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Endocrinology Unit, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. Nicholas M. Morton, Endocrinology Unit, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, Scotland, United Kingdom. E-mail: nik.morton{at}ed.ac.uk.
Abstract
The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1) amplifies intracellular glucocorticoid action in vivo. 11ß-HSD-1 activity is increased in adipose tissues of obese humans and genetically obese rodents, providing a mechanistic basis for the similarities between metabolic disease arising from high circulating glucocorticoids (Cushing’s syndrome) and idiopathic obesity/metabolic syndrome where plasma glucocorticoids are typically unaltered. Fat-specific overexpression of 11ß-HSD-1 produces a metabolic syndrome in mice, whereas 11ß-HSD-1 null mice resist high-fat diet (HF)-induced visceral obesity and its metabolic consequences. Here we compared the effects of chronic (18 wk) HF feeding on adipose 11ß-HSD-1 activity in strains of mice that are either resistant (A/J) or prone (C57BL/6J) to metabolic disease. 11ß-HSD-1 activity was highest in sc fat, followed by epididymal fat, with lowest activity in the mesenteric visceral depot of both strains. 11ß-HSD-1 activity was lower in white adipose tissues of A/J compared with C57BL/6J mice. Chronic HF feeding unexpectedly caused a down-regulation of 11ß-HSD-1 in adipose tissues of both strains, despite comparable adiposity. However, A/J mice down-regulated adipose 11ß-HSD-1 to a significantly lower level than C57BL/6J mice in white and thermogenic brown adipose tissues. We propose that a lower adipose 11ß-HSD-1 set point affords a metabolic protection to A/J mice. Adaptive down-regulation of adipose 11ß-HSD-1 in response to chronic HF represents a novel mechanism that may counteract metabolic disease.
THERE ARE WELL-RECOGNIZED phenotypic parallels between Cushing’s syndrome and idiopathic obesity with or without the coassociated features of the metabolic syndrome (visceral adiposity, insulin resistance, type 2 diabetes mellitus, dyslipidemia, hypertension, and an increased cardiovascular risk profile). However, whereas high circulating glucocorticoid levels cause Cushing’s syndrome, there is no evidence of plasma cortisol excess (1, 2) in subjects with the metabolic syndrome.
The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1) serves as a prereceptor glucocorticoid-amplifying mechanism by catalyzing the activation of inert 11-dehydrocorticosterone (cortisone in humans) into active corticosterone (cortisol) in vivo (3, 4, 5). 11ß-HSD-1 is highly expressed in metabolically active tissues such as liver and adipose. The finding that 11ß-HSD-1 activity is elevated in adipose tissue of obese rodents (5, 6) and humans (7, 8, 9, 10, 11, 12) suggested that increased 11ß-HSD-1-mediated glucocorticoid action within fat could explain the similarities between Cushingoid and idiopathic obesity/metabolic syndrome even in the absence of elevated circulating cortisol in humans. This hypothesis is strongly supported by the finding that transgenic mice overexpressing 11ß-HSD-1 selectively in adipose tissue develop a full metabolic syndrome with visceral obesity, diabetes, dyslipidemia (5), and hypertension (13). The effects of adipose-specific 11ß-HSD-1 overexpression are more pronounced in visceral depots where glucocorticoid receptor levels are higher (5, 14) and where fat accumulation is strongly correlated with metabolic and cardiovascular disease (15). Conversely, 11ß-HSD-1-deficient mice are protected from diet-induced visceral obesity and its metabolic consequences, in part through insulin sensitization in, and redistribution of, adipose tissue to metabolically safer anatomical sites (16).
Although obesity is associated with elevated adipose 11ß-HSD-1 activity in genetically obese rodent models (5, 6), we wished to examine the more physiologically relevant effects of long-term excess dietary fat exposure on 11ß-HSD-1 expression in key metabolic tissues in mouse strains previously demonstrated to reflect the extremes in susceptibility (C57BL/6J) or resistance (A/J) to metabolic disorders with chronic high-fat diet (HF) feeding (17, 18).
Materials and Methods
Animals
C57BL/6J, A/J mice and C57BL/6JLepob/ob mice (Harlan UK Ltd., Bicester, UK) were housed in standard conditions on a 12-h light, 12-h dark cycle (lights on at 0700 h). Adult age-matched male mice (n = 6–10) were given control (11% calories as fat, Research Diets D12328, Research Diets, Inc., New Brunswick, NJ) or HF (58% calories as fat, Research Diets D12331) for 2 or 18 wk, a diet previously optimized for weight gain and insulin resistance (17). Mice were singly housed for the final week of the experiment. Mice were killed by cervical dislocation at around 0800 h within 1 min of disturbing each cage.
Intraperitoneal glucose tolerance test
After 18 wk on control or HF, mice were fasted overnight and then injected ip with 2 mg/g D-glucose (25% stock solution in saline) or 1 mg/g body weight Humulin S (Lilly UK, Basingstoke). Blood samples were taken by tail venesection into EDTA-microtubes (Sarstedt, Leicester, UK) at zero (before injection and within 1 min of disturbing the cage) and at 15-, 30-, 60-, and 120-min intervals after the glucose bolus. Glucose was measured with the Sigma HK assay (Sigma, Poole, UK). Insulin levels were determined by ELISA (Crystalchem, Chicago, IL).
11ß-HSD-1 enzyme activity
Adipose, liver, and muscle samples were dissected and weighed. Tissues were homogenized, as described (19), and incubated (0.2 mg/ml protein) with 10 nM tritiated corticosterone and an excess (400 µM) of the 11ß-HSD-1-specific cofactor nicotinamide adenine dinucleotide phosphate (under in vitro conditions in homogenized tissues, 11ß-HSD-1 is bidirectional, with assay of dehydrogenation more stable). This assay was in the linear range of protein concentration and product formation with relative differences between samples unaffected over a higher range of tritiated corticosterone concentrations. After a 10-min (liver), 1-h (adipose), or 6-h (quadriceps muscle) incubation, steroids were extracted with ethyl acetate, separated by thin-layer chromatography, identified by migration in comparison to unlabeled corticosterone and 11-dehydrocorticosterone standards and quantified with a phosphorimager tritium screen (Fujifilm, Tokyo, Japan). Time points were optimized to achieve linearity of the assay in each tissue, which is dependent upon the level of 11ß-HSD-1 expression within tissues (liver>adipose>muscle).
RNA extraction and analysis
Tissues were snap-frozen in liquid nitrogen and homogenized in Trizol (Life Technologies, Inc., Paisley, UK). Total RNA was blotted according to standard Northern blot procedure, as described (16, 20). Gene expression was analyzed and relative transcript levels expressed in arbitrary units after correction with a U1 RNA loading control (16, 20). 11ß-HSD-1 probes were generated as described (20).
Statistical analyses
Data are expressed as means ± SEM. Results were subjected to two-way ANOVA and post hoc Tukeys’ test for strain and diet. Significance was set at P < 0.05. Daggers indicate a significant difference between strain on the same diet: 
, P < 0.05; , P < 0.01; , P < 0.001; and asterisks indicate a significant effect of diet within a strain: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Results
The previously described adipose and metabolic phenotypes described for C57BL/6J and A/J mice (17, 18) after chronic (18 wk) HF were recapitulated before investigation of 11ß-HSD-1 biology. Briefly, after chronic HF feeding, both C57BL/6J and A/J mice became obese with a marked increase in fat mass to body weight ratio (Fig. 1A
). On control diet, the A/J mice had a higher fat/body weight ratio, but with chronic HF feeding the C57BL/6J strain showed the greater increase in fat mass. As expected, C57BL/6J mice became profoundly hyperinsulinemic, suggestive of insulin resistance (Fig. 1B), and were clearly glucose intolerant (Fig. 1C). In contrast, A/J mice were largely resistant to the development of these metabolic derangements with chronic HF (Fig. 1, B and C), as previously reported (17, 18).
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, A and B). In each depot, 11ß-HSD-1 activity was higher in C57BL/6J than in A/J mice fed control diet (Fig 2A). A similar pattern was broadly observed for 11ß-HSD-1 mRNA (Fig 2B). We also measured 11ß-HSD-1 activity in adipose depots from the C57BL/6JLepob/ob mice (5), as a comparator model of genetic obesity that shares the same genetic background (C57BL/6J) as the HF-induced obesity-susceptible mice in our study (Fig 2A). The markedly elevated adipose 11ß-HSD-1 activity in C57BL/6JLepob/ob mice was confirmed and appeared more pronounced compared with C57BL/6J controls than to the FVB strain described previously (5).
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). Similarly, 11ß-HSD-1 mRNA levels fell in all depots with chronic HF feeding, although in C57BL/6J mice 11ß-HSD-1 activity was significantly lower only in the mesenteric fat (Fig. 2B). Absolute levels of 11ß-HSD-1 activity and mRNA were significantly lower in adipose tissues from A/J mice on both diets compared with C57BL/6J, with the exception of activity in sc adipose tissue (Fig. 2, A and B). The chronic HF-mediated fall in 11ß-HSD-1 was selective for adipose tissue because hepatic 11ß-HSD-1 activity was not significantly affected by chronic HF in either strain (Fig. 2C).
To determine whether 11ß-HSD-1 activity is also negatively regulated as a short-term adaptation to the HF we performed similar studies over a 2-wk exposure. Short-term HF similarly induced a down-regulation of white adipose 11ß-HSD-1 activity in both strains but had no effect on liver 11ß-HSD-1 activity (Table 1). The magnitude of the 2-wk HF-mediated down-regulation of adipose 11ß-HSD1 was similar in sc and epididymal fat in the two strains, but greater in the mesenteric fat of A/J compared with C57BL/6J mice (Table 1).
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In thermogenic brown adipose tissue (BAT), 11ß-HSD-1 activity was down-regulated by both acute and chronic HF feeding. With chronic HF exposure, A/J mice showed a greater down-regulation of 11ß-HSD-1 in BAT (Fig. 2D).
Circulating corticosterone levels were lower in control-fed A/J mice (Fig. 3). HF feeding caused an increase in corticosterone levels in both strains, however, HF-fed AJ mice maintained lower plasma corticosterone levels than C57BL/6J (Fig. 3).
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The major findings in the present study are that metabolic disease-resistant A/J mice (17, 18) have generally lower basal (control fed) adipose tissue 11ß-HSD-1 levels than disease-prone C57BL/6J mice and that chronic HF feeding causes a novel and unexpected down-regulation of adipose 11ß-HSD-1 that gives rise to lower absolute 11ß-HSD-1 levels in the adipose tissues of the metabolic disease-resistant A/J strain.
Obesity (5, 6, 7, 8, 9, 10, 11, 12) and insulin resistance (8, 10, 11, 12) have been associated with elevated adipose 11ß-HSD-1, data we recapitulate in obese Lepob/ob mice. On control diet, adipose tissue 11ß-HSD-1 expression was greater in the metabolic disease prone C57BL/6J strain than the resistant A/J strain in adipose tissue depots. Because both strains gained substantial adipose mass with chronic HF, the association of higher adipose 11ß-HSD-1 activity appears to be with adverse metabolic consequences than with adiposity per se in mice. Interestingly, although visceral adipose tissue is thought to have the greatest impact upon the metabolic consequences of obesity and has the highest density of glucocorticoid receptors (5, 14), 11ß-HSD-1 expression was highest in peripheral adipose depots in both strains. The importance of glucocorticoid action in specific fat depots remains to be explored, but the data suggest complex effects dependent upon the interplay between circulating glucocorticoids, 11ß-HSD-1 activity and glucocorticoid receptor expression.
Unexpectedly, chronic HF feeding caused a striking and tissue-specific down-regulation of 11ß-HSD-1 in all adipose depots. The short-term HF feeding data suggest that the down-regulation happens as an early adaptation and is maintained throughout chronic HF exposure. However, whether or not even longer exposure to HF results in reduced adipose 11ß-HSD-1 is unknown. 11ß-HSD-1 down-regulation occurred in both A/J and C57BL/6J mice, although the latter are much more prone to obesity and diabetes on HF. These data can be interpreted in two ways. First, given that the C57BL/6J mice develop obesity and metabolic disease despite down-regulating adipose 11ß-HSD-1, then regulation of adipose 11ß-HSD-1 by HF has no bearing on the development of obesity and its metabolic consequences. Second, down-regulation of adipose 11ß-HSD-1 is an adaptive response to HF, an attempt to minimize intraadipose glucocorticoid signaling. Thus, because A/J mice resist the metabolic consequences of obesity, the lower adipose 11ß-HSD-1 levels in A/J mice, particularly upon exposure to chronic HF feeding provides a degree of metabolic protection that is impaired in the C57BL/6J metabolic disease-susceptible mice. This adaptive hypothesis is supported by our findings in 11ß-HSD-1 null mice, where 11ß-HSD-1-deficient adipocytes exhibit higher basal and insulin-stimulated glucose uptake (16) suggesting greater insulin sensitivity. Reduced adipose 11ß-HSD-1 may also improve whole body insulin sensitivity in A/J mice by increasing expression of factors that promote insulin sensitization, such as adiponectin (21), and reducing expression of factors that promote insulin resistance, such as resistin (22) and TNF-
(23), as demonstrated in adipose tissue of 11ß-HSD-1 null mice (16). The higher depot-specific 11ß-HSD-1 expression found in sc fat may also have a greater effect on glucocorticoid sensitive transcripts that are more highly expressed in this depot such as leptin (24), as is indeed found for leptin in 11ß-HSD-1 null mice (16). Glucocorticoid action is affected both by circulating hormone levels and by 11ß-HSD-1-mediated tissue-specific reamplification. The higher adipose tissue 11ß-HSD-1 levels in C57BL/6J mice appears to be compounded by higher circulating glucocorticoid levels on both control and HF. In contrast, in A/J mice, the combination of low circulating corticosterone levels and very low tissue reamplification is likely to have a combined protective effect in the adipocyte.
We also observed HF-mediated down-regulation of 11ß-HSD-1 in the thermogenic BAT—a key tissue that counteracts diet-induced obesity and insulin resistance in mice (25). Glucocorticoids inhibit BAT thermogenesis (26). The metabolic disease-resistant A/J mice exhibited a more pronounced down-regulation of BAT 11ß-HSD-1 activity with chronic HF feeding, which is anticipated to reduce glucocorticoid-mediated constraint of diet-induced thermogenesis and offer a further metabolic protection in the HF-fed A/J mice.
We have evidence that HF-mediated down-regulation of 11ß-HSD-1 also occurs in rats (Drake, A. J., N. M. Morton, B. R. Walker, and J. R. Seckl, unpublished data), suggesting the HF-mediated effect is conserved across at least several strains of rodents. We speculate, therefore, that such a mechanism, if present in humans, will have implications for the chronic dietary context within which 11ß-HSD-1 activity is measured in human adipose tissues (6, 7, 8, 9, 10, 11, 12). This is pertinent given that most studies report a positive correlation between obesity and adipose 11ß-HSD-1 (mRNA and activity), whereas one recent study found no such relationship (27). It is further possible that interindividual variation in the ability of humans to invoke the potentially protective down-regulation of adipose 11ß-HSD-1 in response to a diet high in saturated fat (a Western diet) may play a role in determining their overall susceptibility to insulin resistance and type 2 diabetes. Furthermore, the elevated adipose 11ß-HSD-1 levels found in obese rodents and humans could represent escape of this regulatory mechanism that then contributes to the disease process.
The molecular mechanism of 11ß-HSD-1 down-regulation with HF feeding is unknown. High-affinity peroxisome proliferator-activated receptor (PPAR)-
and liver X receptor nuclear receptor ligands can down-regulate 11ß-HSD-1 activity in vivo (28, 29) and could be activated by an HFmediated increase in free fatty acid (30) and cholesterol metabolites (29) in adipose tissue. However, the modest effects of potent PPAR and liver X receptor ligands do not account for the magnitude of HF-mediated down-regulation seen in these studies. Furthermore, if increased free fatty acids were responsible, reduced hepatic 11ß-HSD-1 might also be expected via PPAR- activation (31). A/J mice resist chronic HF-induced obesity partly because of enhanced adrenergic stimulation in adipose tissue (32), suggesting the sympathetic outflow as a novel candidate mechanism inhibiting adipose 11ß-HSD-1 expression. Differences between 11ß-HSD-1 activity and mRNA levels in the present study also suggest the possibility of posttranscriptional regulation of the enzyme. Potential posttrancriptional mechanisms leading to inhibition of 11ß-HSD-1 reductase activity include down-regulation of nicotinamide adenine dinucleotidephosphate cosubstrate generating enzymes (33) and stimulation of the lipid activated protein kinase C (34). Finally, whether adipose 11ß-HSD-1 activity is modulated by hormones such as insulin, corticosterone, and leptin in vivo is unknown. However, individually these hormones are perhaps unlikely to account for the magnitude of the down-regulation observed, considering the tissue specificity of the effect.
Given the protective effects of adipose tissue 11ß-HSD-1 deficiency (16), the detrimental effects of selective adipose tissue 11ß-HSD-1 overexpression (5, 13) and the largely positive correlations between adipose 11ß-HSD-1, obesity, and insulin resistance (6, 7, 8, 9, 10, 11, 12), we hypothesize that HF-mediated down-regulation of adipose 11ß-HSD-1 is a beneficial adaptation, diminished in the metabolic disease-prone, that protects from the metabolic derangements caused by exposure to HF.
Acknowledgments
We would like to thank Drs. Brian Walker, Karen Chapman, and Chris Kenyon for critical discussions and Rachel Kerr for technical assistance.
Footnotes
This work was funded by a Wellcome Trust program grant (to J.R.S.) and a Wellcome Trust Cardiovascular Research Institute Intermediate Fellowship (to N.M.M.).
Abbreviations: BAT, brown adipose tissue; 11ß-HSD-1, 11ß-hydroxysteroid dehydrogenase type 1; HF, high-fat diet; PPAR, peroxisome proliferator-activated receptor.
Received December 10, 2003.
Accepted for publication March 16, 2004.
References
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