrf2 signaling—dampens these protective responses. Nrf2-sufficient (wild type) newborn mice exposed to hyperoxia develop hypoalveolarization, which phenocopies human BPD, and Nrf2 deficiency worsens it. In this study, we used PND1 pups bearing bearing hypomorphic Keap1 floxed alleles (Keap1f/f) with increased levels of Nrf2 to test the hypothesis that constitutive levels of Nrf2 in the premature lung are insufficient to mitigate hyperoxia-induced hypoalveolarization. Both wildtype and Keap1f/f pups at PND1 were exposed to hyperoxia for 72 h and then allowed to recover at room air for two weeks (at PND18), sacrificed, and lung hypoalveolarization and inflammation assessed. Hyperoxia-induced lung hypoalveolarization was remarkably lower in Keap1f/f pups than in wildtype counterparts (28.9% vs 2.4%, wildtype vs Keap1f/f). Likewise, Keap1f/f pups were protected against prolonged (96 h) hyperoxia-induced hypoalveolarization. However, there were no differences in hyperoxia-induced lung inflammatory response immediately after exposure or at PND18. Lack of hypoalveolarization in Keap1f/f pups was accompanied by increased levels of expression of antioxidant genes and GSH as assessed immediately following hyperoxia. Keap1 knockdown resulted in upregulation of lung cell proliferation postnatally but had opposing effects following hyperoxia. Collectively, our study demonstrates that augmenting endogenous Nrf2 activation by targeting Keap1 may provide a physiological way to prevent hypoalveolarization associated with prematurity.

Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver that may progress to hepatic fibrosis and nonalcoholic steatohepatitis (NASH). Mechanisms underlying NAFLD and NASH are not yet fully understood. Dietary cholesterol was recently shown to be a risk factor for the development of NASH, suggesting a role for intestinal handling of cholesterol. One important regulator of cholesterol homeostasis is the sterol response element-binding protein-2 (SREBP-2) transcription factor. We tested the hypothesis that the overactivation of intestinal SREBP-2 increases the susceptibility to diet-induced NASH. A transgenic mouse model with intestine-specific overexpression of active SREBP-2 (ISR2 mice) driven by villin promoter was used. ISR2 mice and their wild-type littermates were fed a regular chow diet or a high-fat, high-cholesterol (HFHC) diet (15% fat, 1% cholesterol) for 15 wk. Results showed that HFHC feeding to ISR2 mice caused hepatic inflammation with increased levels of proinflammatory cytokines. Histological examination demonstrated extensive fibrosis after a HFHC diet associated with a perivascular as well as pericellular collagen deposits in ISR2 mice compared with wild-type littermates. The severe hepatic inflammation and advanced fibrosis in ISR2 mice was not associated with a difference in lipid accumulation in ISR2 mice compared with wild type littermates after HFHC feeding. These data indicate that overactivation of intestinal SREBP2 promotes diet-induced hepatic inflammation with features of human NASH resulting in rapid severe fibrosis and provide a novel link between regulatory processes of intestinal cholesterol and progression of fatty liver. The current study highlights the role of overactivation of intestinal SREBP-2 transcription factor in the progression of hepatic fibrosis associated with diet-induced NASH. Mice with intestine-specific overexpression of SREBP-2 demonstrated more inflammation and severe fibrosis in the liver in response to 15 wk of being fed a high-cholesterol, high-fat diet as compared with their wild-type littermates. These data demonstrate a novel link between intestinal regulatory processes of cholesterol metabolism and the pathogenesis of fatty liver diseases.

Objective: Concerns have been raised regarding the impact of time-restricted eating (TRE) on sex hormones in females. This study examined how TRE affects sex steroids in premenopausal and postmenopausal females.Methods: This is a secondary analysis of an 8-week TRE study (4-to 6-hour eating window) conducted in adults with obesity. Men and perimenopausal females were excluded. Females were classified into two groups based on menstrual status: premenopausal (n = 12) or postmenopausal (n = 11).Results: After 8 weeks, body weight decreased in premenopausal females (À3% AE 2%) and postmenopausal females (À4% AE 2%) (main effect of time, p < 0.001), with no difference between groups (no group  time interaction). Circulating levels of testosterone, androstenedione, and sex hormone binding globulin (SHBG) did not change in either group (no group  time interaction). Dehydroepiandrosterone (DHEA) concentrations decreased (p < 0.05) in premenopausal (À14% AE 32%) and postmenopausal females (À13% AE 34%; main effect of time, p = 0.03), with no difference between groups. Estradiol, estrone, and progesterone were measured only in postmenopausal females, and they remained unchanged.Conclusions: In premenopausal females, androgens and SHBG remained unchanged during TRE, whereas DHEA decreased. In postmenopausal females, estrogens, progesterone, androgens, and SHBG did not change, but DHEA was reduced.