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. 2008 Jan 24;149(5):2584–2591. doi: 10.1210/en.2007-1705

Lack of Hexose-6-Phosphate Dehydrogenase Impairs Lipid Mobilization from Mouse Adipose Tissue

Iwona J Bujalska 1,a, Kylie N Hewitt 1,a, David Hauton 1, Gareth G Lavery 1, Jeremy W Tomlinson 1, Elizabeth A Walker 1, Paul M Stewart 1
PMCID: PMC2329282  PMID: 18218694

Abstract

In adipose tissue, glucocorticoids regulate lipogenesis and lipolysis. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme located in the endoplasmic reticulum that provides a cofactor for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), regulating the set point of its activity and allowing for tissue-specific activation of glucocorticoids. The aim of this study was to examine the adipose tissue biology of the H6PDH null (H6PDH/KO) mouse. Real-time PCR analysis confirmed similar mRNA levels of 11β-HSD1 and glucocorticoid receptor-α in wild-type (WT) and H6PDH/KO mice in liver and gonadal fat depots. Microsomal 11β-HSD1 protein levels shown by Western blot analysis corresponded well with mRNA expression in gonadal fat of WT and H6PDH/KO mice. Despite this, the enzyme directionality in these tissues changed from predominately oxoreductase in WT to exclusively dehydrogenase activity in the H6PDH/KO mice. In the fed state, H6PDH/KO mice had reduced adipose tissue mass, but histological examination revealed no difference in average adipocyte size between genotypes. mRNA expression levels of the key lipogenic enzymes, acetyl CoA carboxylase, adiponutrin, and stearoyl-coenzyme A desaturase-2, were decreased in H6PDH/KO mice, indicative of impaired lipogenesis. In addition, lipolysis rates were also impaired in the H6PDH/KO as determined by lack of mobilization of fat and no change in serum free fatty acid concentrations upon fasting. In conclusion, in the absence of H6PDH, the set point of 11β-HSD1 enzyme activity is switched from predominantly oxoreductase to dehydrogenase activity in adipose tissue; as a consequence, this leads to impairment of fat storage and mobilization.

ADIPOSE TISSUE MASS and its differentiation are key factors that are implicated in the pathogenesis of metabolic syndrome. The dynamics of adipose tissue mass are regulated by two major processes: lipogenesis (triglyceride accumulation) and lipolysis (triglyceride mobilization). Glucocorticoids (GCs) regulate lipid metabolism, exerting diverse effects, depending on the nutritional state; increasing lipogenesis in the fed state (1,2); and increasing lipolysis in the fasted state (3,4,5). In vitro, GCs are essential for terminal differentiation of human and rodent adipocytes (6,7). However, adipose tissue mass depends on not only adipocyte number and size but also availability and recruitment of new cells (preadipocytes) to undergo adipogenesis. Notwithstanding increasing differentiation, GCs are also known to have an antiproliferative effect on preadipocytes (8,9).

GC action is not exclusively dependent on their systemic levels. Our recent studies have shown the pivotal role of two luminal endoplasmic reticulum (ER) enzymes in tissue-specific GC metabolism: 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and hexose-6-phosphate dehydrogenase (H6PDH) (10,11,12). 11β-HSD1, expressed mainly in the liver and adipose tissue (reviewed in Ref. 13), is a bidirectional enzyme that in vivo acts predominantly as an oxoreductase, converting inactive cortisone/11-dehydrocorticosterone to metabolically active glucocorticoids, cortisol/corticosterone in humans and rodents, respectively (14,15,16). Conversely, 11β-HSD1 dehydrogenase inactivates glucocorticoids. H6PDH is a ubiquitously expressed component of an ER pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphogluconate, generating the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (12). The evidence that H6PDH is a key regulator of 11β-HSD1 set point has been obtained from studies on the H6PDH knockout (H6PDH/KO) mouse, in which the lack of H6PDH resulted in a switch in 11β-HSD1 activity from oxoreductase to dehydrogenase (17).

Inhibition of 11β-HSD1 in vitro impairs adipogenesis (7), an observation confirmed in vivo using knockout and transgenic mouse models. 11β-HSD1/KO mice resist diet-induced obesity and have improved glucose tolerance and insulin sensitivity (18,19). Conversely, transgenic mice overexpressing 11β-HSD1 in adipose tissue display features of metabolic syndrome with hepatic steatosis, central obesity, hyperinsulinemia, hyperlipidemia, and hypertension (20,21), whereas the liver-specific 11β-HSD1 overexpression causes metabolic syndrome without obesity (22).

The aim of this study was to use the H6PDH/KO mouse model to investigate the in vivo effects of lack of H6DPH on prereceptor metabolism of GCs on adipose tissue lipid homeostasis.

Materials and Methods

Animals

Studies were performed in accordance with Home Office Guidance (Animals Scientific Procedures Act 1986). The H6DPH/KO mice were generated by the insertion of a neomycin resistance cassette into exons 2 and 3 of the H6PDH gene as reported (17). Homozygous null mice were generated by heterozygous mating. Mice were housed in pathogen-free conditions and had a 12-h light, 12-h dark cycle and unlimited access to standard mouse chow (when not fasting) and water. Twelve- to 14-wk-old male mice were used for this study.

Mouse tissue collection

Liver and gonadal fat (GF) were excised from wild-type (WT) and H6PDH/KO mice. Tissues were snap frozen in either liquid N2 for RNA and lipid extraction or microsomal isolation, fixed in 10% buffered formalin for histology, or used fresh for 11β-HSD activity on GF explants. Where indicated, microsomes from liver and GF were isolated as reported (17) and used for protein expression studies.

GF stromal-vascular cell isolation

Mouse adipose preadipocytes/stromal-vascular (S-V) cells were isolated from GF by collagenase digestion. Briefly, adipose tissue was washed and cut into small (about 2 mm3) pieces and then digested with 2 mg/ml collagenase type 2 (Sigma, Poole, UK) at 37 C in a shaking water bath for 45 min. Adipocytes were separated from the S-V fraction by centrifugation at 100 × g for 5 min, washed with DMEM/F12 media, and strained through a 0.2-μm filter. S-V cells were plated into 24-well tissue culture plates and cultured until confluence in DMEM/F12 media supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen, Paisley, UK).

RNA extraction

Mouse adipose and liver tissues were homogenized with an Ultra-Turrax homogenizer and total RNA extracted using TriReagent (Sigma) according to the manufacturer’s protocol. RNA quality was assessed by 1% agarose gel electrophoresis and quantified spectrophotometrically.

Reverse transcriptase and real-time PCR

Two-step RT-PCR was performed using 1 μg of RNA, random hexamers, and Multiscribe reverse transcriptase kit (Applied Biosystems, Warrington, UK). An ABI 7500 real-time PCR machine was used to amplify mouse transcripts using specific primer pairs and probes, which were quantified relative to ribosomal 18S expression. Real-time primers and probes for mouse H6PDH, 11β-HSD1, glucocorticoid receptor (GR)-α, adiponutrin, lipoprotein lipase (LPL), fatty acid synthase, acetyl CoA carboxylase (ACC)-α, and 18S were purchased as predesigned expression assays (Applied Biosystems). Real-time PCR data are presented as arbitrary units (AU) calculated as AU = 1000 × 2^(−ΔCt). ΔCt values were calculated as a difference between Ct of the target gene minus Ct of the housekeeping gene, where Ct is the cycle number at which logarithmic PCR plots cross a claculated threshold line.

SDS-PAGE gel and Western blot analysis

Microsomes were isolated from liver and GF from WT and H6PDH/ KO mice. Protein concentration was determined using a 96-well plate Bio-Rad protein assay kit (Bio-Rad Laboratories, Hemel Hempstead, UK) with internal standards of BSA ranging from 0.5 to 5 mg/ml. Thirty micrograms of protein were resolved on a 12.5% acrylamide gel and high-precision molecular weight markers (Bio-Rad) were used to assess protein size. The H6PDH antibody is a polyclonal antirabbit antibody produced from a H6PDH peptide sequence (17), which recognizes H6PDH protein at a molecular mass of 89 kDa (17). The 11β-HSD1 antibody is a polyclonal antisheep antibody previously characterized (23) and detects the 11β-HSD1 protein at a molecular mass of 34 kDa. ECL (Amersham, Buckinghamshire, UK) was used as the detection method. Equal loading of protein was confirmed by Ponso S staining of the membrane.

11β-HSD1 activity

Measurement of 11β-HSD1 activity in mouse GF explants and S-V cells was carried out in 0.5 ml of serum-free DMEM/F12 using either 100 nm 11-dehydrocorticosterone (A) or 100 nm corticosterone (B) (for oxoreductase and dehydrogenase reaction directions, respectively). An appropriate tritiated tracer (3HA or 3HB) was added to each well at 0.02 μCi/reaction. Tritiated B was purchased from Sigma, and tritiated A was synthesized in-house as previously described (24). 11β-HSD1 activity was expressed as picomole product converted per gram of wet tissue (or per milligram of protein for cells) in 1 h.

Adipocyte size

GF was fixed in 10% buffered formalin, embedded in paraffin, and sectioned to 10 μm. Sections were then stained in hematoxylin and counterstained with eosin. Sections were visualized microscopically with a microscope (Leica, Milton Keynes, UK) at ×200 magnification and photographed using a digital camera (PowerShot S70; Canon, Surrey, UK). Adipocytes were counted by two independent researchers (masked to genotype) on an equal area (square millimeters, n = 10), and average radius (r, micrometers) was calculated: r = 1000√area/(adipocyte numberπ).

In vivo rates of lipogenesis and lipolysis

Lipogenesis.

Mice were placed into two groups (n = 6), fed, and fasted 16 h. Mice from each group were injected ip with 3H2O (2 mCi/mouse) 2 h before collection. Mice were anesthetized with pentobarbitone and samples of blood drawn from the inferior vena cava. Tissues were excised, weighed, and snap frozen in liquid nitrogen. The amount of specific radioactivity incorporated into fatty acids in the GF was then measured. Briefly, approximately 50 mg of GF was ground to powder in liquid nitrogen and transferred to a glass tube. After addition of 4 ml of 7.5% KOH, samples were heated at 70 C for 2 h before 5 ml of H2O were added with 0.5 g of NaSO4. To adjust for recovery efficiency, an internal standard (2200 dpm of 14C-oleate acid diluted in chloroform) was added to each sample. Then 1 m HCl (1 ml) was added to ensure hydrolysis of fatty acids, and fatty acids were eluted by shaking with 3 × 10 ml of hexane. The samples were dried down overnight under air and fatty acids resuspended in 250 μl chloroform. In vivo lipogenesis rates were calculated as micromoles of fatty acids per gram tissue per 2 h. Serum triglycerides (TAG) were measured in fed and fasted mice using a triglyceride kit (Thermo Fisher Scientific, BL Breda, The Netherlands) according to the manufacturer’s protocol.

Lipolysis.

Blood was collected from the inferior vena cava of the mice in heparinized syringes and centrifuged to separate the serum (n = 5). Serum was snap frozen and stored at −20 C until required. Nonesterified free fatty acids (FFAs) were measured using a nonesterified fatty acid kit (WAKO, Alpha Laboratories, Eastleigh, UK) according to the manufacturer’s protocol. Additionally, GF pads were collected and weighed from WT and H6PDH/KO mice in the fed and fasted (16 h) groups (n = 10 per group).

Statistical analysis

Data were expressed as mean ± sd or mean ± sem. Where data were normally distributed, unpaired Student t test was used to compare single treatments to control. If normality tests failed, then nonparametric ANOVA on ranks was used to compare multiple treatments (SigmaStat 3.1; Systat Software, Inc., Point Richmond, CA) and P < 0.05 was accepted as statistically significant. Statistical analysis on real-time PCR data was performed on mean ΔCt values.

Results

Gene expression of 11β-HSD1, GRα, and H6PDH in metabolic tissues from WT and H6PDH/KO mice

The pattern of expression of 11β-HSD1, GRα, and H6PDH mRNA was measured in liver and GF from WT and H6PDH/KO mice. The highest levels of 11β-HSD1 mRNA were found in the liver, with similar amounts in both genotypes (WT 0.046 ± 0.005 vs. H6PDH/KO 0.046 ± 0.005 AU, mean ± sd, n = 4), and lower levels in GF (WT 0.026 ± 0.009 vs. H6PDH/KO 0.034 ± 0.007 AU, mean ± sd, n = 4) (Fig. 1A). 11β-HSD1 protein levels were assessed by Western blot analysis of protein from isolated microsomes. The pattern of protein expression was similar to that of the mRNA levels, with higher 11β-HSD1 in the liver microsomes, compared with GF. Again, there was no difference in protein expression between genotypes (Fig. 1B).

Figure 1.

Figure 1

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mRNA expression and protein levels of 11β-HSD1 (A and B, respectively), H6PDH (C and D, respectively), and GRα mRNA expression levels (E) in liver and GF in WT and H6PDH/KO mice. Relative mRNA levels were measured by real-time PCR, expressed as arbitrary units and shown as mean ± sd, n = 3. **, P < 0.01; ***, P < 0.001.

In the WT animals, H6PDH mRNA expression was significantly higher in GF, compared with liver (GF 0.090 ± 0.024 vs. liver 0.037 ± 0.007 AU; mean ± sd, P < 0.01), and there was no detectable H6PDH transcript present in the H6PDH/KO mice (Fig. 1C). Again, protein expression followed that of mRNA with higher microsomal H6PDH in GF of WT mice than in liver and no detectable protein expression in H6PDH/KO mice (Fig. 1D). GRα gene expression was higher in GF in both genotypes (WT: GF 0.022 ± 0.002 vs. liver 0.0041 ± 0.0005 AU, mean ± sd, P < 0.001, n = 4; H6PDH/KO: GF 0.024 ± 0.006 vs. liver 0.0044 ± 0.0003 AU mean ± sd, P < 0.001, n = 4) (Fig. 1E).

11β-HSD1 activity in GF explants and S-V cells

In GF explants from WT mice, 11β-HSD1 oxoreductase activity predominated (219 ± 55 pmol/g wet tissue/h) with very low levels of dehydrogenase activity (2.3 ± 3.8 pmol/g wet tissue/h, mean ± sd, P < 0.001, n = 4) (Fig. 2A). Conversely, GF explants from H6PDH/KO mice had significantly reduced 11β-HSD1 oxoreductase activity (18 ± 14 pmol/g wet tissue/h) and high levels of dehydrogenase activity (300 ± 29 pmol/g wet tissue/h, mean ± sd, P < 0.001, n = 4) (Fig. 2A).

Figure 2.

Figure 2

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11β-HSD1 oxoreductase and dehydrogenase activity in GF explants (picomoles per gram wet tissue per hour) (A) and in GF S-V cells (picomoles per milligram protein per hour) (B) from WT and H6PDH/KO mice (mean ± sd, n = 4. **, P < 0.01; ***, P < 0.001).

In the GF S-V cells from WT mice, there were similar levels of 11β-HSD1 oxoreductase and dehydrogenase activities: 25 ± 19 vs. 16 ± 11 pmol/mg·h, respectively, mean ± sd, n = 4 (Fig. 2B). In the GF S-V cells from the H6PDH/KO, 11β-HSD1 dehydrogenase activity was significantly higher (51 ± 14 pmol/mg·h), compared with WT (16 ± 11 pmol/mg·h, mean ± sd, P < 0.01, n = 4) (Fig. 2B). No oxoreductase activity was detected.

Morphometric data

The H6PDH/KO mice weighed significantly less than the WT mice (H6PDH/KO 27 ± 3 g vs. WT 33 ± 5 g, P = 0.001, n = 10). H6PDH/KO mice had significantly less GF, compared with WT mice (H6PDH/KO 0.5 ± 0.2 vs. WT 0.9 ± 0.5 g, mean ± sem, P < 0.01, n = 10). Despite decreased levels of GF in H6PDH/KO mice, this was in proportion to their overall body weight, and there was no difference in average adipocyte size between the genotypes (data not shown).

Rates of lipogenesis

Rates of lipogenesis in vivo were measured by the incorporation of tritium during de novo fatty acid synthesis in GF from WT and H6PDH/KO mice. There was a significant decrease in the rate of lipogenesis in the H6PDH/KO mice, compared with WT mice (WT 12.5 ± 3.9 μmol/g per 2 h vs. H6PDH/KO 8.7 ± 3.3 μmol/g per 2 h, P < 0.05, n = 5) (Fig. 3A). This was concurrent with significantly lower mRNA expression of the fatty acid synthesis rate-limiting enzyme, ACCα (WT 0.040 ± 0.017 vs. H6PDH/KO 0.008 ± 0.003 AU, mean ± sd, P < 0.01, n = 4) (Fig. 3B). Additionally, mRNA expression of adiponutrin, a novel prolipogenic gene, was significantly reduced in H6PDH/KO mice, compared with WT mice (WT 0.1 ± 0.04 vs. H6PDH/KO 0.04 ± 0.01 AU, mean ± sd, P < 0.01, n = 4) (Fig. 3C). mRNA expression of stearoyl-coenzyme A desaturase (SCD)-2, a microsomal enzyme that catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, was significantly decreased in H6PDH/KO mice (WT 0.19 ± 0.07 vs. H6PDH/KO 0.07 ± 0.03 AU, mean ± sd, P < 0.01, n = 4). Fatty acid synthase mRNA was also lower in H6PDH/KO mice, compared with WT (WT 0.29 ± 0.13 vs. H6PDH/KO 0.21 ± 0.07 AU, mean ± sd, n = 4) but did not reach statistical significance. There was no significant difference in LPL mRNA levels (data not shown).

Figure 3.

Figure 3

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Rates of lipogenesis in vivo in the WT and H6PDH/KO mice in fed state (fatty acid, micromole per gram wet tissue per 2 h, n = 5) (A). mRNA expression levels of ACCα (B), adiponutrin (C), and SCD2 (D) mRNA levels in fed H6PDH/KO and WT mice (arbitrary units, mean ± sd, n = 3). *, P < 0.05; **, P < 0.01.

Lipolysis

Overnight fasting (16 h) significantly reduced GF depot mass of WT mice (fed 1.02 ± 0.51 g vs. fasted 0.44 ± 0.34 g, mean ± sem, P < 0.05, n = 6) (Fig. 4A). GF depots of H6PDH/KO mice were smaller than those of WT mice, but importantly, fasting did not significantly alter the depot size in these KO animals (fed 0.49 ± 0.19 g vs. fasted 0.39 ± 0.17 g, mean ± sem, n = 6) (Fig. 4A). Serum TAG levels were not different between WT and H6PDH/KO mice in the fed state (WT 0.54 ± 0.05 mmol/liter vs. H6PDH/KO 0.58 ± 0.06 mmol/liter). Fasting (as expected) decreased serum TAG levels in WT mice (fed 0.54 ± 0.05 mmol/liter vs. fasted 0.43 ± 0.03 mmol/liter, n = 6, P < 0.05), but there was no change in H6PDH/KO mice (fed 0.58 ± 0.06 vs. fasted 0.67 ± 0.04 mmol/liter, n = 6) (Fig. 4B). GF histology showed that in WT mice, overnight fasting decreased average adipocyte size (84 ± 3 vs. 73 ± 3 μm, mean ± sem, n = 10, P < 0.05). Conversely, lipids were not mobilized from adipocytes of H6PDH/KO mice because the average adipocyte size did not change (83 ± 2 vs. 85 ± 3 μm, mean ± sem, n = 10) (Fig. 4C). Representative histology of the GF from fasted WT and H6PDH/KO is shown in Fig. 4D.

Figure 4.

Figure 4

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A, GF pad weight in fed and fasted (for 16 h) WT and H6PDH/KO mice (grams, mean ± sd, n = 10). B, Serum TAG in fed and fasted WT and H6PDH/KO mice (millimoles per liter, mean ± sem, n = 6). C, Average adipocyte size (radius, micromoles, mean ± sem, n = 4) in fed and fasted WT and H6PDH/KO. D, Representative histology of GF of fasted WT and H6PDH/KO mice. *, P < 0.05; **, P < 0.01.

In overnight fasted WT mice, serum FFA concentrations were significantly elevated, compared with fed WT mice (fed 0.9 ± 0.2 mmol/liter vs. fasted 1.4 ± 0.1 mmol/liter mean ± sem, P < 0.05, n = 5) (Fig. 5A). In contrast, serum FFA concentrations did not differ between the fed and fasted H6PDH/KO mice. Serum FFA concentrations in fasted WT mice were also significantly higher than in fasted H6PDH/KO mice (P < 0.05, Fig. 5A).

Figure 5.

Figure 5

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A, Serum FFA levels in fed and fasted WT and H6PDH/KO mice (millimoles per liter, mean ± sem, n = 5). B, HSL mRNA expression levels in fed and fasted WT and H6PDH/KO mice (arbitrary units ± sd, n = 4). *, P < 0.05; ***, P < 0.001.

In line with increased in vivo lipolysis in GF of WT mice, we observed increased mRNA expression of hormone-sensitive lipase (HSL) enzyme (fed 0.48 ± 0.08 vs. fasted 1.55 ± 0.22 AU, mean ± sd, P < 0.001, n = 4) (Fig. 5B). Moreover, expression of HSL in H6PDH/KO mice was low and did not alter with nutritional status.

Discussion

GCs are implicated in the pathogenesis of obesity and are known to be regulated at the prereceptor level by the enzymes 11β-HSD1 and H6PDH. Previously we have shown that H6PDH is the key regulator of the set point of 11β-HSD1 activity by producing the reduced form of NADPH within the ER, a necessary cofactor for 11β-HSD1 oxoreductase activity (11,17). In the present study, we investigated the adipose tissue phenotype of the H6PDH/KO mouse.

In the H6PDH/KO mouse, we have shown that in the absence of H6PDH, GF explants and isolated S-V cells have a change in 11β-HSD1 enzyme directionality from predominately oxoreductase to dehydrogenase. This results in local GC inactivation within this depot, regardless of circulating GCs. Within adipose tissue, depending on nutritional state, GCs exert diverse effects on lipid metabolism, increasing lipogenesis in the fed state (1) and increasing lipolysis in the fasted state (3,4,5). Two major determinants of lipogenesis are first de novo FFA synthesis and the hydrolysis of triglyceride component of circulating chylomicrons and very low-density lipoproteins in the serum (25,26) and second, the reesterification of FFA within adipocyte. ACC enzyme converts acetyl CoA to malonyl CoA, and is the rate-limiting step of fatty acid biosynthesis (27). It exists as two isoforms: cytosolic ACCα, expressed mainly in liver and adipose tissue, and mitochondrial ACCβ, expressed in heart and muscle (28). GCs have previously been shown to alter the expression pattern of this gene within adipose tissue: enhancing the effects of insulin by increasing active form of ACCα in ovine sc adipose tissue explants (29) as well as increasing mRNA transcription (30). In GF of H6PDH/KO mouse, attenuated GC regeneration decreased de novo fatty acid synthesis together with reducing ACCα expression and hence further defined the key role of intracellular GCs on lipid synthesis.

LPL facilitates triglyceride hydrolysis by allowing uptake of circulating lipids into adipocytes, therefore contributing significantly into lipogenesis (31). These two mechanisms, de novo FFA synthesis and hydrolysis, are finely synchronized because increase in the first one can compensate for the lack of LPL in adipose-specific LPL knockout mice (32). In human adipose tissue, GCs increased LPL activity in vitro and in vivo (33,34), whereas in rat adipocytes, dexamethasone decreased LPL activity and mRNA but had no effect when adipocytes were cotreated with insulin (35). We observed no difference in TAG levels between the WT and H6PDH/KO mice in the fed state; this may be explained by no difference in LPL mRNA expression. This, simultaneously with similar serum TAG levels in both genotypes, could result in the redirection of fat storage into liver or muscle, but this hypothesis needs to be further evaluated.

Adiponutrin is an adipose-specific, transmembrane protein regulated by energy balance (36). It is highly expressed in the fed state, decreasing to undetectable levels in fasting and has been negatively correlated with insulin sensitivity in humans (37), and because small interfering RNA knockdown did not alter lipolysis in 3T3-L1 cells (38), it has been postulated to be the part of the adipose-specific energy homeostasis sensor (39,40). Our study confirmed much higher adiponutrin expression in WT than H6PDH/KO mice in the fed state and undetectable levels in both genotypes in fasted state (data not published). Biological function of adiponutrin is still unclear; nevertheless, it would be interesting to find out what effect glucocorticoids have on the function of this protein.

SCD is a microsomal, rate-limiting enzyme involved in the biosynthesis of monounsaturated fatty acids from saturated fatty acyl CoAs (41) and absolutely required for adipogenesis (42). The saturated to monounsaturated fatty acid ratio affects membrane phospholipid composition, and alteration in this ratio has been implicated in a variety of disease states, including diabetes, obesity, cardiovascular disease, neurological disease, skin disorders, and cancer (43,44,45). In mice, SCD exists as four isozymes (46), of which SCD2 is expressed mainly in liver and adipose tissue and recently was shown to play an important role in early liver and skin development in mice (47). Increased SCD2 expression by dexamethasone was demonstrated in rat testis (48); our study showed the effect of intracellular GCs on SCD2 expression in adipose tissue. SCD2 is a reduced form of nicotinamide adenine dinucleotide-dependent enzyme; however, its orientation toward cytosolic site (49) would imply the effect of reduced GCs rather than alteration in nicotinamide adenine dinucleotide hydroxide levels within the ER lumen of H6PDH/KO mice.

Additionally, we have shown that in acute situation of fasting, lack of GC availability to adipose tissue impairs the ability to mobilize lipid. A significant decrease in adipose tissue mass in fasted WT mice occurs due to hydrolysis of TAG, resulting in increased serum FFA concentrations. Conversely, in the H6PDH/KO mice, the weight of adipose tissue depots and serum FFA levels were unchanged and the adipocyte remains large. This finding is in keeping with human studies in which treatment with dexamethasone increased serum glycerol levels (3,4) and limiting GC availability to adipose tissue decreases glycerol release (5). In isolated rat adipocytes, GCs increase mRNA levels of HSL, the key enzyme that regulates lipolysis (50); lack of GCs in GF of H6PDH/KO mice resulted in attenuated HSL mRNA expression. Lower expression of HSL might lead to an obese phenotype in the H6PDH/KO mice; however, this was not observed in these relatively young mice on a regular diet. Further studies are required to assess the affect the ageing process and/or high-fat diet on body composition.

Recently 11β-HSD1-specific inhibitors emerged as a potential therapy to improve the triglyceride profile (51,52). A beneficial effect of 11β-HSD1-specific inhibition was to reduce mesenteric fat accumulation in obese rats by decreasing gene expression of enzymes involved in lipid synthesis, increasing those associated with lipid oxidation (51), and improving triglyceridemia (52). Lack of H6PDH and 11β-HSD1 have only partially comparable outcome on lipid homeostasis, given that in the latter, tissues still will be exposed to systemic GCs. In fact, apparent cortisone reductase deficiency patients are mainly obese and have impaired lipid mobilization after exercise, indicating that in humans H6PDH inhibition has a deleterious effect on lipid mobilization.

In summary, using a mouse model that has impaired intracellular generation of GC within adipose tissue, we have demonstrated wide-ranging metabolic effects on lipogenesis and lipolysis. In humans, disturbance of lipid metabolism and use, especially increased FFA release from the adipose tissue into the liver, is a major risk factor for many diseases. Pharmacological modulation of local GC levels by either 11β-HSD1- or H6PDH-specific inhibitors may provide a novel approach for the treatment of the dyslipidemia of the metabolic syndrome.

Acknowledgments

We thank Keith L. Parker and Perrin C. White for helpful discussions.

Footnotes

This work was supported by Wellcome Trust Program Grant 066357 (to K.N.H., I.J.B., and P.M.S.), National Institutes of Health Grant DK068101 (to G.G.L. and P.M.S.), and Wellcome Trust Project Grant 074088 (to P.M.S. and E.A.W.).

Disclosure Statement: I.J.B., K.N.H., D.H., G.G.L., J.W.T., and E.A.W. have nothing to declare. P.M.S. is on the advisory board for Pfizer Global Research and Development.

First Published Online January 24, 2008

Abbreviations: ACC, Acetyl CoA carboxylase; AU, arbitrary unit; ER, endoplasmic reticulum; FFA, free fatty acid; GC, glucocorticoid; GF, gonadal fat; GR, glucocorticoid receptor; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; H6PDH, hexose-6-phosphate dehydrogenase; H6PDH/KO, H6PDH knockout; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; SCD, stearoyl-coenzyme A desaturase; S-V, stromal-vascular; TAG, triglycerides; WT, wild type.

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