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J. Biol. Chem., Vol. 276, Issue 44, 41293-41300, November 2, 2001

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Improved Lipid and Lipoprotein Profile, Hepatic Insulin Sensitivity, and Glucose Tolerance in 11-Hydroxysteroid Dehydrogenase Type 1 Null Mice^*

Nicholas M. Morton^§, Megan C. Holmes^¶, Catherine Fiévet, Bart Staels^**, Anne Tailleux^**, John J. Mullins, and Jonathan R. Seckl

From  Molecular Endocrinology and the ^¶ Department of Clinical Neurosciences, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Rd. S., Edinburgh EH4 2XU, United Kingdom, the  Molecular Physiology Laboratory, University of Edinburgh, Wilkie Building, Teviot Place, Edinburgh EH8 9AG, United Kingdom,  Département d'Athérosclérose, U.545 INSERM, Institut Pasteur de Lille, 1 Rue du Prof. Calmette B.P.245, 59019 Lille Cedex, France, and ^** Université de Lille II, 59019 Lille Cedex, France

Received for publication, April 25, 2001, and in revised form, August 22, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Excess tissue glucocorticoid action may underlie the dyslipidemia, insulin resistance, and impaired glucose tolerance of the metabolic syndrome. 11-Hydroxysteroid dehydrogenase type 1 (11-HSD-1) catalyzes conversion of circulating inert 11-dehydrocorticosterone into active corticosterone, thus amplifying local intracellular glucocorticoid action, particularly in liver. The importance of 11-HSD-1 in glucose homeostasis is suggested by the resistance of 11-HSD-1^/ mice to hyperglycemia upon stress or obesity, due to attenuated gluconeogenic responses. The present study further investigates the metabolic consequences of 11-HSD-1 deficiency, focusing on the lipid and lipoprotein profile. Ad lib fed 11-HSD-1^/ mice have markedly lower plasma triglyceride levels. This appears to be driven by increased hepatic expression of enzymes of fat catabolism (carnitine palmitoyltransferase-I, acyl-CoA oxidase, and uncoupling protein-2) and their coordinating transcription factor, peroxisome proliferator-activated receptor- (PPAR). 11-HSD-1^/ mice also have increased HDL cholesterol, with elevated liver mRNA and serum levels of apolipoprotein AI. Conversely, liver A-fibrinogen mRNA levels are decreased. Upon fasting, the normal elevation of peroxisome proliferator-activated receptor- mRNA is lost in 11-HSD-1^/ mice, consistent with attenuated glucocorticoid induction. Despite this, crucial oxidative responses to fasting are maintained; carnitine palmitoyltransferase-I induction and glucose levels are similar to wild type. Refeeding shows exaggerated induction of genes encoding lipogenic enzymes and a more marked suppression of genes for fat catabolism in 11-HSD-1^/ mice, implying increased liver insulin sensitivity. Concordant with this, 24-h refed 11-HSD-1^/ mice have higher triglyceride but lower glucose levels. Further, 11-HSD-1^/ mice have improved glucose tolerance. These data suggest that 11-HSD-1 deficiency produces an improved lipid profile, hepatic insulin sensitization, and a potentially atheroprotective phenotype.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glucocorticoids are important regulators of glucose and lipid homeostasis, acting largely via intracellular glucocorticoid receptors in the liver, adipose tissue, and muscle. Glucocorticoid excess, epitomized by Cushing's syndrome in humans, leads to insulin resistance/type 2 diabetes, dyslipidemia, and a redistribution of fat to visceral depots associated with increased cardiovascular risk (1). The metabolic syndrome (syndrome X) resembles Cushing's syndrome, although plasma glucocorticoid levels are typically normal. It has therefore been suggested that increased glucocorticoid action within specific cells may occur in the metabolic syndrome (2-4). Indeed, there is now evidence for a novel and important level of control of glucocorticoid action, prereceptor metabolism by 11-hydroxysteroid dehydrogenases (11-HSDs).¹ 11-HSDs catalyze the interconversion of active 11-hydroxyglucocorticoids (cortisol in most mammals, corticosterone in rats and mice) and their inert 11-keto forms (cortisone, 11-dehydrocorticosterone). There are two isozymes of 11-HSD, the products of distinct genes (5, 6). 11-HSD type 2 is a high affinity dehydrogenase that rapidly inactivates corticosterone in kidney and colon, thus excluding glucocorticoids from inherently nonselective mineralocorticoid receptors in vivo (7, 8). However, white adipose tissue (9) and the liver, where the enzyme is particularly abundant (10, 11), solely express 11-HSD type 1.

11-HSD-1, while bidirectional in tissue homogenates, is a predominant 11-reductase, thus regenerating active glucocorticoids within most intact cells in culture including hepatocytes (12), adipocytes (13), and neurons (14) and in the isolated liver ex vivo (15). Mice homozygous for targeted disruption of the 11-HSD-1 gene cannot regenerate corticosterone from inert 11-dehydrocorticosterone, indicating that this isozyme is the unique 11-reductase in vivo (16). Strikingly, 11-HSD-1 null animals exhibit attenuated gluconeogenic responses upon stress and resist hyperglycemia induced by chronic high fat feeding (16), suggesting that 11-HSD-1 reductase activity is an important amplifier of intrahepatic glucocorticoid action in vivo. Intriguingly, tissue-specific alterations in 11-HSD-1 activity have been implicated in the development of obesity and insulin resistance in obese Zucker rats (4) and in humans (2-3, 9).

The metabolic syndrome is characterized by hypertriglyceridemia and an aberrant lipoprotein and cholesterol profile with elevated VLDL but reduced "cardioprotective" HDL cholesterol (17). The lipid profile is determined to a large extent by expression of liver genes involved in lipid metabolism, synthesis, packaging, and export, many of which are glucocorticoid-sensitive. Administration of glucocorticoids (18, 19) or elevated endogenous glucocorticoid levels promotes hyperlipidemia and induces insulin resistance (1). Consequently, in liver, there is elevated VLDL secretion (20) as well as increased glucose output. Adrenalectomy corrects the aberrant lipid and insulin sensitivity profile of rodent obesity models (21), an effect reversed by glucocorticoid replacement (22). Glucocorticoids also have important indirect effects on hepatic lipid metabolism through regulation of key transcription factors. Notably, glucocorticoids induce the peroxisome proliferator-activated receptor- (PPAR) (23, 24), which drives the oxidative adaptation to fasting (25, 26) and serves as the molecular target for the hypolipidemic fibrate drugs (27, 28). However, the precise contribution of glucocorticoids to altered hepatic lipid metabolism during pathogenic states such as the metabolic syndrome is unclear. Indeed, modulation of intrahepatic glucocorticoid actions by 11-HSD-1, and its physiological impact in regulating lipid metabolism is unexplored.

Here we investigate liver-dependent changes in lipid and lipoprotein metabolism in 11-HSD-1^/ mice. To address this, we have analyzed circulating lipids and lipoproteins and assessed expression of hepatic genes involved in lipid metabolism. A-fibrinogen expression levels have also been measured as an independent cardiovascular risk marker. Since elevated glucocorticoids play a crucial role in the metabolic adaptation to fasting, we have also investigated the effects of 11-HSD-1 deficiency on this process. Furthermore, we have assessed glucose tolerance and the relative hepatic insulin sensitivity of 11-HSD-1^/ mice through their transcriptional response to refeeding after a fast.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- Male 11-HSD-1^/ mice on a MF1/129 background and their age-matched wild type controls, bred as previously described (16), were housed in standard conditions on a 12-h light/12-h dark cycle (lights on at 7 a.m.). For fasting/refeeding experiments, animals were housed singly and allowed to acclimatize for at least 2 days. Animals were allocated at random (n = 6 per group) to receive either ad lib access to chow, a 24-h fast, a 24-h fast with a 4-h refeed, or a 24-h fast with a 24-h refeed. All fasting commenced at 8 a.m. Animals were killed by decapitation in an adjacent room from their housing within 1 min of their cage being disturbed.

11-HSD-1 Enzyme Activity-- Liver samples were homogenized and assayed for 11-dehydrogenase activity, as described (12). The reaction included 0.1 mg/ml protein, 25 nM [³H]corticosterone, and an excess (2 µM) of the 11-HSD-1-specific co-factor NADP. After a 10-min incubation, steroids were extracted with ethyl acetate and analyzed with thin layer chromatography and high pressure liquid chromatography against known standards (12).

Plasma and Serum Parameters-- Trunk blood was collected rapidly, plasma or serum was separated, and samples were kept on ice until measurement of triglyceride, total cholesterol, and HDL cholesterol. Total triglyceride was measured with a triglyceride kit (Roche Molecular Biochemicals), and true triglyceride without glycerol interference was measured with a GPO-TRINDER kit (Sigma). Plasma total and HDL cholesterol were measured using the CHOL and HDL C-Plus kits (Roche Molecular Biochemicals). The cholesterol and triglyceride distribution among the lipoproteins was determined by FPLC as previously described (28). Glucose was measured with a Glucose HK kit (Sigma). Plasma insulin was measured by enzyme-linked immunosorbent assay (Crystalchem, Chicago, IL). Corticosterone levels were determined by radioimmunoassay, as described (29). ApoAI, apoAII, apoB, and total apoCIII were measured by nephelometry using specific anti-apolipoprotein antibodies (28). Non-HDL (triglyceride-associated lipoproteins) and HDL lipoprotein fractions were isolated from 200 µl of a representative pooled sample from each genotype by sequential ultracentrifugation at d < 1.063 and 1.063 < d < 1.21, respectively (28). The apolipoprotein composition of isolated lipoproteins was analyzed by nonreducing PAGE (4-20%; Novex, San Diego, CA), as described (30).

RNA Extraction and Analysis-- Tissues were snap-frozen in liquid nitrogen and homogenized in Trizol (Life Technologies, Inc.). Total RNA was purified with a binding matrix (RNaid Plus kit, BIO 101, Anachem, UK) and eluted in diethylpyrocarbonate-pretreated water containing 400 units/ml RNAsin (Promega, Southampton, UK) and 10 mM dithiothreitol. RNA (5-20 µg) was resolved on a MOPS/formaldehyde, 1% agarose gel and blotted according to standard Northern blot procedures in 20× SSC onto Hybond N⁺ membranes (Amersham Pharmacia Biotech). Probes were labeled with [³²P]dCTP (Amersham Pharmacia Biotech) using a random primed labeling kit (Roche Molecular Biochemicals), purified through nick columns (Amersham Pharmacia Biotech), and hybridized overnight in buffer (0.2 M NaH₂PO₄, 0.6 M Na₂HPO₄, 5 mM EDTA) containing 6% SDS and 0.5 mg/ml denatured salmon testes DNA (Sigma) at 65 °C. Blots were washed at 65 °C to a stringency of 0.5× SSC, 0.1% SDS, exposed to imaging film (FLA2000; Fujifilm, London, UK), and analyzed by quantitative imaging software (Aida, Raytek Scientific, Sheffield, UK). Blots were also exposed to film (Biomax-MR; Eastman Kodak Co.). 11-HSD-1 cDNA was as described (11). All other probes were generated by PCR using the following primer pairs: apolipoprotein AI forward (5'-TGG GTT CAA CCG TTA GTC-3') and reverse (5'-GTG GGG TTG CTC TTG AGC-3'), fatty acid synthase forward (5'-AAG CGG CCA TTT CCA TTG-3') and reverse (5'-CGT ACC TGG ACA AGG ACT TTG-3'), glycerolphosphate acyltransferase forward (5'-TGA TCA GCC AGG AGC AGC TG-3') and reverse (5'-AGA CAG TAT GTG GCA CTC TC-3'), hydroxymethylglutaryl CoA-reductase forward (5'-AAC TAT TGC ACC GAC AAG-3') and reverse (5'-TTG CTG AGG TAG AAG GTT G-3'), sterol receptor element-binding protein-1c forward (5'-ATC GGC GCG GAA GCT GTC GGG GTA GCG TC-3') and reverse (5'-ACT GTC TTG GTT GTT GAT GAG CTG GAG CAT-3'), carnitine palmitoyltransferase forward (5'-GTC CCA GCT GTC AAA GAT AC-3') and reverse (5'-GGA AGT ATT GAA GAG TCG C-3'), acyl-CoA oxidase forward (5'-ATG AAT CCC GAT CTG CGC AAG GAG C-3') and reverse (5'-AAA GGC ATG TAA CCC GTA GCA CTC C-3'), uncoupling protein-2 forward (5'-GCA TTG CAG GTC TCA TCA C-3') and reverse (5'-CTT GGT GTA GAA CTG TTT GAC-3'), and PPAR forward (5'-ATC CAG ATG ACA CCT TCC TC-3') and reverse (5'-CCG TTG TCT GTC ACT GTC TG-3'). All probe identities were confirmed by sequencing using the Thermosequenase kit (U.S. Biochemical Corp.) on standard 8% acrylamide sequencing gels. Gene expression levels were corrected for RNA loading using a probe for U1 RNA as an internal control and are presented as arbitrary units. U1 levels do not change with genotype or the dietary manipulation described.

Intraperitoneal Glucose Tolerance Test-- In a separate experiment, transgenic and wild type mice were fasted overnight and then injected intraperitoneally with 2 mg/g of body weight D-glucose (25% stock solution in saline). Blood samples were taken by tail venesection into EDTA-microtubes (Sarstedt, Leicester, UK) at 0 min (just before glucose injection) and at 5-, 15-, 30-, 60-, and 120-min intervals after the glucose load.

Statistical Analyses-- Data are expressed as means ± S.E. Results were subjected to two-way ANOVA (factors: genotype and feeding status) or repeated measures two-way ANOVA (factors: genotype and time) for glucose tolerance using a Sigmastat program (Jandel Corp., San Rafael, CA). Where feeding status, genotype, or an interaction between these two factors was found by two-way ANOVA, significant differences between relevant groups were determined with post hoc Tukey multiple comparisons tests. In Figs. 1-4 and 6-8, asterisks represent significant differences between genotypes at a given feeding status (*, **, *** = p < 0.05, p < 0.01, and p < 0.001, respectively); daggers indicate significant differences due to feeding status within a genotype compared with the ad lib fed level (, , , = p < 0.05, p < 0.01, and p < 0.001, respectively). Where indicated, lipid and lipoprotein parameters measured after FPLC fractionation of serum from ad lib fed animals has been compared with Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Effects of Feeding Status on Circulating Lipids and Lipoproteins in 11-HSD-1^/ Mice-- Plasma triglycerides were 42% lower in ad lib fed 11-HSD-1^/ mice compared with wild type (Fig. 1A). FPLC analysis indicated that the difference in triglycerides between genotypes in animals fed ad lib was associated with the VLDL fraction and excluded any contribution from glycerol interference to this difference (Fig. 1B). True triglyceride measured after FPLC fractionation in serum from ad lib fed animals was as follows: wild type, 1.71 ± 0.2 g/liter, n = 7; 11-HSD-1^/, 1.04 ± 0.08 g/liter, n = 7 (p < 0.02). Consistent with reduced plasma triglycerides, apoCIII, one of the main apolipoproteins involved in regulating triglyceride levels (31), was preferentially increased in the HDL fraction and decreased in the non-HDL (triglyceride-rich) fraction in 11-HSD-1^/ mice (data not shown). Serum apoCIII was unchanged overall in 11-HSD-1^/ mice.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   The effects of dietary status on plasma triglyceride levels in 11-HSD-1/ mice. A, triglyceride levels in wild type (solid bars) versus 11-HSD-1/ (open bars) animals that are ad lib fed (AL), 24-h fasted (F), fasted with a 4-h refeed (4RF), or fasted with a 24-h refeed (24RF). Results were tested with two-way ANOVA and post hoc Tukey tests as described under "Experimental Procedures." Significant differences between genotypes at a given feeding state are represented by asterisks (*, p < 0.05; **, p < 0.01), and key significant differences due to feeding status within a genotype compared with the ad lib fed level are represented by daggers (, p < 0.01). B, FPLC profile of triglyceride from ad lib fed wild type () and 11-HSD-1/ mice (). The VLDL triglyceride peak is found at fractions 30-35, and the glycerol peak is at fractions 65-70.

We also investigated the effects of 11-HSD-1 deficiency on regulation of triglycerides with fasting and refeeding. Plasma triglycerides fell upon fasting in both genotypes (Fig. 1A). In wild type animals, triglyceride levels returned to ad lib fed values with 24 h of refeeding. In contrast, in 11-HSD-1^/ mice, triglycerides returned to ad lib levels by 4 h of refeeding and exhibited an overshoot at 24 h to levels significantly higher than in the ad lib fed state (Fig. 1A).

The effect of 11-HSD-1 deficiency on plasma total and HDL cholesterol across changing dietary status was also investigated. Feeding status had no effect on total and HDL cholesterol. However, 11-HSD-1^/ mice had 30% higher HDL cholesterol (significant effect of genotype: p < 0.001) (Fig. 2A). FPLC analysis of serum from ad lib fed animals additionally showed increased total cholesterol (wild type, 1.06 ± 0.11 g/liter, n = 7; 11-HSD-1^/, 1.45 ± 0.1 g/liter, n = 7 (p < 0.005)) and confirmed increased HDL cholesterol in 11-HSD-1^/ mice (wild type, 0.71 ± 0.09 g/liter, n = 6; 11-HSD-1^/, 1.17 ± 0.08 g/liter, n = 7 (p < 0.005) (Fig. 2B)). Apolipoprotein AI mRNA, encoding the major component of the HDL particle (32), was significantly elevated in liver from fed 11-HSD-1^/ mice (Fig. 2C). ApoAI mRNA, a glucocorticoid-inducible transcript in rodents and humans (33), is induced on fasting in wild type mice (Fig. 2C). This effect is attenuated in 11-HSD-1^/ mice (Fig. 2C). Further, serum apoAI levels were higher in ad lib fed 11-HSD-1^/ mice: wild type, 0.2 ± 0.02 g/liter, n = 6; 11-HSD-1^/, 0.54 ± 0.03 g/liter, n = 7 (p < 0.001). Serum levels of apoAII and apoB were not different between genotypes (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   The effects of dietary status on the cholesterol profile of 11-HSD-1/ mice. Shown are plasma cholesterol levels in wild type (solid bars) versus 11-HSD-1/ (open bars) animals that are ad lib fed (AL), 24-h fasted (F), fasted with a 4-h refeed (4RF), or fasted with a 24-h refeed (24RF). A, HDL cholesterol. Two-way ANOVA reveals a significant effect of genotype (***, p < 0.001) but not feeding status on HDL cholesterol. B, FPLC profile of cholesterol from ad lib fed wild type () and 11-HSD-1/ mice (). The HDL cholesterol peak is found at fractions 55-60. C, hepatic apolipoprotein AI mRNA levels. Transcript levels were analyzed by Northern blot and corrected for RNA loading by using a cDNA probe for the U1 small ribonucleoprotein as described under "Experimental Procedures." Significant differences between genotypes at a given feeding state are represented by asterisks (*, p < 0.05), and significant differences due to feeding status within a genotype are represented by daggers (, p < 0.05;  = p < 0.01).

The Effects of Feeding Status on Hepatic Expression of Genes for Lipogenesis in 11-HSD-1^/ Mice-- To investigate the origins of the reduction in plasma triglycerides, expression of mRNA transcripts encoding enzymes of lipid synthetic pathways were examined by Northern analyses (Fig. 3). Fatty acid synthase (FAS) (Fig. 3A) and glycerol phosphate acyltransferase (GPAT) (Fig. 3B), enzymes involved in triglyceride synthesis and esterification, respectively, were similarly expressed in ad lib fed 11-HSD-1^/ and wild type mice. Indeed, levels of the crucial lipogenic transcription factor SREBP-1c, which drives expression of FAS, GPAT, and other enzymes in the lipid synthesis pathway (34, 35), were comparable between genotypes (Fig. 3C). Furthermore, mRNA encoding the rate-limiting enzyme in cholesterol synthesis, hydroxymethylglutaryl-CoA reductase (HMG-CoAR) was also expressed at similar levels in both genotypes in the fed state (Fig. 3D).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The effects of feeding status on hepatic mRNA levels encoding lipogenic genes in 11-HSD-1/ mice. mRNA levels encoding proteins of the lipogenic (A-C) and cholesterol biosynthesis pathways (D) in livers of wild type (solid bars) versus 11-HSD-1/ (open bars) animals that are ad lib fed (AL), 24-h fasted (F), fasted with a 4-h refeed (4RF), or fasted with a 24-h refeed (24RF). Transcript levels were analyzed by Northern blot and corrected for RNA loading by using a cDNA probe for the U1 small ribonucleoprotein as described under "Experimental Procedures." A, FAS transcript levels; B, GPAT; C, SREBP-1c; D, HMG-CoAR. Significant differences between genotypes at a given feeding state are represented by asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001), and significant differences due to feeding status within a genotype compared with the ad lib fed level are represented by daggers (, p < 0.05; , p = < 0.01; , p < 0.001).

Lipogenic enzyme mRNA transcripts are repressed on fasting and are induced by insulin on refeeding, the degree of induction being a measure of relative hepatic insulin sensitivity. Following a 24-h fast, FAS, SREBP-1c, and HMG-CoAR transcript levels clearly fell in both genotypes (Fig. 3, A, C, and D). However, in 11-HSD-1^/ mice, GPAT mRNA did not fall with fasting (Fig. 3B). Upon refeeding SREBP-1c, FAS, GPAT, and HMG-CoAR were more rapidly and/or markedly induced in 11-HSD-1^/ mice (Fig. 3, A-D), implying the 11-HSD-1^/ liver has enhanced insulin sensitivity.

The Effects of Feeding Status on Hepatic Expression of Genes for Lipid Catabolism in 11-HSD-1^/ Mice-- Given unchanged lipogenic enzyme mRNAs, we investigated whether increased hepatic lipid catabolism may be driving the reduced plasma triglyceride levels. 11-HSD-1^/ liver had elevated levels of mRNAs encoding liver mitochondrial carnitine palmitoyltransferase-I (mCPT-I), a rate-limiting enzyme in the mitochondrial -oxidation pathway (36), peroxisomal acyl-CoA oxidase (ACO), another enzyme of fatty acid oxidation (37), and uncoupling protein-2 (UCP-2), a protein implicated in elimination of oxidants produced during fat metabolism (38) and known to be expressed in hepatocytes (39) (Fig. 4, A-C). Moreover, PPAR mRNA, the key hepatic transcription factor that promotes expression of genes for lipid catabolism, was elevated in ad lib fed 11-HSD-1^/ mice (Fig. 4D).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   The effects of feeding status on hepatic mRNA levels encoding genes for lipid catabolism in 11-HSD-1/ mice. Shown are mRNA levels encoding proteins in the fat catabolism pathway in livers of wild type (solid bars) versus 11-HSD-1/ (open bars) animals that are ad lib fed (AL), 24-h fasted (F), fasted with a 4-h refeed (4RF), or fasted with a 24-h refeed (24RF). Transcript levels were analyzed by Northern blot and corrected for RNA loading by using a cDNA probe for the U1 small ribonucleoprotein as described under "Experimental Procedures." A, liver mCPT-I. B, ACO; C, UCP-2; D, PPAR. Significant differences between genotypes at a given feeding state are represented by asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001), and significant differences due to feeding status within a genotype compared with the ad lib fed level are represented by daggers (, p < 0.05; , p = <0.01; , p < 0.001).

Fasting induces mRNA encoding enzymes of fatty acid oxidation, and refeeding represses their expression. Fasting indeed produced significant increases in mCPT-I, ACO, and UCP-2 mRNAs as well as PPAR mRNA in wild type liver (Fig. 4), consistent with reports that this transcription factor mediates glucocorticoid-induced fatty acid oxidation during a fast (25, 26). However, upon fasting, induction of PPAR and ACO mRNAs in 11-HSD-1^/ liver was abolished (Fig. 4D), and UCP-2 induction was attenuated (Fig. 4C), whereas induction of mCPT-I mRNA was similar to wild type (Fig. 4A).

With refeeding, mCPT-I, ACO, and UCP-2 mRNAs were suppressed more rapidly and/or to a greater extent in 11-HSD-1^/ mice than in wild type (Fig. 4, A-C), observations also consistent with increased hepatic insulin sensitivity. PPAR was suppressed similarly in both genotypes by 4 h of refeeding, but by 24 h of refeeding 11-HSD-1^/ PPAR levels were significantly higher than wild type, reestablishing the ad lib fed pattern (Fig. 4D).

The Effects of Feeding Status on Lipid Accumulation in 11-HSD-1^/ Liver-- Oil red O histology of liver showed increased accumulation of lipid in fasted 11-HSD-1^/ mice compared with wild type (Fig. 5, B versus D). This lipid accumulation resolved upon refeeding (data not shown).

View larger version (159K): [in this window] [in a new window]   Fig. 5.   Fasting 11-HSD-1/ liver shows pronounced lipid accumulation. Shown is Oil red O histology of liver sections from ad lib fed wild type mice (A), 24-h fasted wild type mice (B), ad lib fed 11-HSD-1/ mice (C), and 24-h fasted 11-HSD-1/ mice (D). Liver sections were viewed with a light microscope at × 40 magnification. Blue hematoxylin counterstaining indicates nuclei, and red staining indicates fat deposition.

11-HSD-1 Does Not Respond Acutely to Fasting/Refeeding in Wild Type Mice-- We also investigated whether altered circulating corticosterone levels or regulation of wild type 11-HSD-1 with fasting and refeeding may account for the differences in gene expression and lipid profile between the genotypes. Plasma corticosterone levels were higher in ad lib fed 11-HSD-1^/ mice, as previously reported (16), but the rise and fall in plasma corticosterone with fasting and refeeding, respectively, were similar to wild type (Fig. 6A). Neither 11-HSD-1 mRNA nor activity levels were altered by feeding status in wild type mice (Fig. 6, B and C).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   The effects of feeding status on plasma corticosterone levels in 11-HSD-1/ mice and on wild type 11-HSD-1 mRNA and activity. Panel A, corticosterone (B) levels; panel B, 11-HSD-1 mRNA; panel C, 11-HSD-1 activity (expressed as percentage conversion of corticosterone to 11-dehydrocorticosterone) in wild type (solid bars) and 11-HSD-1/ (open bars) animals that are ad lib fed (AL), 24-h fasted (F), fasted with a 4-h refeed (4RF), or fasted with a 24-h refeed (24RF). Significant differences between genotypes at a given feeding state are represented by asterisks (*, p < 0.05), and significant differences due to feeding status within a genotype compared with the ad lib fed level are represented by daggers (, p < 0.05; , p < 0.01).

11-HSD-1^/ Mice Have Improved Glucose Tolerance-- After 24 h of refeeding, plasma glucose was significantly lower in 11-HSD-1^/ mice than in wild type (Fig. 7A). Otherwise, glucose and insulin levels were similar across feeding states (Fig. 7, A and B). Intraperitoneal glucose tolerance tests indicated that 11-HSD-1^/ mice have improved glycemic control (Fig. 7C). Note that glucose levels are lower after a 24-h fast (Fig. 7A) than after a 16-h fast performed before a glucose tolerance test (Fig. 7C). This is probably due to the increased length of food restriction and the greater contribution of stress to plasma glucose in fasted wild type mice compared with 11-HSD-1^/ mice during glucose tolerance tests. Lower fasting glucose levels have previously been described in stressed 11-HSD-1^/ mice (16).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   The effects of dietary status and 11-HSD-1 deficiency on plasma glucose, insulin, and dynamic glucose disposal after intraperitoneal glucose administration. Shown are plasma glucose (A) and plasma insulin (B) in wild type (solid bars) and 11-HSD-1/ (open bars) animals that are ad lib fed (AL), 24-h fasted (F), fasted with a 4-h refeed (4RF), or fasted with a 24-h refeed (24RF). Significant differences between genotypes at a given feeding state are represented by asterisks (*, p < 0.05), and significant differences due to feeding status within a genotype compared with the ad lib fed level are represented by daggers (, p < 0.01). C, dynamics of glucose disposal upon intraperitoneal glucose load (2 mg/g of body weight) following a 16-h fast in wild type () and 11-HSD-1/ mice (). For intraperitoneal GTT, repeated measures two-way ANOVA shows that 11-HSD-1/ mice have significantly lower glucose levels over time compared with wild type (**, p < 0.01).

11-HSD-1^/ Mice Have a Favorable Fibrinogen Expression Profile-- To assess a hepatic transcript unrelated to lipoproteins or lipid metabolism, we investigated A-fibrinogen mRNA. A-fibrinogen is a glucocorticoid-sensitive plasma factor (40) that is an independent cardiovascular risk factor (41). A-fibrinogen transcript levels were reduced by 25% in fed 11-HSD-1^/ mice (Fig. 8).

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Hepatic A-fibrinogen expression in 11-HSD-1/ mice. Shown are mRNA levels of A-fibrinogen in livers of wild type (solid bars) and 11-HSD-1/ (open bars) animals that are ad lib fed (AL) or 24-h fasted (F). Transcript levels were analyzed by Northern blot and corrected for RNA loading by using a cDNA probe for the U1 small ribonucleoprotein as described under "Experimental Procedures." Significant differences between genotypes at a given feeding state are represented by asterisks (**, p < 0.01), and significant differences due to feeding status within a genotype are represented by daggers (, p < 0.05; , p < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we show that 11-HSD-1 deficiency in mice opposes the pathogenic lipid and lipoprotein profile found in disease states such as the metabolic syndrome. The distinctive phenotype of 11-HSD1^/ mice during different feeding states (summarized in Table I) indicates at least three, potentially interrelated hepatic mechanisms contributing to their "favorable" metabolic profile: increased lipid catabolism (ad lib fed), reduced intracellular glucocorticoid concentrations (fasting), and increased hepatic insulin sensitivity (refeeding).

View this table: [in this window] [in a new window]   Table I Summary of the effects of 11-HSD-1 deficiency on metabolism Arrows depict the direction of change (up or down) in the parameter shown. In the ad lib fed state, this indicates relative to wild type. In fasted or refed animals, the arrows indicate both the direction of change and the relative degree of change compared to the wild type response. Note, in 11-HSD-1/ mice, first, the reduced ad lib fed triglyceride (TG) and increased HDL cholesterol/ApoAI, the former driven by increased lipid oxidation and PPAR; second, the attenuated fasting responses, suggesting reduced glucocorticoid action; and third, the exaggerated refeeding responses compatible with increased insulin sensitivity.

In the ad lib fed state, 11-HSD-1^/ mice show reduced circulating triglyceride. This may be in part due to reduction of non-HDL-associated (triglyceride-rich) apoCIII in 11-HSD-1^/ mice. ApoCIII increases triglyceride levels by inhibiting the binding and intravascular lipolysis of triglyceride-rich particles at the endothelium and by interfering with apoE-directed clearance of these particles (31, 42, 43). We also find evidence that altered hepatic lipid metabolism is likely to play a key role in the plasma triglyceride profile. Thus, while mRNAs encoding key rate-limiting lipogenic enzymes and the lipogenic transcription factor SREBP-1c were maintained at wild type levels, mRNAs encoding key enzymes of hepatic fat oxidation were elevated in the 11-HSD-1^/ liver. This is compatible with increased triglyceride catabolism and/or diminished VLDL triglyceride secretion as a mechanism driving reduced plasma triglycerides in 11-HSD-1^/ mice.

Glucocorticoids mediate the physiological adaptation of an animal to starvation by driving hepatic glucose production and fatty acid oxidation. In the fasted state, 11-HSD-1^/ mice show attenuated induction of genes that are directly (PPAR, apoAI) or indirectly (ACO, UCP-2) glucocorticoid-sensitive. These results agree with previous findings of attenuated induction of gluconeogenic enzymes on fasting in 11-HSD-1^/ mice (16). The data support the idea that 11-HSD-1^/ mice are relatively deficient in intracellular glucocorticoids, particularly when glucocorticoid levels are rising (fasting or stress). Despite abolished PPAR induction, 11-HSD-1^/ mice appear to maintain hepatic fatty acid oxidation, perhaps due to the already elevated ad lib fed PPAR levels. Thus, mCPT-I mRNA is fully induced, and fasting plasma glucose levels are similar to wild type animals. Up-regulation of mCPT-I therefore appears independent of PPAR under fasting conditions, in agreement with studies in PPAR null mice (44). In contrast, the abolished ACO and attenuated UCP-2 induction may reflect the relatively greater sensitivity of these promoters to PPAR regulation on fasting. Alternatively, PPAR target gene induction (mCPT-I, UCP-2), despite unchanged PPAR expression, may also be due to increased provision of endogenous fatty acid activators of the transcription factor (27) with fasting.

We find pronounced lipid accumulation in 11-HSD-1^/ liver on fasting, reminiscent of the fatty liver observed in fasted PPAR null mice (25, 26). However, hepatic lipid accumulation readily resolves in 11-HSD-1^/ mice upon refeeding. Lower PPAR-mediated fat catabolism may contribute to liver lipid accumulation, as supported by the attenuated fasting ACO and UCP-2 responses. In addition, failure to suppress GPAT through attenuated glucocorticoid action and/or inappropriate insulin-mediated GPAT induction could contribute to excessive lipid esterification on fasting. Despite the relatively small changes in mRNA level observed here, it is likely that insulin will have post-transcriptional effects on enzyme expression and activity that could account for the marked morphological changes observed in liver.

Refeeding after a fast is characterized by an insulin-mediated overshoot of lipogenic gene expression (45) and repression of oxidative processes (46). We have used this as a measure of relative hepatic insulin sensitivity. 11-HSD-1^/ mice manifest increased hepatic insulin sensitivity, since upon refeeding there is an exaggerated induction of lipogenic genes (SREBP-1c, FAS, GPAT, and HMG-CoAR) and suppression of oxidative genes (mCPT-I, UCP-2, and ACO). This provides an explanation for the more rapid recovery of triglycerides to ad lib fed levels, as well as the 24-h triglyceride "overshoot" upon refeeding 11-HSD-1^/ mice. The contention that 11-HSD-1^/ mice have increased hepatic insulin sensitivity is supported by their enhanced glucose disposal despite similar fasted insulin levels. Recent evidence supports the idea that insulin sensitivity in the liver (where 11-HSD-1 is most highly expressed) is a major determinant of glucose homeostasis; knockout of the insulin receptor in the liver results in profound glucose intolerance in mice (47) in contrast to analogous studies in muscle (48). However, the contribution of muscle and adipose tissues to the improved glucose tolerance of the 11-HSD-1^/ mice has yet to be determined.

Increased hepatic PPAR may play a key role in the 11-HSD-1^/ metabolic phenotype. PPAR activation by fibrates lowers plasma triglyceride (28) and represses apoCIII (28) and A-fibrinogen expression (49). We observed increased expression of the PPAR target genes mCPT-I (50), ACO (51), and UCP-2 (39, 52) in liver of the 11-HSD-1^/ mice. Moreover, the 25% reduction in A-fibrinogen mRNA levels in 11-HSD-1^/ mice is similar to the effect produced by fibrates (49). 11-HSD-1^/ mice thus mimic, in part, the phenotype of a fibrate-treated animal. It is intriguing that a recent report suggests that fibrates reduce hepatic 11-HSD-1 expression (53), which may mediate part of their metabolic actions. This could also indicate a negative feedback loop in wild type animals whereby amplification of glucocorticoid levels through 11-HSD-1 increases PPAR expression, which in turn down-regulates expression of 11-HSD-1.

While elevated PPAR levels may be a mechanism driving the ad lib fed lipid phenotype of 11-HSD-1^/ mice, its increased expression in this model of intracellular glucocorticoid deficiency presents an apparent paradox. PPAR is induced by glucocorticoids within the physiological range (24). One potential explanation for increased PPAR expression, therefore, could be the subtly elevated circulating corticosterone levels in 11-HSD-1^/ mice caused by impaired (11-HSD-1-mediated) negative feedback upon the hypothalamic-pituitary-adrenal axis (16, 29). Nevertheless, 11-HSD-1^/ mice have reduced intracellular glucocorticoid levels and responses in the brain in the face of such increased plasma corticosterone (54). The lack of PPAR induction with fasting is compatible with reduced intrahepatic glucocorticoid levels. Furthermore, hepatic glucocorticoid-sensitive genes such as corticosterone-binding globulin (55) and glucocorticoid receptor (56) are not down-regulated in ad lib fed 11-HSD-1^/ mice (29), despite the modestly higher corticosterone. This suggests that factors other than plasma corticosterone may be responsible for the elevated PPAR expression. Indeed, PPAR is regulated by many factors including other steroids (57), lipids (58), retinoids (59), and hormones (60) including insulin, as indicated in the present study. The precise determinants of elevated basal PPAR in this model of chronic subtle glucocorticoid depletion in the liver remain to be determined.

The complex relationship between glucocorticoid and PPAR signaling is also reflected by the expression profile of genes regulated by both transcription factors. For example, fibrinogen levels are positively regulated by glucocorticoids (40, 61) and negatively regulated by PPAR (49). In ad lib fed 11-HSD-1^/ mice, lower A-fibrinogen expression suggests that PPAR-mediated repression appears to dominate at the fibrinogen promoter. In addition, apolipoprotein AI is induced by glucocorticoids (33) and insulin (62) but is repressed by PPAR in mice (28). Our observation of elevated apoAI transcript levels in ad lib fed 11-HSD-1^/ mice implies that PPAR does not dominate on the apoAI promoter. Increased apoAI expression upon fasting in wild type mice is consistent with the glucocorticoid induction observed in rats and humans (33). The attenuated induction of ApoAI on fasting in 11-HSD1^/ mice perhaps reflects their reduced intrahepatic glucocorticoid action. These findings illustrate the interdependence of the three principle mechanisms that govern the overall metabolic phenotype of 11-HSD-1^/ mice, each emphasized during a different feeding state: increased ad lib fed PPAR expression, intrahepatic glucocorticoid deficiency with fasting, and hepatic insulin sensitization upon refeeding.

Mice with a targeted disruption in the gene encoding the 11-HSD-1 enzyme represent a model that lacks a crucial intracellular glucocorticoid reamplifying mechanism but maintains adequate circulating corticosterone levels. Previous studies show that adrenalectomy can ameliorate all of the metabolic abnormalities produced by high fat feeding including hypertriglyceridemia and insulin resistance (21). However, adrenalectomy causes complete glucocorticoid deficiency and is not therapeutically relevant. The current work demonstrates that intracellular glucocorticoid reactivation by 11-HSD-1 represents a powerful mechanism regulating triglyceride metabolism in mice on a standard diet. Moreover, expression of 11-HSD-1 in normal liver was unaffected by the dietary manipulations in vivo, suggesting that, as a putative drug target, its expression is maintained over commonly occurring changes in feeding status. The data presented here suggest that inhibitors of the enzyme may have favorable effects on several metabolic and cardiovascular risk factors. Thus, elevated triglycerides, apoCIII (63) and high fibrinogen levels (41) are associated with increased risk for cardiovascular disease, whereas mice lacking 11-HSD-1 activity show an amelioration of all of these parameters. Null mice also have increased HDL cholesterol and apoAI levels that are associated with a reduced cardiovascular risk (32). Finally, the improved hepatic insulin sensitivity and glucose tolerance exhibited by 11-HSD-1^/ mice support the notion of a favorable metabolic profile. However, experimental manipulation through high fat feeding to induce the metabolic syndrome will be necessary to test any true disease-resisting potential of the 11-HSD-1^/ mouse phenotype. Further, the data suggest that a combination of an 11-HSD-1 inhibitor, perhaps with a fibrate, could represent a useful therapeutic strategy.

	ACKNOWLEDGEMENTS

We thank Dr. Philip Wenham and Jacques Fremaux for lipid and lipoprotein analyses, Keith Chalmers and Lynne Ramage for excellent technical assistance, and Drs. Karen Chapman, Brian Walker, and Chris Kenyon for helpful discussions on the manuscript.

	FOOTNOTES

^* This work was supported by a Wellcome Trust program grant (to J. R. S. and J. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 44-131-651-1030; Fax: 44-131-651-1085; E-mail: nik.morton@ed.ac.uk.

Published, JBC Papers in Press, August 23, 2001, DOI 10.1074/jbc.M103676200

	ABBREVIATIONS

The abbreviations used are: 11-HSD-1, 11-hydroxysteroid dehydrogenase type 1; VLDL, very low density lipoprotein; HDL, high density lipoprotein; apo, apolipoprotein; SREBP-1c, sterol regulatory element-binding protein-1c; FPLC, fast protein liquid chromatography; PPAR, peroxisome proliferator-activated receptor-; MOPS, 4-morpholinepropanesulfonic acid; FAS, fatty acid synthase; GPAT, glycerol phosphate acyltransferase; HMG-CoAR, hydroxymethylglutaryl-CoA reductase; mCPT-I, mitochondrial carnitine palmitoyltransferase-I; ACO, acyl-CoA oxidase; UCP-2, uncoupling protein-2; ANOVA, analysis of variance.

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				C. M. Alexander The Metabolic Syndrome: The Emperor Needs Some Consistent Clothes: Response to Vinicor and Bowman Diabetes Care, May 1, 2004; 27(5): 1244 - 1244. [Full Text] [PDF]

				N. M. Morton, J. M. Paterson, H. Masuzaki, M. C. Holmes, B. Staels, C. Fievet, B. R. Walker, J. S. Flier, J. J. Mullins, and J. R. Seckl Novel Adipose Tissue-Mediated Resistance to Diet-Induced Visceral Obesity in 11{beta}-Hydroxysteroid Dehydrogenase Type 1-Deficient Mice Diabetes, April 1, 2004; 53(4): 931 - 938. [Abstract] [Full Text] [PDF]

				J. Justesen, L. Mosekilde, M. Holmes, K. Stenderup, J. Gasser, J. J. Mullins, J. R. Seckl, and M. Kassem Mice Deficient in 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Lack Bone Marrow Adipocytes, but Maintain Normal Bone Formation Endocrinology, April 1, 2004; 145(4): 1916 - 1925. [Abstract] [Full Text] [PDF]

				J. R. Seckl, N. M. Morton, K. E. Chapman, and B. R. Walker Glucocorticoids and 11beta-Hydroxysteroid Dehydrogenase in Adipose Tissue Recent Prog. Horm. Res., January 1, 2004; 59(1): 359 - 393. [Abstract] [Full Text]

				C. G. Perry, A. Spiers, S. J. Cleland, G. D. O. Lowe, J. R. Petrie, and J. M. C. Connell Glucocorticoids and Insulin Sensitivity: Dissociation of Insulin's Metabolic and Vascular Actions J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6008 - 6014. [Abstract] [Full Text] [PDF]

				L. Liu, D. M. Hawkins, S. Ghosh, and S. S. Young Robust singular value decomposition analysis of microarray data PNAS, November 11, 2003; 100(23): 13167 - 13172. [Abstract] [Full Text] [PDF]

				P. Alberts, C. Nilsson, G. Selen, L. O. M. Engblom, N. H. M. Edling, S. Norling, G. Klingstrom, C. Larsson, M. Forsgren, M. Ashkzari, et al. Selective Inhibition of 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Improves Hepatic Insulin Sensitivity in Hyperglycemic Mice Strains Endocrinology, November 1, 2003; 144(11): 4755 - 4762. [Abstract] [Full Text] [PDF]

				J. Westerbacka, H. Yki-Jarvinen, S. Vehkavaara, A.-M. Hakkinen, R. Andrew, D. J. Wake, J. R. Seckl, and B. R. Walker Body Fat Distribution and Cortisol Metabolism in Healthy Men: Enhanced 5{beta}-Reductase and Lower Cortisol/Cortisone Metabolite Ratios in Men with Fatty Liver J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4924 - 4931. [Abstract] [Full Text] [PDF]

				C. Mattsson, M. Lai, J. Noble, E. McKinney, J. L. Yau, J. R. Seckl, and B. R. Walker Obese Zucker Rats Have Reduced Mineralocorticoid Receptor and 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Expression in Hippocampus--Implications for Dysregulation of the Hypothalamic-Pituitary-Adrenal Axis in Obesity Endocrinology, July 1, 2003; 144(7): 2997 - 3003. [Abstract] [Full Text] [PDF]

				R. S. Lindsay, D. J. Wake, S. Nair, J. Bunt, D. E. W. Livingstone, P. A. Permana, P. A. Tataranni, and B. R. Walker Subcutaneous Adipose 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Activity and Messenger Ribonucleic Acid Levels Are Associated with Adiposity and Insulinemia in Pima Indians and Caucasians J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2738 - 2744. [Abstract] [Full Text] [PDF]

				Z. Chai, P. Brereton, T. Suzuki, H. Sasano, V. Obeyesekere, G. Escher, R. Saffery, P. Fuller, C. Enriquez, and Z. Krozowski 17{beta}-Hydroxysteroid Dehydrogenase Type XI Localizes to Human Steroidogenic Cells Endocrinology, May 1, 2003; 144(5): 2084 - 2091. [Abstract] [Full Text] [PDF]

				D. E. W. Livingstone and B. R. Walker Is 11beta -Hydroxysteroid Dehydrogenase Type 1 a Therapeutic Target? Effects of Carbenoxolone in Lean and Obese Zucker Rats J. Pharmacol. Exp. Ther., April 1, 2003; 305(1): 167 - 172. [Abstract] [Full Text]

				R. C. Andrews, O. Rooyackers, and B. R. Walker Effects of the 11{beta}-Hydroxysteroid Dehydrogenase Inhibitor Carbenoxolone on Insulin Sensitivity in Men with Type 2 Diabetes J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 285 - 291. [Abstract] [Full Text] [PDF]

				R. C. Andrews, O. Herlihy, D. E. W. Livingstone, R. Andrew, and B. R. Walker Abnormal Cortisol Metabolism and Tissue Sensitivity to Cortisol in Patients with Glucose Intolerance J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5587 - 5593. [Abstract] [Full Text] [PDF]

				T. M. Stulnig, U. Oppermann, K. R. Steffensen, G. U. Schuster, and J.-A. Gustafsson Liver X Receptors Downregulate 11{beta}-Hydroxysteroid Dehydrogenase Type 1 Expression and Activity Diabetes, August 1, 2002; 51(8): 2426 - 2433. [Abstract] [Full Text] [PDF]

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