Search

11β-hydroxysteroid dehydrogenase type 1 (11HSD1) regenerates cortisol from cortisone within adipose tissue and liver. 11HSD1 inhibitors may enhance insulin sensitivity in type 2 diabetes and be most efficacious in obesity when 11HSD1 is increased in subcutaneous adipose biopsies. We examined the regeneration of cortisol in vivo in obesity, and the effects of the 11HSD1 inhibitor carbenoxolone. We compared six lean and six obese men and performed a randomized, placebo-controlled crossover study of carbenoxolone in obese men. The obese men had no difference in their whole-body rate of regenerating cortisol (measured with 9,11,12,12-[²[H.sub.4]]cortisol tracer), but had more rapid conversion of [³H]cortisone to [³H]cortisol in abdominal subcutaneous adipose tissue (measured with microdialysis). During insulin infusion, adipose 11HSD1 activity fell markedly in lean but not in obese men. Carbenoxolone inhibited whole-body cortisol regeneration, but did not significantly inhibit adipose 11HSD1 and had no effects on insulin sensitivity (measured by [²[H.sub.2]]glucose infusion with or without hyperinsulinemia). Thus, in vivo cortisol generation is increased selectively within adipose tissue in obesity, perhaps reflecting resistance to insulin-mediated downregulation of 11HSD1. However, obese men are less susceptible than lean men to the insulin-sensitizing effects of carbenoxolone. To be useful in obese patients, 11HSD1 inhibitors will need to inhibit the enzyme more effectively in adipose tissue., 11β-Hydroxysteroid dehydrogenase type 1 (11HSD1) is a microsomal enzyme expressed in many tissues, including liver and adipose tissue (1). It catalyzes the regeneration of the active glucocorticoid cortisol from its [...]

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitrobut does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Kynurenine monooxygenase (KMO) blockade protects against multiple organ failure caused by acute pancreatitis (AP), but the link between KMO and systemic inflammation has eluded discovery until now. Here, we show that the KMO product 3-hydroxykynurenine primes innate immune signaling to exacerbate systemic inflammation during experimental AP. We find a tissue-specific role for KMO, where mice lacking Kmosolely in hepatocytes have elevated plasma 3-hydroxykynurenine levels that prime inflammatory gene transcription. 3-Hydroxykynurenine synergizes with interleukin-1β to cause cellular apoptosis. Critically, mice with elevated 3-hydroxykynurenine succumb fatally earlier and more readily to experimental AP. Therapeutically, blockade with the highly selective KMO inhibitor GSK898 rescues the phenotype, reducing 3-hydroxykynurenine and protecting against critical illness and death. Together, our findings establish KMO and 3-hydroxykynurenine as regulators of inflammation and the innate immune response to sterile inflammation. During critical illness, excess morbidity and death from multiple organ failure can be rescued by systemic KMO blockade.

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead–based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein–compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO.

Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst−/−mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst−/−mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst−/−mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.

Increased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated estradiol in men and postmenopausal women. Pulmonary arteries synthesise estradiol via aromatase and metabolise it via CYP1B1 to mitogenic metabolites; SNPs in aromatase and CYP1B1 have been associated with PAH. This suggests that estradiol metabolism could be altered in iPAH. This proof-of-concept study profiles estradiol and several metabolites of estradiol simultaneously in serum from iPAH patients and controls. We show that the estradiol and metabolite profile is altered in iPAH and that 16-hydroxyestrone and 16-hydroxyestradiol accumulate in iPAH patients with 16-hydroxyestrone levels relating to disease severity.