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Endocrinology, doi:10.1210/en.2006-1472
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Endocrinology Vol. 148, No. 5 2487-2495
Copyright © 2007 by The Endocrine Society

The Glucose-6-Phosphate Transporter-Hexose-6-Phosphate Dehydrogenase-11ß-Hydroxysteroid Dehydrogenase Type 1 System of the Adipose Tissue

Paola Marcolongo1, Simona Piccirella1, Silvia Senesi1, Livius Wunderlich, Isabelle Gerin, József Mandl, Rosella Fulceri, Gábor Bánhegyi and Angelo Benedetti

Department of Pathophysiology, Experimental Medicine, and Public Health (P.M., S.P., S.S., R.F., G.B., A.B.), University of Siena, Siena 53100, Italy; Department of Medical Chemistry, Molecular Biology, and Pathobiochemistry (J.M., G.B.), Semmelweis University, and Endoplasmic Reticulum Research Group (L.W., J.M., G.B.), Hungarian Academy of Sciences, H-1088 Budapest, Hungary; and Laboratoire de Chimie Physiologique (I.G.), Universite Catholique de Louvain, B-1200 Brussels, Belgium

Address all correspondence and requests for reprints to: Gábor Bánhegyi, Dipartimento di Fisiopatologia, Medicina Sperimentale e Sanità Pubblica, Via A. Moro no. 1, 53100 Siena, Italy. E-mail: banhegyi{at}unisi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
11ß-Hydroxysteroid dehydrogenase type 1, expressed mainly in the endoplasmic reticulum of adipocytes and hepatocytes, plays an important role in the prereceptorial activation of glucocorticoids. In liver endoplasmic reticulum-derived microsomal vesicles, nicotinamide adenine dinucleotide phosphate reduced supply to the enzyme is guaranteed by a tight functional connection with hexose-6-phosphate dehydrogenase and the glucose-6-phosphate transporter (G6PT). In adipose tissue, the proteins and their activities supporting the action of 11ß-hydroxysteroid dehydrogenase type 1 have not been explored yet. Here we report the occurrence of the hexose-6-phosphate dehydrogenase in rat epididymal fat, as detected at the level of mRNA, protein, and activity. In the isolated microsomes, the activity was evident only on the permeabilization of the membrane because of the poor permeability to the cofactor nicotinamide adenine dineucleotide phosphate (NADP+), which is consistent with the intralumenal compartmentation of both the enzyme and a pool of pyridine nucleotides. In fat cells, the access of the substrate, glucose-6-phosphate to the intralumenal hexose-6-phosphate dehydrogenase appeared to be mediated by the liver-type G6PT. In fact, the G6PT expression was revealed at the level of mRNA and protein. Accordingly, the transport of glucose-6-phosphate was demonstrated in microsomal vesicles, and it was inhibited by S3483, a prototypic inhibitor of G6PT. Furthermore, isolated adipocytes produced cortisol on addition of cortisone, and the production was markedly inhibited by S3483. The results show that adipocytes are equipped with a functional G6PT-hexose-6-phosphate dehydrogenase-11ß-hydroxysteroid dehydrogenase type 1 system and indicate that all three components are potential pharmacological targets for modulating local glucocorticoid activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
11ß-HYDROXYSTEROID DEHYDROGENASE type 1 (11ßHSD1) regenerates active glucocorticoids from inactive forms by catalyzing the conversion of 11-ketoglucocorticoids (cortisone or 11-dehydrocorticosterone) to 11ß-hydroxy derivatives (cortisol or corticosterone). Therefore, it is currently assumed that 11ßHSD1 plays a central role in regulating local, intracellular concentrations of active glucocorticoids (see Refs. 1, 2, 3, 4, 5 for recent reviews).

In the liver endoplasmic reticulum (ER), 11ßHSD1 can be considered as a component of a complex system, which is also comprised of the hexose-6-phosphate dehydrogenase enzyme (H6PDH) and the glucose-6-phosphate transporter (G6PT). Whereas in vitro 11ßHSD1 can act either as a dehydrogenase or a reductase, accepting both nicotinamide adenine dinucleotide phosphate (NADP+) and reduced (NADPH) as cofactor, in vivo conditions favor the reductase activity (6 and references therein). In liver microsomes, we previously forwarded direct evidence that NADP+ reduction can be ensured by H6PDH activity, which in turn depends on the G6PT-mediated entry into the microsomal lumen of its substrate glucose-6-phosphate (G6P) (7). Indeed, the 11ßHSD1 and the H6PDH enzyme are both compartmentalized within the liver ER lumen (8, 9), and G6PT allows the entry of G6P, produced in the cytosol, in the ER space (7, 10). Recent data in H6PDH knockout mice (11) also indicate that H6PDH supplies the cofactor NADPH to 11ßHSD1 in liver microsomes. With respect to the cofactor availability for both 11ßHSD1 and H6PDH within the ER lumen, the existence of a separate ER intraluminal pyridine nucleotide pool appears also to be required (12, 13, 14, 15). In fact, the activity of both 11ßHSD1 and H6PDH of liver microsomal vesicles is latent (i.e. well evident upon membrane disruption) because of the limited membrane permeability of the ER/microsomal membrane to pyridine nucleotides (12, 13, 15).

H6PDH is the microsomal counterpart of the cytosolic glucose-6-phosphate dehydrogenase; both enzymes use G6P and NADP+. However, H6PDH is a bifunctional enzyme that catalyzes the first two reactions of the pentose phosphate pathway (10, 16, 17, 18). It contains hexose-6-phosphate dehydrogenase and 6-phosphogluconolactonase activities on a single polypeptide chain.

G6PT is highly represented in gluconeogenic tissues (19, 20 and references therein), and a variant (vG6PT) has been also described in brain, heart, and skeletal muscle (21, 22). On the other hand, cells that do not appear to express (v)G6PTs, still possess a microsomal G6P transport activity, which is eventually less specific, i.e. operative with phosphoesters other that G6P, and insensitive to G6PT inhibitors, such as chlorogenic acid and its derivatives (23 and references therein).

From a pathophysiological point of view, the functioning of 11ßHSD1 enzyme in the adipose tissue should be at least as important as in the liver. It is well established that 11ßHSD1 is expressed and active in adipocytes, and a variety of studies proposed the involvement of 11ßHSD1 in adipocyte proliferation and differentiation (24), idiopathic obesity (4), corticosteroid treatment and the metabolic syndrome (25, 26, 27).

Nonetheless, little information exists on the mechanisms, enzymes, and transporters, which can allow the functioning of the 11ßHSD1 enzyme in the ER compartment of adipocytes. In particular, it has not been explored yet the expression/activity of H6PDH in the ER compartment, G6P transport across the ER membrane, and functional connection of these components with 11ßHSD1. In the present study, we addressed the above-mentioned topics using rat epididymal fat pads. Taken together, the present results clearly indicate the existence of a G6PT-H6PDH-11ßHSD1 system in the adipose cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental methods
Alamethicin, cortisone, cortisol, G6P, D-[14C]glucose-6-phosphate (230 mCi/mmol), 6-phosphoglucosamine, glucose-1-phosphate, NAD+, NADH, NADP+, NADPH, 6-phosphogluconic acid dehydrogenase (type IV from yeast), collagenase (type II), polyethylene glycol (molecular mass 8000 Da), and 4-morpholinepropanesulfonic acid (Mops) were purchased from Sigma Chemical Co. (St. Louis, MO). S3483 was kindly supplied by Aventis Pharma (Frankfurt, Germany). [1,2(n)-3H]cortisone (specific activity 40 Ci/mmol) was from Amersham (Aylesbury, UK). Cellulose acetate/nitrate filter membranes (pore size 0.22 µm) were from Millipore (Bedford, MA). All other reagents and solvents were of analytical grade.

Subcellular fractions and cells
Microsomal fractions were prepared from epididymal fat pads and from livers of overnight fasted male Sprague Dawley rats (180–230 g), as reported elsewhere (Refs. 28 and 7 , respectively). Microsomes were washed and resuspended in KCl/Mops buffer [100 mM KCl, 20 mM NaCl, 1 mM MgCl2, 20 mM Mops (pH 7.2)] and then immediately frozen and kept in liquid nitrogen until use (within 6 months). A cocktail of protease inhibitors (1 mM phenylmethylsulfonylfluoride, 1 µM pepstatin A, 1 µM leupeptin, and 1 µg/ml aprotinin) was added to the homogenization medium as well as in the KCl/Mops buffer. The protein concentration in microsomal suspensions was determined using the method of Lowry et al. (29) with BSA as a standard. Just before each experiment, microsomes were thawed and rapidly washed to further remove possible cytosolic contaminants, particularly the glucose-6-phosphate dehydrogenase enzyme. To this end, microsomes were rapidly pelleted as previously reported in (30). Briefly, microsomal suspensions [0.5–1 mg protein/ml, in the KCl/Mops buffer containing 4.5% polyethylene glycol (wt/vol)] were centrifuged at 1800 x g for 30 sec, and the supernatant was removed immediately. The microsomal pellets were then resuspended in the KCl/Mops buffer, but without polyethylene glycol, and used for the subsequent assays.

Freshly isolated adipocytes were prepared from epididymal fat pads of male Sprague Dawley rats (180–200 g) by the collagenase digestion method, essentially as reported by Rodbell (31). Briefly, isolated adipose tissue was minced and adipocytes were released by digestion at 37 C in a water bath shaker (60 rpm) for 40 min in Krebs-Ringer solution [(pH 7.4), 118 mM NaCl, 4.7 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 24.8 mM NaHCO3, 1.2 mM KH2PO4, 25 mM HEPES] including 5.6 mM glucose, 4% fatty acid-free BSA, and 1 mg/ml collagenase. Dispersed cells were filtered through nylon mesh (200 µm) and adipocytes were washed with fresh Krebs-Ringer solution containing 4% fatty acid-free BSA and centrifuged for 2 min at 400 x g. The floating fat cell layer was also washed twice in Krebs Ringer solution. Isolated adipocytes (0.5 x 106 cells/ml) were equilibrated at 37 C in a humidified atmosphere of 5% CO2-95% air for 1 h in Krebs-Ringer solution with insulin from bovine pancreas 10 µg/ml and in the presence of 5 mM glucose. Cell viability was higher than 82% as evaluated by a propidium iodide exclusion test.

Enzyme assays
H6PDH activity and 11ßHSD1 activity (as cortisol dehydrogenase) were evaluated by measuring NADPH formation upon the addition of 2 mM NADP+ and 10 µM cortisol or 1 mM G6P (unless otherwise stated) to microsomes that had been permeabilized with alamethicin (0.1 mg/mg protein) to allow the free access of the cofactor to the intraluminal enzymes (32). In some experiments, 6-phosphogluconic acid was measured enzymatically with 6-phosphogluconate dehydrogenase on the basis of NADPH formation. NADPH fluorescence was monitored at 350 nm excitation and 460 nm emission wavelengths by using a Cary Eclipse fluorescence spectrophotometer (Varian, Woburn, MA).

Measurement of cortisol production in isolated adipocytes
Freshly isolated adipocytes (0.5 x 106 cells/ml) were incubated in the presence of cortisone, either at 5 or 0.5 µM concentration (for details, see Fig. 6Go). Steroids were extracted from cells as reported elsewhere (33), and the extracted material was separated on a HPLC column (Luna 5µ CN, 150 x 4.6 mm; Phenomenex, Torrance, CA) with a mobile phase consisting of a mixture of heptane (A), methylene chloride/methanol (B) (80:20), and A/B (80:20), at flow rate of 2.0 ml/min. Detector wavelength was 250 nm. Cortisol (and cortisone) HPLC peaks were identified with pure standard compounds. Known amounts of cortisol were also added to parallel incubates but without cortisone addition, and the recovery of the steroid, after the extraction procedure, was always higher than 85%. The HPLC chromatograms, derived from control and experimental incubates, were free of peaks with a retention time equal or close to that of (added) cortisone or (formed) cortisol. Moreover, the use of cortisone as substrate for 11ßHSD1 in rat adipocytes allowed us to exclude any contribution of the endogenous glucocorticoids to the measured ones. Indeed, the rat analogs of the human cortisone/cortisol are dehydrocorticosterone/corticosterone, but cortisone is also a good substrate for the rat 11ßHSD1 enzyme (34). Furthermore, the coinjection of suitable amounts of cortisol (as an internal standard) with experimental samples confirmed the identification of the cortisol peak. In the experiments with 0.5 µM cortisone as substrate, the amounts of cortisol produced were too low to be confidently measured by the UV detector. Therefore, adipocytes were incubated with 0.5 µM cortisone including 3H-cortisone (0.85 x 106 dpm/ml), and the radiolabeled cortisol was recovered after the HPLC separation as above, except that 1 µM cold cortisol was added to each sample at the time of extraction, to visualize the cortisol peak and to allow the collection of the corresponding eluted material for liquid-scintillation counting.


Figure 6
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  		FIG. 6. Effect of the inhibitor of G6PT, S3483, on the production of cortisol by isolated adipocytes treated with cortisone. Adipocytes were isolated from rat epididymal fat pad and incubated at 37 C as detailed in Experimental methods. Adipocytes were preincubated for 30 min in the presence or absence of 200 µM S3483. Cells were then incubated for 2 h in the presence of cortisone at the concentration of 5 µM (A) and 0.5 µM (B). In the experiments with 0.5 µM cortisone, trace amounts of 3H-cortisone (0.85 x 106 dpm/ml) were also present in the incubation mixture. Cortisol production was measured by an HPLC based method by detecting the absorbance at 250 nm (A) or measuring the radioactivity of the eluted steroid (B), as described in Experimental methods. Data are means ± SD of five separate experiments.

 
G6P uptake
In rapid filtration assay, microsomes (1 mg of protein per milliliter) were incubated in the KCl/Mops buffer including 1 mM G6P plus [14C]G6P (25 µCi/ml). To distinguish the intravesicular and the bound radioactivity, 0.1% deoxycholate was added to the incubation mixture (32). The deoxycholate-releasable portion of radioactivity was regarded as intravesicular. At the indicated time intervals, aliquots (0.05 ml) were rapidly withdrawn and filtered through cellulose acetate/nitrate filters (pore size 0.22 µm). Filters were washed with 4 ml of ice-cold HEPES (20 mM) buffer (pH 7.2), containing 250 mM sucrose and 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The radioactivity associated with microsomes retained by filters was measured by liquid scintillation counting (15).

The permeability of the microsomal membranes toward various compounds was also measured by the continuous detection of the osmotically induced changes in size and shape of microsomal vesicles by light scattering (35 and references therein). Briefly, microsomal vesicles (50 µg protein per milliliter) were equilibrated for 1 h in a hypotonic medium [5 mM 1,4-piperazinediethanesulfonic acid potassium salt (pH 7.0), containing 1 mM EGTA] at 22 C. Light scattering of microsomal suspensions was monitored at 550 nm at right angles to the incoming light beam using a Cary Eclipse fluorescence spectrophotometer (Varian), equipped with a temperature-controlled cuvette holder (22 C) and magnetic stirrer. Investigated compounds were added as a small volume (<5% of the total incubation volume) of a concentrated solution.

Western blot analysis
Microsomal proteins were resolved on polyacrylamide gels and blotted on nitrocellulose. Immunoblots were probed with the different antibodies and analyzed by enhanced chemiluminescence. Horseradish peroxidase-conjugated antirabbit Ig-specific secondary antibody (sc-2004) and an enhanced chemiluminescence kit were from Amersham Biosciences (Piscataway, NJ). The H6PDH protein was immunoreacted with a rabbit polyclonal antiserum against the lactonase domain (residues 539–791) of human H6PDH kindly provided by Dr. E. van Schaftingen (Laboratoire de Chimie Physiologique, ICP and UCL, Brussels, Belgium). For G6PT, rabbit polyclonal antibodies against the amino acid residues 123–134 or 415–429 of the human hepatic protein (referred as anti-P2 and anti-P3, respectively; see Ref. 20) were used. The 11ßHSD1 protein was immunorevealed with rabbit polyclonal antibodies recognizing a 14 amino acid sequence common to the human and rat protein (Cayman Chemical, Ann Arbor, MI).

RT-PCR assays
RNA from rat livers and epididymal fat pads was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA), and contaminant DNA was removed by using Turbo DNA-free (Ambion, Austin, TX). RNA concentration and quality were checked by spectrometry and electrophoresis on 1% agarose/formamide gels. Two micrograms of total RNA were reverse transcribed in a final volume of 20 µl, by using the SuperScript first-strand synthesis system (Invitrogen) and Oligo(dT)12–18.

Conventional PCR.
In the case of H6PDH, the primers, corresponding to nucleotides 763–780 and 3053–3070 of predicted rat sequence (GenBank accession no. XM_233688) and covering the coding sequence, were: sense, 5'-CAG GCA CAG GAA TTC AGG-3'; antisense, 5'-AGT CCA TGT ACC AAA CCA-3'. Amplification protocol was: 94 C (2 min); cycles 1–10, 94 C (0.5 min), 56 C (1 min), 68 C (2 min); cycles 11–30, 94 C (0.5 min), 56 C (1 min), 68 C (2 min, with an increase of 5 sec by each cycle); 68 C (7 min). In case of G6PT, the following primers were used: sense, 5'-ATG GCA GCC CAG GGC TAT GGC-3'; antisense, 5'-G TCA CTC AGC CTT CTT GGA CAC-3'; 1.5 U Taq DNA polymerase (Roche, Indianapolis, IN) were used. Amplification protocol was: 94 C (3 min); cycles 1–35, 94 C (1 min), 55 C (1 min), 72 C (1 min); 72 C (7 min). To discriminate between the liver-type G6PT mRNA (without exon 7) and the brain-type splicing variant vG6PT (including exon 7), RT-PCR assay was performed with primer pairs spanning the sequence from exon 4 to exon 8. The primers were: sense, 5'-TGT CCC CTT ACC TGT GGG TGC TCT C-3'; antisense, 5'-CCA AAT ACA GCT CCC AAT ACC AGG A-3'. Amplification protocol was: 94 C (3 min); cycles 1–35, 94 C (0.5 min), 62 C (0.5 min), 72 C (0.5 min); 72 C (7 min). PCR products were separated on a 1 or 1.5% agarose gel. The identity of the RT-PCR products was confirmed by DNA sequencing.

Expression levels of G6PT and H6PDH mRNA were quantified by fluorescent real-time PCR with an Opticon Monitor 4 (MJ Research, Waltham, MA). Analyses were performed in triplicate in a 25 µl reaction mixture. cDNA (1 µl) was amplified with Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) and 50 nM of the sense and antisense primers. The primers were: G6PT sense, 5'-TGT CCC CCT ATC TCT GGG TGC TCT C-3'; G6PT antisense, 5'-AAG GGC CGA CTG CCC TCT CTC CTG G-3'; H6PDH sense, 5'-GGA GCT GAT CTC CAA GCT GGC-3'; H6PDH antisense, 5'-CCC TGA CAG TGC CAG GTG GAA-3'. Amplification protocol was: 95 C (15 min), 45 cycles of 95 C (20 sec), 56.5 C (20 sec), 72 C (20 sec). The PCR amplification efficiency was evaluated by serial (10-fold) dilutions of the rat liver cDNA. Diluted and undiluted samples were then analyzed in duplicate. Amplification efficiency was calculated as reported in (36). The amplification efficiency for G6PT and H6PDH was 90 and 89%, respectively. As a reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used. GAPDH primers were: sense, 5'-TCC ATC TTC CAG GAG CGA GAT C-3'; antisense, 5'-GAG CCC CAG CCT TCT CCA TGG T-3'. Amplification protocol was: 95 C (15 min), 45 cycles of 95 C (20 sec), 56 C (20 sec), 72 C (20 sec). Amplification efficiency was 82%. Every assay was run in triplicate and negative controls (no template, template produced with no reverse transcriptase enzyme) were always included. In the negative controls, no signal was detected in the investigated amplification range (45 cycles).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and activity of microsomal H6PDH
The expression of H6PDH was detected at the level of mRNA and protein in epididymal fat as well as in rat liver as a control. The analysis of RT-PCR products showed a band whose apparent size (~2.4 kb) is consistent with a mRNA coding for a protein of approximately 90 kDa, which is the molecular mass of the H6PDH protein (17, 18), both in adipose tissue and liver (Fig. 1AGo). In additional experiments, the mRNA level was evaluated by real-time RT-PCR. The mRNA level appeared to be lower in adipose tissue than in liver, although the difference was not statistically significant (Table 1Go).


Figure 1
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  		FIG. 1. Expression of H6PDH at mRNA (A) and protein (B) level and 11ßHSD1 protein (C) in rat epididymal fat. A, Total RNA was isolated from adipose and hepatic tissue (as a control), and aliquots (2 µg) were reverse transcribed and subjected to PCR with a primer pair covering the whole H6PDH coding sequence (see Experimental methods for details). A representative 1% agarose gel electrophoresis of the PCR products (revealed with ethidium bromide) of three is shown. The M lane contains a mixture of DNA fragments of different size (in kilobase pairs). B, Microsomes were prepared from adipose and hepatic tissue (as a control), and microsomal proteins were separated by 8% SDS-PAGE. Gels were blotted on a nitrocellulose membrane and the H6PDH protein was immunorevealed with antibodies toward the lactonase domain of the enzyme (see Experimental methods). The amount of microsomal proteins (micrograms) applied was: liver 10, epididymal fat 20 (left panel), and 40 (right panel). On the right side, the size of molecular mass markers (in kilodaltons) is shown. A representative experiment of five is shown. C, Microsomal proteins from adipose and hepatic tissue (as a control) were separated by 12% SDS-PAGE. Gels were blotted on a nitrocellulose membrane, and the 11ßHSD1 protein was immunorevealed as reported in Experimental methods. The amount of microsomal proteins (micrograms) applied was: liver 30, epididymal fat 20 (left panel), and 80 (right panel). On the right side, the size of molecular mass markers (in kilodaltons) is shown. A representative experiment of three is shown.

 

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  		TABLE 1. Quantitative, real-time RT-PCR analysis of H6PDH and G6PT mRNA in rat epididymal fat pad and liver

 
In Western blot experiments, antibodies toward the lactonase domain of human H6PDH revealed a band at an apparent molecular mass of approximately 90 kDa in rat fat and liver microsomes (Fig. 1BGo, arrowhead). Because the antibodies used were against the human protein, we preliminarily verified whether they can also immunoreveal the rat protein. Western blot analyses of human and rat liver microsomes (20 µg of microsomal protein each) revealed a major band at an apparent molecular mass of approximately 90 kDa in both instances and virtually with the same intensity (data not shown). Another immunoreactive band, at a lower apparent molecular mass, was evident particularly in fat microsomes (Fig. 1BGo). However, the possibility for a tissue-specific isoform is unlikely. Indeed, the analysis of RT-PCR products with a primer pair spanning the whole coding sequence (see Fig. 1AGo) appears to rule out the possibility for a splicing variant-derived isoform. Moreover, this immunoreactive band was occasionally present also in liver microsomes, and it was differently represented in different fat microsomal preparations (data not shown), which suggests a proteolytic degradation. We thus concluded that the band at an apparent molecular mass of approximately 90 kDa likely represents the rat H6PDH protein. The expression of the H6PDH protein was approximately 4.8-fold lower in fat microsomes than liver microsomes, as revealed by densitometry measurements of Western blots like those shown in Fig. 1BGo.

Figure 2AGo shows that H6PDH activity was also present in epididymal fat microsomes. In the presence of NADP+, a marked G6P-dependent NADPH formation was evident on permeabilization of the microsomal membrane (addition of alamethicin), whereas a little activity was present before permeabilization. This was expected because NADP+ cannot easily cross the ER membrane (15) and indicates the predicted intralumenal compartmentation of the enzyme. Under the experimental conditions of Fig. 2Go, the H6PDH activity was 10.1 ± 3.3 nmol of NADPH per minute per milligram of microsomal protein (mean ± SD of five separate experiments). In liver microsomes, a higher H6PDH activity was measured: 16.02 ± 2.31 nmol of NADPH per minute per milligram of microsomal protein (mean ± SD of three separate experiments). This was consistent with the higher expression of the H6PDH mRNA and protein in liver, compared with adipose tissue. In additional experiments, the H6PDH activity has been evaluated with substrates other than G6P, namely glucose-1-phosphate and glucosamine-6-phosphate. Virtually no activity was present with glucose-1-phosphate as substrate (even at a 5-fold higher concentration than G6P, 5 instead of 1 mM) both in intact and permeabilized microsomes (Fig. 2BGo). It has been reported that the purified hepatic enzyme oxidizes other hexose-6-phosphates including G6P, although at a lower efficiency (18). With G6P as substrate (5 mM), no activity was present in intact microsomes, whereas a relatively low activity, compared with the experiments with G6P, was observed (Fig. 2CGo). Under the experimental conditions of Fig. 2CGo, NADPH formation (nanomoles per minute per milligram protein, mean ± SD of three separate experiments) was 0.39 ± 0.09.


Figure 2
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  		FIG. 2. Activity of H6PDH in microsomes from rat epididymal fat. Microsomes, prepared as reported in Experimental methods, were incubated at 22 C in the KCl/Mops buffer in a fluorometer cuvette at a protein concentration of 0.1 mg/ml. A, The activity of H6PDH was measured on the basis of NADPH formation (excitation and emission wavelengths at 350 and 460 nm, respectively) after the subsequent addition (arrows) of 2 mM NADP, 1 mM G6P, and alamethicin (Alam, 0.1 mg/mg of microsomal protein). NADPH (5 µM, arrowheads) was subsequently added for calibration. B, Additions were as in A, except that glucose-1-phosphate (G1P, 5 mM, arrow) replaced G6P. C, Additions were as in A, except that glucosamine-6-phosphate (GN6P, 5 mM, arrow) replaced G6P. D, A control sample performed as above but without the addition of substrates. A representative experiment of five (A) or three (B–D) is shown.

 
The ER H6PDH is a dual enzyme possessing both G6P dehydrogenase and 6-phosphogluconolactonase activity (10). Therefore, 6-phosphogluconolactone, derived from G6P dehydrogenation, should be further metabolized to 6-phosphogluconate, and radiolabeled 6-phosphogluconate has been recovered from liver microsomes incubated with radiolabeled G6P (10, 19). As shown in Fig. 3Go, 6-phosphogluconate was indeed formed by adipocyte microsomes. In fact, the addition of 6-phosphogluconate dehydrogenase to microsomal incubates, in which G6P has been already oxidized to 6-phosphogluconolactone (as revealed by the slow increase of NADPH), resulted in a further, rapid increase in NADPH level. The exogenously added 6-phosphogluconate dehydrogenase should oxidize 6-phosphogluconate and produce NADPH because excess NADP+ was present in the system. From different traces as in Fig. 3Go, it can be calculated that 84 ± 5% (means ± SD, n = 3) of the 6-phosphogluconolactone (produced by G6P dehydrogenase activity) was recovered as 6-phosphogluconate (produced by the lactonase activity). This indicates that 6-phosphogluconate accumulates in the incubation system, possibly because downstream enzymes of the pentose pathway are poorly represented in the adipocyte microsomes.


Figure 3
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  		FIG. 3. The H6PDH-dependent dehydrogenation of G6P is associated with the formation of 6-phosphogluconate in microsomes from rat epididymal fat. Microsomes, prepared as reported in Experimental methods were incubated at 22 C in the KCl/Mops buffer in a fluorometer cuvette at a protein concentration of 0.1 mg/ml. Dehydrogenation of G6P was monitored on the basis of NADPH formation (excitation and emission wavelengths at 350 and 460 nm, respectively) after the subsequent addition (arrows) of 2 mM NADP, 10 µM G6P (to traces referred as 1) and alamethicin (Alam, 0.1 mg/mg of microsomal protein). Eighteen minutes after G6P addition, the production of 6-phosphogluconate was measured on the basis of the rapid, further increase in NADPH signal, on the addition of 6-phosphogluconic acid dehydrogenase (6PGDH, 1.5 U/ml, indicated by arrows) to the reaction mixture. Pulse additions of NADPH (2 µM each, arrowheads) were subsequently done for calibration. Control traces (referred as 2), but without the addition of G6P, have been also run. A representative experiment of three is shown.

 
The 11ßHSD1 colocalized with the H6PDH protein in the microsomal fractions employed (detected by Western blotting, Fig. 1CGo). The expression of the 11ßHSD1 protein was approximately 7.7-fold lower in fat microsomes than liver microsomes, as revealed by densitometry measurements of Western blots like those shown in Fig. 1CGo. As in the case of H6PDH activity (see Fig. 2AGo), the 11ßHSD1 activity of fat microsomes appeared to be measurable in permeabilized microsomal vesicles only (data not shown). Again, the latency of the enzyme was likely due to the poor permeability of the microsomal membrane to pyridine nucleotides (12, 13, 15). In permeabilized fat and liver microsomes, incubated in the presence of 2 mM NADP+ and 10 µM cortisol, the dehydrogenase activity of 11ßHSD1 (NADPH formation as nanomoles per minute per milligram protein, mean ± SD of five separate experiments) was 0.29 ± 0.04 and 2.7 ± 0.31, respectively.

Liver-type G6PT is functionally expressed in fat microsomes
The microsomal intralumenal activity of H6PDH requires the transport of the substrate G6P across the microsomal membrane. Therefore, we first investigated the transport of G6P in fat-derived microsomal vesicles by two different methods: a radioisotopic rapid filtration assay and a light-scattering technique (35, 37). As shown in Fig. 4AGo, a time-dependent uptake of radioactivity was present in microsomal vesicles incubated in the presence of radiolabeled G6P, and the uptake was markedly reduced by the prototypic inhibitor of G6PT, S3483 (Refs. 19 and 38 and references therein). At 60 µM S3483, the inhibition of the initial rate (1 min, Fig. 4AGo) was approximately 90%. Higher concentrations of the inhibitor (up to 100 µM) did not result in larger inhibition of G6P uptake.


Figure 4
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  		FIG. 4. Transport of G6P in microsomal vesicles from rat epididymal fat. A, The microsomal uptake of G6P was measured by a rapid filtration technique with [14C]G6P as reported in Experimental methods. The transport of G6P was measured in the absence (empty circles) and presence of 30 µM (dark circles) or 60 µM (dark triangles) of the G6PT inhibitor S3483. Data are means ± SD of three separate experiments. B, The influx of G6P into microsomal vesicles was evaluated by a light-scattering technique as described in Experimental methods. Osmotically induced changes in microsomal vesicle size and shape were initiated by adding a concentrated solution (5% of the total incubation volume) of G6P (arrow) to have a 25 mM final concentration of the compound. In trace 2, microsomes were also treated with the G6PT inhibitor S3483 (20 µM, arrow). Alamethicin (0.1 mg/mg of microsomal protein, arrowheads) was then added to fully permeabilize microsomal vesicles. Traces are representative of four separate experiments.

 
The transport of G6P was also demonstrated by a light-scattering technique. As shown by trace 1 in Fig. 4BGo, the addition of 25 mM G6P to microsomal suspensions resulted in a rapid increase, followed by a relatively slow decrease, in the light-scattering signal: a pattern of variations expected in the case of a permeant compound (35). Upon the addition of G6P, the rapid increase should reflect shrinkage of microsomal vesicles caused by the abrupt increase of extravesicular osmolality, whereas the subsequent decrease reflects the vesicular swelling due to permeation of G6P (35). The pretreatment of microsomes with the G6PT inhibitor, S3483 resulted in a large reduction of the swelling phase, i.e. of the rate of G6P entry into vesicles (trace 2, Fig. 4BGo). The complete permeabilization of microsomal vesicles by the pore-forming peptide, alamethicin (Fig. 4BGo, arrowheads) resulted in maximal swelling [i.e. in maximal decrease in the light-scattering signal (35)]. In control experiments (not shown), we observed that the permeant KCl (35) behaved essentially as G6P, whereas the poorly permeant compound sucrose (35) resulted in a sustained shrinking phase. Moreover, adipose tissue microsomes appeared to be poorly permeable to glucose 1-phosphate (not shown), which is consistent with the selectivity toward substrates of the liver-type G6PT (10, 19, 22, 23, 35, 37).

The pharmacological feature of adipocyte microsomal G6P transport strongly suggests the involvement of the microsomal transporter G6PT (or of its variant vG6PT), which has not been previously characterized in the ER of adipocytes. Therefore, the expression of G6PT/vG6PT was investigated at the level of both mRNA and protein in rat epididymal fat. As shown in Fig. 5AGo, the analysis of RT-PCR products, with a primer pair spanning the coding sequence, showed a band whose apparent size (~1300 bp) is consistent with mRNA(s) coding for the whole G6PT protein(s) (19). Indeed, the mRNA of the isoform vG6PT has 66 bp additional sequence [exon 7 (21)], whereas the mRNA of liver-type G6PT has not, and this size difference cannot be appreciated in the experimental conditions as above. Therefore, we also analyzed RT-PCR products obtained with primers suitable to discriminate the mRNA for liver-type G6PT and its variant vG6PT (see Experimental methods). As shown in Fig. 5BGo, adipose tissue appeared to contain almost exclusively the liver-type mRNA. As shown in Table 1Go, the level of G6PT mRNA, evaluated by real-time RT-PCR, was approximately 100-fold lower in adipose tissue than liver.


Figure 5
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  		FIG. 5. Expression of G6PT at the mRNA (A and B) and protein (C) level in rat epididymal fat. A, Total RNA was isolated from adipose and hepatic tissue (as a control), and aliquots (2 µg) were reverse transcribed and subjected to PCR for the full-length G6PT mRNA, as detailed in Experimental methods. A representative 1% agarose gel electrophoresis of the PCR products (revealed with ethidium bromide) of three is shown. The M lane contains a mixture of DNA fragments of different size (in kilobase pairs). B, Total RNA was isolated from adipose and hepatic tissue as well as rat brain. RT-PCR analysis was run as in A, except that the portion of the mRNA excluding the exon 7 (liver-type G6PT) or including the exon 7 (brain-type vG6PT) was amplified, as detailed in Experimental methods. The M lane contains a mixture of DNA fragments of different size (in kilobase pairs). C, Microsomes were prepared from adipose and hepatic tissue (as a control), as reported in Experimental methods. Microsomal proteins were separated by 12% SDS-PAGE, gels were blotted on a nitrocellulose membrane, and the G6PT protein was immunorevealed with antibodies (Ab to P2 and Ab to P3) toward two different epitopes of the G6PT protein (see Experimental methods). The amount of microsomal proteins (micrograms) applied was: liver 5 (left side) and 1 (left side) and epididymal fat 40. On the right side, the size of molecular mass markers (in kilodaltons) is shown. A representative experiment of four is shown.

 
Not only the mRNA, but also the microsomal G6PT protein was expressed in adipocytes. Western blot analysis of microsomal proteins was performed with two different polyclonal antibodies (anti-P2 and anti-P3) toward two different epitopes of the G6PT protein as reported elsewhere (20). In fat microsomes, a band corresponding to the G6PT protein of liver microsomes was immunorevealed by both the antibodies (Fig. 5CGo). Consistently with the mRNA analyses, the expression level of the G6PT protein was lower in epididymal fat microsomes, compared with liver microsomes (consider the amounts of microsomal proteins analyzed in Fig. 5CGo). Densitometry analysis of Western blots like those shown in Fig. 5CGo revealed that G6PT expression was approximately 110- or 85-fold higher in liver than adipose tissue microsomes, with anti-P2 and anti-P3 antibodies, respectively.

The G6PT inhibitor S3483 inhibits the reduction of cortisone to cortisol in isolated intact adipocytes
Microsomal vesicle obtained from adipose tissue not only contain 11ßHSD1 and H6PDH activities/proteins but also appear to contain the liver-type G6PT activity/protein. If G6PT-mediated G6P entry in the ER compartment is indeed a prerequisite for the reduction of keto- to hydroxyglucocorticoids, the inhibition of G6PT should result in an inhibition of hydroxy-glucocorticoids generation. To verify this hypothesis, isolated intact adipocytes were treated with the G6PT prototypic inhibitor S3483. This compound has been previously shown to inhibit G6PT in vivo: in isolated hepatocytes and the liver of rats (39, 40).

Isolated epididymal adipocytes, after the addition of at 5 or 0.5 µM cortisone, produced cortisol at a rate of 170 or 5.7 pmol/h per 106 cells, respectively (Fig. 6Go). We should note that the reductase activity of 11ßHSD1 was even higher at the higher substrate concentration used (5 µM), which is consistent with the kinetic properties of the enzyme (see4). The treatment with S3483 (200 µM) markedly reduced cortisol production by 75 or 79% at 5 (Fig. 6AGo) or 0.5 µM (Fig. 6BGo) cortisone, respectively. Higher doses of S3483 (up to 500 µM) did not result in higher inhibition of cortisol production. The inhibition of cortisol production by the G6PT inhibitor was not merely due either to a direct effect on 11ßHSD1/H6PDH enzymes or a loss in cell viability. S3483, in fact, did not inhibit (up to 500 µM) either the 11ßHSD1 or H6PDH enzyme (measured as mentioned above; not shown). Moreover, the viability in the adipocyte suspensions ranged from 82 to 87% in any case, prior or after the incubation, with or without cortisone and/or S3483.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present paper demonstrates the presence of the G6PT-H6PDH-11ßHSD1 microsomal system in adipose cells. H6PDH protein and activity as well as the occurrence of G6PT are first shown in the ER compartment of adipocytes.

H6PDH activity was almost completely latent, in not only liver microsomes (7, 15) but also adipose tissue microsomes, in accordance with its proposed topology, i.e. the catalytic site in the lumen of the ER/microsomes. Moreover, the product of G6P dehydrogenation, 6-phosphogluconolactone, was further converted to 6-phosphogluconate, as expected because the ER H6PDH is a dual enzyme possessing both G6P dehydrogenase and 6-phosphogluconolactonase domains (18).

11ßHSD1 activity was also almost completely latent, which is again consistent with the proposed intraluminal topology of the enzyme (8, 41). The latency of the activity of both H6PDH and 11ßHSD1 is much likely due to the negligible permeability of pyridine nucleotides across the microsomal membrane (12, 13, 15). In rat liver microsomes, we previously evidenced a cooperation of H6PDH and 11ßHSD1 based on the colocalization and the use of a common intravesicular pyridine nucleotide pool (14, 15). In microsomes from adipose tissue, we were unable to obtain similar evidence, presumably because of a lower intraluminal concentration of pyridine nucleotides and/or lower enzyme activities, compared with liver microsomes. Nonetheless, in adipocytes, the compartmentalization of H6PDH and 11ßHSD1 activities in the ER lumen and the observed decrease in cortisone reduction on the addition of the G6PT inhibitor to intact cells (Fig. 6Go) strongly suggest that the redox state of an intraluminal pyridine nucleotide pool is the main determinant of the 11ßHSD1-catalyzed cortisone reduction.

With respect to the substrate supply to H6PDH, the transport of G6P (produced in the cytosol) across the ER membrane is needed. Microsomal G6P transport is mediated by the genetically characterized G6PT in the gluconeogenic tissues (19) or its variant vG6PT in brain, heart, and skeletal muscle (21, 22). Moreover, transporters of G6P other than G6PTs may exist (23). In adipose tissue, the presence of the liver-type G6PT was revealed by both RT-PCR and immunoblotting of microsomal proteins. However, the representation of G6PT mRNA and protein was much lower than in the liver. In the liver ER, a high capacity for the G6P transport is required because of the high Michaelis constant of the glucose-6-phosphatase enzyme (19). This is admittedly assured by a high number of transporters per membrane surface unit. In the adipocyte ER, there is no glucose-6-phosphatase activity, and a relatively low number of G6PT proteins per membrane surface unit is logically sufficient for supplying the substrate to a low Michaelis constant enzyme such as H6PDH (42).

In agreement with the expression of G6PT, its specific inhibitor chlorogenic acid derivative S3483 markedly inhibited the rate of G6P transport in adipose tissue microsomes. Importantly, the same drug also markedly inhibited the reduction of cortisone to cortisol by intact adipocytes challenged with cortisone (Fig. 6Go). In both instances the S3483 inhibition was not complete (from 80 to 90%), which might suggest that glucose-6-phosphate transporters, other than G6PT, are also operative. Alternatively, the G6PT-H6PDH couple might be partially circumvented by a direct communication between the redox state of cytosolic and intraluminal pyridine nucleotides. Indeed, a recent paper suggested that also NADPH generation by the (cytosolic) pentose phosphate pathway might influence the rate of 11ßHSD1-mediated cortisone reduction in adipocytes (43).

In the inherited G6PT deficiency, in addition to the lack of liver glucose-6-phosphatase activity, one would expect that local cortisol production is also impaired in the adipose tissue. Direct evidence for this, to our knowledge, is not present in the literature. As apparent indirect evidence, high levels of circulating cortisol have been reported in type 1 glycogen storage disease patients (44, 45). This might be interpreted as adaptation to the defect in local cortisol production [similar to the case of the H6PDH knockout mice (11)]. However, the altered glucose homeostasis per se can also result in increased levels of circulating cortisol (44). We should note that in these studies the subtype of the investigated patients, if the type 1 glycogenosis is due to a defect in G6PT or in the glucose-6-phophatase enzyme, has not been characterized. Further work, eventually in G6PT knockout mice (46), is needed to clarify this point.

Taken together, our results first demonstrate the existence of the G6PT-H6PDH-11ßHSD1 axis in the adipose tissue. The concerted action of the three proteins, not only in the liver but also in the adipose tissue, likely has an important role in the pathogenesis of obesity, metabolic syndrome, and insulin resistance. The presence of the system might favor prereceptorial glucocorticoid activation in postprandial state, especially on a carbohydrate-rich meal in the (visceral) adipose tissue. Consequently, the G6PT-H6PDH-11ßHSD1 axis can promote the glucocorticoid-stimulated proliferation and differentiation of preadipocytes (6). Glucocorticoids reactivated in the visceral adipose tissue may also have an endocrine role by being delivered to the liver (47 and references therein).

The knowledge that not only 11ßHSD1 but also H6PDH and G6PT can be regarded as common pharmacological targets in the liver and in the fat cells may serve a basis for therapeutic considerations. It has been speculated that, besides known inhibitors of the 11ßHSD1 enzyme, putative inhibitors of H6PDH might also be useful in treatment of patients with insulin resistance, obesity, or metabolic syndrome (48). Moreover, a variety of G6PT inhibitors has been proposed and investigated in experimental models (see Ref. 49 and references therein) to reduce hepatic glucose overproduction in type 2 diabetes. It can be speculated that the simultaneous inhibition of liver and adipocyte G6PT should result in an antidiabetic effect via two synergic mechanisms: inhibition of liver G6PT reduces hepatic glucose production, and inhibition of adipocyte G6PT should reduce the flow of cortisol from the visceral fat tissue to the liver.


   Acknowledgments
 
We thank Aventis Pharma Deutschland (Frankfurt, Germany) for providing the S3483 compound.


   Footnotes
 
This work was supported by the Italian Ministry of University and Research (Grant RBAU014PJA), a Hungarian-Italian Bilateral Intergovernmental S&T Cooperation grant, the Hungarian Scientific Research Fund (Grants F37484, T48939, F46740, and T38312) by the Hungarian Academy of Sciences, the Ministry of Health and Welfare, Hungary (ETT 613/03), the Hungarian National Research Initiative (NKFP-1A/056/2004 and KKK-0015/3.0). I.G. is Charge de Recherches of the Belgian Fonds National de la Recherche Scientifique.

Present address for I.G.: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109.

First Published Online February 15, 2007

1 P.M., S.P., and S.S (listed in alphabetical order) contributed equally to this work. Back

Abbreviations: ER, Endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6P, glucose-6-phosphate; G6PT, G6P transporter; H6PDH, hexose-6-phosphate dehydrogenase enzyme; 11ßHSD1, 11ß-hydroxysteroid dehydrogenase type 1; Mops, 4-morpholinepropanesulfonic acid; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, NADP+ reduced; vG6PT, vG6PT variant.

Received November 3, 2006.

Accepted for publication February 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Seckl JR, Walker BR 2004 11ß-Hydroxysteroid dehydrogenase type 1 as a modulator of glucocorticoid action: from metabolism to memory. Trends Endocrinol Metab 15:418–424[CrossRef][Medline]
  2. Andrew R, Westerbacka J, Wahren, J, Yki-Jarvinen H, Walker BR 2005 The contribution of visceral adipose tissue to splanchnic cortisol production in healthy humans. Diabetes 54:1364–1370[Abstract/Free Full Text]
  3. Seckl JR, Morton NM, Chapman KE, Walker BR 2004 Glucocorticoids and 11ß-hydroxysteroid dehydrogenase in adipose tissue. Recent Prog Horm Res 59:359–393[Abstract/Free Full Text]
  4. Draper N, Stewart PM 2005 11ß-Hydroxysteroid dehydrogenase and the pre-receptor regulation of corticosteroid hormone action. J Endocrinol 186:251–271[Abstract/Free Full Text]
  5. Tomlinson JW, Walker EA, Bujalska IJ, Draper N, Lavery GG, Cooper MS, Hewison M, Stewart PM 2004 11ß-Hydroxysteroid dehydrogenase type 1: a tissue specific regulator of glucocorticoid response. Endocr Rev 25:831–866[Abstract/Free Full Text]
  6. Atanasov AG, Nashev LG, Schweizer RA, Frick C, Odermatt A 2004 Hexose-6-phosphate dehydrogenase determines the reaction direction of 11ß-hydroxysteroid dehydrogenase type 1 as an oxidoreductase. FEBS Lett 571:129–133[CrossRef][Medline]
  7. Bánhegyi G, Benedetti A, Fulceri R, Senesi S 2004 Cooperativity between 11ß-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the lumen of the endoplasmic reticulum. J Biol Chem 279:27017–27021[Abstract/Free Full Text]
  8. Ozols J 1995 Lumenal orientation and post-translational modifications of the liver microsomal 11ß-hydroxysteroid dehydrogenase. J Biol Chem 270:2305–2312[Abstract/Free Full Text]
  9. Ozols J 1993 Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase. Proc Natl Acad Sci USA 90:5302–5306[Abstract/Free Full Text]
  10. Gerin I, van Schaftingen E 2002 Evidence for glucose-6-phosphate transport in rat liver microsomes. FEBS Lett 517:257–260[CrossRef][Medline]
  11. Lavery GG, Walker EA, Draper N, Jeyasuria P, Marcos J, Shakleton CH, Parker KL, White PC, Stewart PM 2006 Hexose-6-phosphate dehydrogenase knock-out mice lack 11ß-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation. J Biol Chem 281:6546–6551[Abstract/Free Full Text]
  12. Takahashi T, Hori SH 1978 Intramembraneous localization of rat liver microsomal hexose-6-phosphate dehydrogenase and membrane permeability to its substrates. Biochim Biophys Acta 524:262–276[Medline]
  13. Bublitz C, Lawler CA 1987 The levels of nicotinamide nucleotides in liver microsomes and their possible significance to function of hexose phosphate dehydrogenase. Biochem J 245:263–277[Medline]
  14. Czegle I, Piccirella S, Senesi S, Csala M, Mandl J, Bánhegyi G, Fulceri R, Benedetti A 2005 Cooperativity between 11ß-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase is based on a common pyridine nucleotide pool in the lumen of the endoplasmic reticulum. Mol Cell Endocrinol 248:24–25[CrossRef][Medline]
  15. Piccirella S, Czegle I, Lizak B, Margittai E, Senesi S, Papp E, Csala M, Fulceri R, Csermely P, Mandl J, Benedetti A, Banhegyi G 2006 Uncoupled redox systems in the lumen of the endoplasmic reticulum. Pyridine nucleotides stay reduced in an oxidative environment. J Biol Chem 281:4671–4677[Abstract/Free Full Text]
  16. Mason PJ, Stevens D, Diez A, Knight SW, Scopes DA, Vulliamy TJ 1999 Human hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) encoded at 1p36: coding sequence and expression. Blood Cells Mol Dis 25:30–37[CrossRef][Medline]
  17. Collard F, Collet JF, Gerin I, Veiga-da-Cunha M, van Schaftingen E 1999 Identification of the cDNA encoding human 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway. FEBS Lett 459:223–226[CrossRef][Medline]
  18. Clarke JL, Mason PJ 2003 Murine hexose-6-phosphate dehydrogenase: a bifunctional enzyme with broad substrate specificity and 6-phosphogluconolactonase activity. Arch Biochem Biophys 415:229–234[CrossRef][Medline]
  19. van Schaftingen E, Gerin I 2002 The glucose-6-phosphatase system. Biochem J 362:513–532[CrossRef][Medline]
  20. Senesi S, Marcolongo P, Kardon T, Bucci G, Sukhodub A, Burchell A, Benedetti A, Fulceri R 2005 Immunodetection of the expression of microsomal proteins encoded by the glucose-6-phosphate transporter gene. Biochem J 389:57–62[CrossRef][Medline]
  21. Middleditch C, Clottes E, Burchell A 1998 A different isoform of the transport protein mutated in the glycogen storage disease 1b is expressed in brain. FEBS Lett 433:33–36[CrossRef][Medline]
  22. Shieh JJ, Pan CJ, Mansfield BC, Chou JY 2004 A potential new role for muscle in blood glucose homeostasis. J Biol Chem 279:26215–26219[Abstract/Free Full Text]
  23. Leuzzi R, Fulceri R, Marcolongo P, Bánhegyi G, Zammarchi E, Stafford K, Burchell A, Benedetti A 2001 Glucose-6-phosphate transport in fibroblast microsomes from glycogen storage disease 1b patients: evidence for multiple glucose-6-phosphate transport systems. Biochem J 357:557–562[CrossRef][Medline]
  24. Tomlinson JW, Stewart PM 2002 The functional consequences of 11ß-hydroxysteroid dehydrogenase expression in adipose tissue. Horm Metab Res 34:746–751[CrossRef][Medline]
  25. Masuzaki H, Paterson J, Shinyama H, Morton NM, Mullins JJ, Seckl JR, Flier JS 2001 A transgenic model of visceral obesity and the metabolic syndrome. Science 294:2166–2170[Abstract/Free Full Text]
  26. Putignano P, Pecori Giraldi F, Cavagnini F 2004 Tissue specific dysregulation of 11ß-hydroxysteroid dehydrogenase type 1 and pathogenesis of the metabolic syndrome. J Endocrinol Invest 27:969–974[Medline]
  27. Paterson JM, Morton NM, Fievet C, Kenyon CJ, Holmes MC, Staels B, Seckl JR, Mullins JJ 2004 Metabolic syndrome without obesity: hepatic overexpression of 11ß-hydroxysteroid dehydrogenase type 1 in transgenic mice. Proc Natl Acad Sci USA 101:7088–7093[Abstract/Free Full Text]
  28. Simpson IA, Yver DR, Hissin PJ, Wardzala LJ, Karnieli E, Salans LB, Cushman SW 1983 Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochim Biophys Acta 763:393–407[Medline]
  29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  30. Banhegyi G, Braun L, Marcolongo P, Csala M, Fulceri R, Mandl J, Benedetti A 1996 Evidence for an UDP-glucuronic acid/phenol glucuronide antiport in rat liver microsomal vesicles. Biochem J 315:171–176[Medline]
  31. Rodbell M 1964 Localization of lipoprotein lipase in fat cells of rat adipose tissue. J Biol Chem 239:375–380[Free Full Text]
  32. Fulceri R, Bánhegyi G, Gamberucci A, Giunti R, Mandl J, Benedetti A 1994 Evidence for the intraluminal positioning of p-nitrophenol UDP-glucuronosyltransferase activity in rat liver microsomal vesicles. Arch Biochem Biophys 309:43–46[CrossRef][Medline]
  33. Alberts P, Rönquist-Nii Y, Larsson C, Klingström G, Engblom L, Edling N, Lidell V, Berg I, Edlund PO, Ashkzari M, Sahaf N, Norling S, Berggren V, Bergdahl K, Forsgren M, Abrahmsén L 2005 Effect of high-fat diet on KKAy and ob/ob mouse liver and adipose tissue corticosterone and 11-dehydrocorticosterone concentrations. Horm Metab Res 37:402–407[CrossRef][Medline]
  34. Arampatzis S, Kadereit B, Schuster D, Balazs Z, Schweizer RA, Frey FJ, Langer T, Odermatt A 2005 Comparative enzymology of 11ß-hydroxysteroid dehydrogenase type 1 from six species. J Mol Endocrinol 35:89–101[Abstract/Free Full Text]
  35. Fulceri R, Bellomo G, Gamberucci A, Scott HM, Burchell A, Benedetti A 1992 Permeability of rat liver microsomal membrane to glucose-6-phosphate. Biochem J 286:813–817[Medline]
  36. Pfaffl MW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007
  37. Bánhegyi G, Marcolongo P, Fulceri R, Hinds C, Burchell A, Benedetti A 1997 Demonstration of a metabolically active glucose-6-phosphate pool in the lumen of liver microsomal vesicles. J Biol Chem 272:13584–13590[Abstract/Free Full Text]
  38. Leuzzi R, Bánhegyi G, Kardon T, Marcolongo P, Capecchi PL, Burger HJ, Benedetti A, Fulceri R 2003 Inhibition of microsomal glucose-6-phosphate transport in human neutrophils results in apoptosis: a potential explanation for neutrophil dysfunction in glycogen storage disease 1b. Blood 101:2381–2387[Abstract/Free Full Text]
  39. Herling AW, Burger HJ, Schwab D, Hemmerle H, Below P, Schubert G 1998 Pharmacodynamic profile of a novel inhibitor of the hepatic glucose-6-phosphatase system. Am J Physiol 274:G1087–G1093
  40. van Dijk TH, van der Sluijs FH, Wiegman CH, Baller JF, Gustafson LA, Burger HJ, Herling AW, Kuipers F, Meijer AJ, Reijngoud DJ 2001 Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048. J Biol Chem 276:25727–25735[Abstract/Free Full Text]
  41. Odermatt A, Atanasov AG, Balazs Z, Schweizer RAS, Nashev LG, Schuster D, Langer T 2006 Why is 11ß-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11ß-HSD1. Mol Cell Endocrinol 248:15–23[CrossRef][Medline]
  42. Buetler E, Morrison M 1967 Localization and characteristics of hexose 6-phosphate dehydrogenase (glucose dehydrogenase). J Biol Chem 242:5280–5293
  43. McCormick KL, Wang X, Mick GJ 2006 Evidence that the 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) is regulated by pentose pathway flux. Studies in rat adipocytes and microsomes. J Biol Chem 281:341–347[Abstract/Free Full Text]
  44. Dunger DB, Holder AT, Leonard JV, Okae J, Preece MA 1982 Growth and endocrine changes in the hepatic glycogenoses. Eur J Pediatr 138:226–230[CrossRef][Medline]
  45. Wolfsdorf JI, Ehrlich S, Landy HS, Crigler Jr JF 1992 Optimal daytime feeding regimen to prevent postprandial hypoglycemia in type 1 glycogen storage disease. Am J Clin Nutr 56:587–592[Abstract/Free Full Text]
  46. Chen LY, Shieh JJ, Lin B, Pan CJ, Gao JL, Murphy PM, Roe TF, Moses S, Ward JM, Lee EJ, Westphal H, Mansfield BC, Chou JY 2003 Impaired glucose homeostasis, neutrophil trafficking and function in mice lacking the glucose-6-phosphate transporter. Hum Mol Genet 12:2547–2558[Abstract/Free Full Text]
  47. Andrew R, Walker BR 2006 Tissue production of cortisol by 11ß-hydroxysteroid dehydrogenase type 1 and metabolic disease. Ann NY Acad Sci 1083:165–184[CrossRef][Medline]
  48. Hewitt KN, Walker EA, Stewart PM 2004 Hexose-6-phosphate dehydrogenase and redox control of 11ß-hydroxysteroid dehydrogenase type 1 activity. Endocrinology 146:2539–2543[CrossRef]
  49. Brauer S, Almstetter M, Antuch W, Behnke D, Taube R, Furer P, Hess S 2005 Evolutionary chemistry approach toward finding novel inhibitors of the type 2 diabetes target glucose-6-phosphate translocase. J Comb Chem 7:218–226[CrossRef][Medline]



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