11 b-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11b-hydroxysteroid dehydrogenases (11b-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11bHSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11bHSD-1) may function as an 11b-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo, mice were produced with targeted disruption of the 11b-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo. Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11b-HSD-1 2y2 mice were found to resist hyperglycamia provoked by obesity or stress. Attenuation of hepatic 11b-HSD-1 may provide a novel approach to the regulation of gluconeogenesis. Glucocorticoids (cortisol in humans, corticosterone in mice and rats) are an important class of adrenocorticosteroids that regulate many metabolic and homeostatic processes and form a key component of the response to stress (1). Glucocorticoids act via intracellular glucocorticoid receptors and, in some tissues, mineralocorticoid receptors; both are nuclear transcription factors (2). Recently, it has become apparent that glucocorticoid action on target tissues depends not only on circulating steroid concentrations and the cellular expression of receptors, but also on intracellular enzymes that critically determine if glucocorticoids gain access to receptors in active forms. 11b-hydroxysteroid dehydrogenase (11b-HSD) catalyzes the interconversion of cortisol and corticosterone with their inert 11-keto forms [cortisone and 11-dehydrocorticosterone (11-DHC), respectively]. There are two isozymes. 11b-HSD type 2 (11b-HSD-2) is a high affinity (nanomolar KM), NADdependent exclusive 11b-dehydrogenase (3, 4), which excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in aldosterone target tissues, such as the distal nephron (5, 6), and from the feto-placental unit (4). Mutations of the gene encoding 11b-HSD-2 are responsible for the human syndrome of ‘‘apparent mineralocorticoid excess,’’ characterized by illicit activation of renal mineralocorticoid receptors by cortisol, producing sodium retention, severe hypertension, and hypokalemia (7–9). In contrast, the function(s) of 11b-HSD type 1 (11b-HSD1), initially purified and cloned from rat liver (10, 11), remain obscure. 11b-HSD-1 shares less than 30% homology with 11b-HSD-2, has a lower affinity for glucocorticoids (micromolar KM), and uses NADP(H) as cosubstrate. High expression of 11b-HSD-1 is found in the liver, lung, adipose tissue, kidney, and brain, largely localized to cells expressing glucocorticoid receptors, but not mineralocorticoid receptors, suggesting that 11b-HSD-1, by analogy to 11b-HSD-2, modulates glucocorticoid access to glucocorticoid receptors (12). 11b-HSD-1 in tissue homogenates and when purified is bidirectional, exhibiting both 11b-dehydrogenase and 11breductase reactions, with greater stability of the dehydrogenase activity (10). However, though transfection of Chinese hamster ovary cells with 11b-HSD-1 cDNA encodes bidirectional activity (11), transfection of the same cDNA into other cell lines encodes a predominant 11b-reductase (13, 14), which regenerates active glucocorticoids from inert 11-keto forms. The reductase reaction predominates in intact cells, whereas broken cells, homogenates, and microsomes all show bidirectional activity. Importantly, primary cultures of rat hepatocytes, lung cells, hippocampal cells, and vascular smooth muscle cells (15–18) also show predominant 11b-reductase activity, which parallels 11b-HSD-1 mRNA expression (15). Such glucocorticoid regeneration, if manifest in vivo, might increase effective intracellular glucocorticoid levels, amplifying glucocorticoid activity. To explore this notion further we generated mice bearing targeted disruption of the 11b-HSD-1 gene. MATERIALS AND METHODS Cloning and Mapping Mouse 11b-HSD-1 Gene. A 129yOla lGEM-12 genomic library was screened with a rat 11b-HSD-1 cDNA probe. A 14-kb 11b-HSD-1 genomic fragment encompassing exons 2–5 was cloned and mapped by partial restriction digestion, subcloning of gene fragments, and crosshybridization of purified fragments. Sequencing of exons 2, 3, 4, and 5 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9414924-6$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations: 11b-HSD-1, 11b-hydroxysteroid dehydrogenase type 1; 11-DHC, 11-dehydrocorticosterone; ES, embryonic stem; PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose 6-phosphatase. †To whom reprint requests should be addressed. e-mail: Yuri. Kotelevtsev@ed.ac. uk.