11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has been implicated in the pathogenesis of human obesity and insulin resistance through 11-oxoreductase activation of cortisol (F) from cortisone (E) stimulating adipocyte differentiation and hepatic glucose output. In its purified state however, 11beta-HSD1 is a dehydrogenase inactivating F to E. In patients with cortisone reductase deficiency we recently identified two inactivating mutations, delta29bp and R453Q, within exon 5 of the gene encoding NADPH-regenerating hexose-6-phosphate dehydrogenase (H6PDH) (1). This suggests a pivotal role for H6PDH in conferring oxoreductase activity upon 11beta-HSD1. Human 11beta-HSD1 and H6PDH wild-type and mutant cDNAs were cloned into pYeDP10 and pYcDE2 yeast expression vectors for co-transformation of S. cerevisiae. Yeast expression of 11beta-HSD1 yielded considerable dehydrogenase and some oxoreductase activity (whole cell assays; 50 nM potassium phosphate, 100 nM F or E). Similarly, a colorimetric assay employing 2-deoxy-glucose confirmed functional expression of H6PDH, with significant activity of wild-type, but grossly reduced activity of mutant proteins (p<0.01). Co-expression of 11beta-HSD1 and H6PDH revealed significantly higher 11beta-HSD1 oxoreductase activity in yeast expressing wild-type H6PDH than in H6PDH mutants (p<0.01). For bacterial expression, we generated a truncated H6PDH construct in pACYC-Duet vector, and used a previously characterised 11beta-HSD1 construct (2). Following purification of bacterially expressed enzymes by Ni-NTA metal affinity chromatography, we were able to recombine H6PDH and 11beta-HSD1 proteins in controlled environment. In our specific oxoreductase activity assay (100nM E, 100microM NADPH), 11beta-HSD1 alone did not exhibit conversion of E to F. However, addition of H6PDH, to regenerate NADPH, resulted in significant conversion of E to F (478 pmol/h/microgram). These findings provide compelling proof of concept of the critical role of H6PDH in regulating 11beta-HSD1 activity; in its own right, H6PDH might represent a novel mechanism in the pathogenesis of insulin resistance and obesity. (1) Nat Genet 2003, 34:434. (2) JBC 2001, 276:21343.
22 - 24 Mar 2004
British Endocrine Societies