Marinobufagenin, a mammalian endogenous cardiotonic steroid, is synthesized from the intermediates in the classical bile acid pathway in Cyp27A1 knockout mice

Abstract

ObjectivesThe bioactive steroidal Na/K‐ATPase ligand marinobufagenin (MBG) participates in renal Na+ transport, cell homeostasis, regulates blood pressure and arterial wall collagen production. MBG biosynthesis in mammals is controlled by the Cyp27a1 enzyme (cytochrome P450, family 27, subfamily a, polypeptide 1), which initiates the acidic bile acid (BA) pathway in rodent adrenal cortex. Nevertheless, the knockout of Cyp27a1 in mice did not reduce MBG production. In addition, the homozygous Cyp27a1(−/−) mice (HO) exhibited an upregulation of adrenal Akr1d1 enzyme (aldo‐keto reductase family 1, member D1) vs. wild type mice (WT). Akr1d1 participates in the final steps of both acidic and classical BA pathways. We hypothesized (i) that MBG may be synthesized from the intermediates in the classical BA pathway under Akr1d1 control, and (ii) that inhibition of Akr1d1 in HO will decrease MBG production.MethodsAll‐trans retinoic acid (ATRA) suppresses Akr1d1 and the enzymes controlled classical BA pathway. Four‐to‐six months old male HO and wild type mice (WT) were fed ATRA diet (150 mg/kg of diet) or control diet (CTRL) for 10 days (n=8/group). Adrenal Cyp27a1 and Akr1d1, and hepatic Cyp27a1, Akr1d1, Cyp7a1, and Cyp8b1 mRNA (by qPCR), and MBG excretion were assessed before (baseline, BL) and after ATRA diet. Data were analyzed by t‐test or 2‐way ANOVA and are presented as mean±SE.ResultsAdrenal and hepatic Cyp27a1 mRNAs were down‐regulated by 80% in HO vs. WT (p<0.01). Adrenal Akr1d1 mRNA was 3.5‐fold overexpressed in HO vs. WT (p<0.01) (Figure 1A). Hepatic mRNAs were overexpressed in HO vs. WT (Figure 1B; Table). ATRA diet decreased expression of hepatic Akr1d1 mRNA in both strains vs. CTRL (HO: by 180%, p<0.01; WT: by 170%, p<0.01). Hepatic Cyp7a1 and Cyp8b1 mRNAs were also significantly down‐regulated by ATRA in both WT and HO but remained significantly higher in HO than in WT (Table). Baseline urine MBG level was 2‐fold higher in HO vs. WT (p<0.05); inhibition of Akr1d1 by ATRA was associated with a decrease in urine MBG in HO (5.3±0.5 vs. 2.9±0.2 pmol/24hr; BL vs. ATRA; p<0.05), but not in WT (2.1±0.4 vs. 2.6±0.9 pmol/24hr; BL vs. ATRA, n/s).ConclusionsThe up‐regulation of hepatic Akr1d1, Cyp7a1 and Cyp8b1 enzymes in Cyp27a1(−/−) mice may compensate for a lack of Cyp27a1 and promote production of a bioactive steroid MBG from the intermediates in the classical BA pathway. Inhibition of Akr1d1 resulted in a decrease of MBG production in Cyp27a1(−/−) mice only. MBG production was not changed by ATRA in WT with normal expression of Cyp27a1, which indicates that Cyp27a1 is a predominant enzyme in MBG biosynthesis.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.