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Endocrinology Vol. 148, No. 10 5072-5080
Copyright © 2007 by The Endocrine Society

Abnormalities of Glucose Homeostasis and the Hypothalamic-Pituitary-Adrenal Axis in Mice Lacking Hexose-6-Phosphate Dehydrogenase

Daniela Rogoff, Jeffrey W. Ryder, Kelli Black, Zheng Yan, Shawn C. Burgess, D. Randy McMillan and Perrin C. White

Departments of Pediatrics (D.R., K.B., D.R.M., P.C.W.) and Physiology (J.W.R.), and Advanced Imaging Research Center (Z.Y., S.C.B.), Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Perrin C. White, M.D., University of Texas Southwestern Medical Center, 5223 Harry Hines Boulevard, Dallas, Texas 75390-9063. E-mail: Perrin.White{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hexose-6-phosphate dehydrogenase (EC 1.1.1.47) catalyzes the conversion of glucose 6-phosphate to 6-phosphogluconolactone within the lumen of the endoplasmic reticulum, thereby generating reduced nicotinamide adenine dinucleotide phosphate. Reduced nicotinamide adenine dinucleotide phosphate is a necessary cofactor for the reductase activity of 11ß-hydroxysteroid dehydrogenase type 1 (EC 1.1.1.146), which converts hormonally inactive cortisone to active cortisol (in rodents, 11-dehydrocorticosterone to corticosterone). Mice with targeted inactivation of hexose-6-phosphate dehydrogenase lack 11ß-hydroxysteroid dehydrogenase type 1 reductase activity, whereas dehydrogenase activity (corticosterone to 11-dehydrocorticosterone) is increased. We now report that both glucose output and glucose use are abnormal in these mice. Mutant mice have fasting hypoglycemia. In mutant primary hepatocytes, glucose output does not increase normally in response to glucagon. Mutant animals have lower hepatic glycogen content when fed and cannot mobilize it normally when fasting. As assessed by RT-PCR, responses of hepatic enzymes to fasting are blunted; enzymes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase) are not appropriately up-regulated, and expression of glucokinase, an enzyme required for glycolysis, is not suppressed. Corticosterone has attenuated effects on expression of these enzymes in cultured mutant primary hepatocytes. Mutant mice have increased sensitivity to insulin, as assessed by homeostatic model assessment values and by increased glucose uptake by the muscle. The hypothalamic-pituitary-adrenal axis is also abnormal. Circulating ACTH, deoxycorticosterone, and corticosterone levels are increased in mutant animals, suggesting decreased negative feedback on the hypothalamic-pituitary-adrenal axis. Comparison with other animal models of adrenal insufficiency suggests that many of the observed abnormalities can be explained by blunted intracellular corticosterone actions, despite elevated circulating levels of this hormone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOSE IS THE major source of energy in most mammalian cells, and its plasma levels depend on a delicate balance between uptake, storage, release, and de novo synthesis. This balance is tightly regulated by several hormones, including insulin, glucocorticoids, glucagon, and epinephrine (1, 2). Glucocorticoids stimulate hepatic gluconeogenesis and antagonize actions of insulin in liver and muscle, thus tending to increase glucose levels (1, 2, 3, 4).

Glucocorticoid actions in target tissues are determined not only by circulating hormone levels, but also by intracellular concentrations, which can be modulated by interconversion between active 11-hydroxylated steroids (cortisol in humans, corticosterone in mice) and their inactive 11-keto forms (cortisone and 11-dehydrocorticosterone, respectively) (5, 6). These reactions are catalyzed by isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) types 1 and 2 (7, 8, 9). The predominant isozyme in glucocorticoid target tissues such as liver, adipose tissue, and muscle is 11ß-HSD1. This enzyme can catalyze both dehydrogenase (11-hydroxyl to 11-keto) and reductase reactions, using nicotinamide adenine dinucleotide phosphate (NADP+) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as cofactors for these reactions, respectively. Owing to the greater availability of NADPH compared with NADP+, the reductase reaction normally predominates in vivo and in most cultured cells (9, 10, 11).

The active site of 11ß-HSD1 is oriented into the lumen of the endoplasmic reticulum (ER). Because the ER is almost impermeable to NADPH, the reductase activity of 11ß-HSD1 depends on generation of NADPH within the ER lumen by hexose-6-phosphate dehydrogenase (H6PD), which converts glucose 6-phosphate (G6P) to 6-phosphogluconolactone (12, 13, 14, 15). This enzyme is distinct from G6P dehydrogenase, which catalyzes the same reaction but exclusively within the cytoplasm.

Several lines of evidence support the dependence of 11ß-HSD1 reductase activity on H6PD. Increasing H6PD levels in cultured cells by transfection, or decreasing expression with short interfering RNA, causes corresponding changes in reductase activity (10). Mice with a targeted inactivation of H6PD have markedly abnormal corticosteroid metabolism, with a shift toward the dehydrogenase direction in isolated liver microsomes, and they predominantly excrete oxidized 11-keto metabolites rather than reduced 11ß-hydroxyl metabolites in the urine (16).

We now report that these mutant mice have abnormal glucose homeostasis with significant fasting hypoglycemia. This is caused by dysregulation of both hepatic glucose output and muscle glucose uptake, which occurs despite elevated circulating levels of the active glucocorticoid, corticosterone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adult H6PD –/– mice on a hybrid C57BL/6J / 129 x 1 background were compared with wild-type (WT) controls, matched by age, sex, genetic background, and generation. Animals were housed in standard conditions on a 12-h light/12-h dark cycle, and given standard chow and water ad libitum. All studies were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center.

Glucose homeostasis
Glucose and insulin measurement.
Plasma samples for glucose and insulin were collected simultaneously by retro-orbital puncture in animals lightly anesthetized by isoflurane inhalation. Samples in fed and 24-h fasting conditions were taken from the same animals with 1 wk between bleeds.

Glucose was measured using a One Touch Ultra Glucometer (LifeScan; Johnson & Johnson, Milpitas, CA). Plasma insulin levels were determined using a commercial kit (Ultrasensitive Mouse Insulin ELISA; ALPCO Diagnostics, Salem, NH) following the manufacturer’s instructions.

As a measurement of insulin sensitivity, we used the homeostasis assessment model (HOMA) index, defined as: fasting glucose (mg/dl) x fasting insulin (mU/ml)/405.

Ex vivo studies.
Fed and 24-h fasted animals were quickly anesthetized by isoflurane inhalation. Livers were removed and pieces immediately homogenized in a phenol/guanidine thiocyanate/chloroform mixture (RNA Stat-60; Tel-Test, Inc., Friendswood, TX) for RNA extraction or snap frozen in liquid nitrogen for glycogen and enzymatic activity assays.

Liver glycogen content was determined as previously described (17). Liver pieces were digested in KOH at 95 C for 30 min and neutralized with HCl. The homogenate was incubated at 50 C for 2 h with sodium acetate (pH 4.8) containing 70U/ml amyloglucosidase (Sigma-Aldrich, St. Louis, MO). After centrifugation the produced glucose was measured colorimetrically using a commercial kit based on an enzymatic method [Glucose (GO) Assay Kit; Sigma-Aldrich].

Glycogen synthase (GS) activity was measured in livers from fed and fasted animals as previously reported, with minor modifications (18). Briefly, liver pieces were homogenized in ice-cold GS homogenization buffer [500 mM Tris-HCl (pH 8.0), 200 mM EDTA, 250 mM KF plus mammalian proteinase inhibitor cocktail (Sigma-Aldrich)]. Tissue homogenates were centrifuged at 12,000 rpm in a refrigerated microcentrifuge for 20 min. GS activity was measured in 50 µl supernatant incubated at 30 C for 15 min with 50 µl buffer containing 10% GS homogenization buffer, 7.5 mg/ml glycogen, and 1.25 µCi/ml UDP-[14C]glucose (400 mCi/mmol) in the presence and absence of 10 mM G6P. Reactions were stopped by adding 75% ethanol, spotted on glass microfiber filters, and washed twice with 75% ethanol. After drying, the filters were placed in scintillation vials, and UDP-[14C]glucose incorporated into glycogen was measured in a scintillation counter. GS activity was expressed as the ratio of the activity measured in the absence and presence of 10 mmol/liter G6P, respectively.

Phosphoenolpyruvate carboxykinase (PEPCK) enzymatic activity was measured in liver homogenates as previously described (19). Results were expressed as nmol of NADH oxidized/min·mg of protein.

Muscle glucose uptake.
We evaluated insulin sensitivity by determining basal and insulin-stimulated glucose transport in extensor digitorum longus (EDL) and soleus muscle, as previously described (20).

Ex vivo gluconeogenesis.
To investigate different glucose metabolic pathways, nuclear magnetic resonance (NMR) spectroscopy using 2H and 13C stable isotopes was performed in isolated livers from 24-h fasted H6PD mutant animals and control mice, as described previously (21).

In vitro studies
Hepatocyte glucose output in response to glucagon stimulation.
Glucose output from hepatocytes was measured in vitro as previously described, with modifications (22). Hepatocytes were obtained from mice fasted for 24 h. Livers were first perfused with Krebs-Henseleit bicarbonate buffer [1 mM MgCl2, 5 mM KCl, 100 mM NaCl, 3 mM CaCl2, 20 mM NaHCO3, 8 mM HEPES, and 0.25% BSA (pH 7.35)] supplemented with 11 mM glucose, 5 mM pyruvate, and 5 mM glutamate, followed by digestion with perfusion buffer, including 100 U/ml collagenase type 1 (Sigma-Aldrich). Cells were initially incubated in Krebs-Henseleit bicarbonate buffer with constant gassing with 95% O2/5% CO2, without BSA, pyruvate, glutamate, or glucose. After 10 min, 100 nM glucagon was added. Incubations were continued for 30 min, and cells were removed by centrifugation. Released glucose was measured in the supernatant and expressed as mg of glucose released after glucagon stimulation – mg of glucose released from unstimulated cells per g of protein.

Modulation of gene expression by glucocorticoids in primary cultured hepatocytes.
Primary hepatocytes were obtained from mice using a collagenase perfusion method (23) better suited to longer term culture than the method cited in the previous section. Briefly, the liver was perfused in situ through the portal vein with Liver Perfusion Medium (Life Technologies, Inc., Invitrogen, Carlsbad, CA) at 37 C, followed by Liver Digest Medium containing collagenase (Life Technologies, Inc., Invitrogen). The liver was removed and mechanically disrupted. Hepatocytes were isolated by centrifugation (50 x g for 3 min) and washed three times with cold Hepatocyte Wash Medium (Life Technologies, Inc.). Cell viability (~80–90%) was determined by trypan blue staining. Cells were plated in collagen-coated 12-well plates at a density of 0.5 x 106 cells per well and allowed to attach for 2 h in HI/WO/BA medium [Waymouth’s MB medium 752/1 (Life Technologies, Inc.), plus 20 mM HEPES, 0.5 mM serine, 0.5 mM alanine, and 0.2% BSA (pH 7.4)]. They were then changed to Williams medium (Life Technologies, Inc.) and incubated for 20–24 h with and without 10 µM corticosterone or 1 µM dexamethasone. After incubation, cells were disrupted in RNA Stat-60 for RNA extraction.

Quantitative RT-PCR.
Total RNA was extracted from liver pieces and cultured hepatocytes and reverse transcribed using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions.

An ABI 7000 Sequence Detection System (Applied Biosystems) was used for real-time mRNA quantitation. Reactions were performed with 100 ng cDNA as template and TaqMan Universal PCR Master mix. For PEPCK, glucose 6-phosphatase (Glu-6-Pase), glucokinase (GCK), and tyrosine aminotransferase (TAT) quantification, premade TaqMan gene expression assays were used (Mm00440636_m1, Mm00839363_m1, Mm00439129_m1, and Mm00455392_m1, respectively; Applied Biosystems). These primers and probe mixtures, which cross exon-exon junctions, only amplify target cDNA. Gene expression levels were normalized for RNA loading using 18S (Predeveloped TaqMan Assay Reagents; Applied Biosystems) as an internal control.

Hypothalamic-pituitary-adrenal (HPA) axis
Corticosterone and ACTH were measured from blood samples collected by retro-orbital puncture at 0800 and 1800 h. Samples were collected from each animal with a 1-wk interval between bleeds. Response to ACTH was determined 1 h after ip injection of ACTH1–24 (Cortrosyn; Organon, West Orange, NJ) 1 U/100 g body weight (1 U = 10 µg).

Corticosterone and ACTH were measured by RIA (ACTH 125I-RIA Kit, Diasorin, Stillwater, MN; Corticosterone 125I RIA Kit for rat and mice, MP Biomedicals, Orangeburg, NY). Deoxycorticosterone (DOC) levels were measured by Quest Diagnostics Nichols Institute (San Juan Capistrano, CA).

Statistical analysis
Data are expressed as mean ± SE. Results of the plasma levels of glucose, insulin, ACTH, corticosterone, and liver glycogen content were log transformed and subjected to multifactorial ANOVA. Preplanned comparisons within each experiment used t tests. Similar results were obtained using nonparametric Mann-Whitney U tests on untransformed data.

{Delta} Ct values (average of Ct for each gene – average of Ct for 18S rRNA) from RT-PCR were analyzed by multifactorial ANOVA with post hoc tests to determine the differences between groups. Differences were considered significant with P values ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glucose homeostasis
Glucose levels.
H6PD mutant animals had lower glucose levels (genotype effect by ANOVA, P = 0.02). This difference was significant in fasted (WT 1.71 ± 0.2 mg/ml; H6PD 0.98 ± 0.19; P = 0.04) but not fed animals (WT 1.93 ± 0.62; H6PD 1.62 ± 0.64) (Fig. 1Go, left panel). WT animals maintained stable glucose levels when fasted, but glucose levels fell significantly in fasted mutant animals (P = 0.002).


Figure 1
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  		FIG. 1. Glucose (left) and insulin (right) levels in fed and 24-h fasted mice (WT: eight males, age 14.5 ± 1 wk; mutant: eight males, age 14.01 ± 1.2 wk). In this and all subsequent figures, error bars represent 1 SE. P values in the text are from log-transformed data (data not shown). Fasted H6PD mutant mice have lower glucose (+, P = 0.04) and insulin levels (+, P = 0.02) when compared with WT mice. WT animals maintain normal glucose levels when fasting, whereas in mutant animals plasma glucose concentration decreases significantly (*, P = 0.002).

 
Insulin levels.
Insulin levels were also lower in mutant compared with WT animals (WT fed 1.93 ± 0.62 µg/liter, fasted 1.71 ± 0.2; H6PD fed 1.2 ± 0.3, fasted 0.17 ± 0.63; genotype effect by ANOVA, P = 0.02). As with glucose, this difference was significant only in fasting animals (P = 0.02) (Fig. 1Go, right panel). When we analyzed insulin sensitivity using the HOMA index, we found that mutant animals (1.1 ± 0.5) were more insulin sensitive than WT mice (5.3 ± 1.3; P = 0.01).

Glucagon stimulated hepatocyte glucose output in vitro.
As a first step in understanding why H6PD mutant mice developed fasting hypoglycemia, we determined the regulation of hepatocyte glucose production by glucagon. In primary culture, hepatocytes from WT animals released glucose into the buffer over 30 min of incubation with glucagon, but mutant hepatocytes did not (WT 0.10 ± 0.059 mg glucose/G protein; H6PD –0.021 ± 0.01; P = 0.006 by Mann-Whitney U test; Fig. 2Go). In fact, the glucose concentration in the medium tended to decrease slightly from baseline after incubation with mutant hepatocytes (this difference was not statistically significant), possibly reflecting glucose consumption by the cells.


Figure 2
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  		FIG. 2. Hepatocyte glucose output in response to glucagon stimulation. Glucose released into the buffer after 30' incubation with glucagon – glucose released by cells with no stimulation was determined in primary hepatocytes from WT (three females, three males, age 14.9 ± 1.4 wk) and mutant mice (three females, two males age 14.7 ± 2.4 wk). Hepatocytes from WT animals responded to glucagon by increasing the glucose released to the medium (+, P = 0.02). This response was not observed in cells from mutant animals (P = 0.3). Glucose output in WT animals was significantly higher than in H6PD mutant mice (*, P = 0.006).

 
Ex vivo gluconeogenesis.
To evaluate whether abnormal regulation of gluconeogenesis contributed to fasting hypoglycemia in mutant mice, we used stable isotope techniques as detected by NMR (21) to determine glucose production from various gluconeogenic pathways in the intact liver. Total glucose production was almost identical between WT and H6PD mutant mice fasted for 24 h. No differences were found in gluconeogenesis from the tricarboxylic acid cycle (pyruvate, lactate) (P = 0.3) or from glycerol (P = 0.4) (Fig. 3Go).


Figure 3
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  		FIG. 3. Glucose production from isolated perfused livers determined from stable isotope NMR data. No differences were observed in total glucose production and in gluconeogenesis from either pyruvate, lactate, or glycerol. GP, Glucose production; GNG, gluconeogenesis; PEP, phosphoenolpyruvate.

 
Liver glycogen content.
To determine whether abnormal glycogen metabolism contributed to fasting hypoglycemia, liver glycogen content was determined in fed and fasted animals. There was a strong genotype/feeding status interaction by ANOVA (P = 0.003) (WT fed 5.2 ± 0.6 mg glucose/g wet weight of tissue, fasted 1.8 ± 0.3; H6PD fed 2.9 ± 0.3, fasted 2.7 ± 0.4); in other words, WT mice responded to fasting with a reduction in glycogen content (P < 0.0001), but mutant mice did not (P = 0.49) (Fig. 4AGo). Fed H6PD mutant animals had lower liver glycogen content than WT (P = 0.003), but there was no difference between genotypes in fasted mice (P = 0.1). We also observed a strong genotype/feeding status interaction (P = 0.0007 by ANOVA) in GS activity (Fig. 4BGo). Fed mutant mice had lower enzymatic activity than fed WT mice (P = 0.02), whereas fasting mutant mice had greater activity than fasting WT mice (P = 0.01). In both genotypes, GS was more active in fasting than in fed mice (P = 0.004 for WT and <0.0001 for mutant animals), as has been previously described (22, 24).


Figure 4
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  		FIG. 4. A, Liver glycogen content in fed and fasted animals (WT: nine females, 14 males, age 14.7 ± 3.1 wk, 12 fed 11 fasted; mutant: eight females, 14 males, age 15.6 ± 2.3 wk, 11 fed, 11 fasted). Mutant animals have lower glycogen content in fed status (*, P = 0.003) and cannot mobilize it adequately during fasting (P = 0.7 mutant mice fed vs. fasted). In contrast, WT mice responded to fasting with a significant reduction of glycogen deposits (***, P < 0.0001). B, GS activity is expressed as percentage of maximum (% max.) activity in the presence of 10 mM G6P. The lower glycogen content in fed mutant mice, compared with wild type, correlates with reduced GS activity (+, P = 0.02). In contrast, fasting H6PD mutant animals have higher GS activity than wild type (*, P = 0.01). Both genotypes respond to fasting by increasing GS activity (*, P = 0.004; ***, P < 0.0001).

 
Changes in expression of hepatic enzymes with fasting.
Although gluconeogenesis did not appear to be grossly reduced in H6PD mutant mice, we could not exclude subtler abnormalities in regulation of hepatic glucose metabolism in response to fasting. Therefore, we used RT-PCR to determine gene expression of liver enzymes involved in glucose production and use, including PEPCK, Glu-6-Pase, GCK, and TAT, in fed and fasted animals. WT animals responded to fasting with a significant induction of PEPCK (P = 0.0002), TAT (P = 0.008), and Glu-6-Pase (P = 0.04), and with suppression of GCK (P = 0.05). These responses were all blunted (P > 0.1) in H6PD mutant animals (Fig. 5AGo). When interactions between genotype and feeding status were explored by ANOVA, there was a significant genotype/feeding status interaction for PEPCK (P = 0.04) and a trend (P = 0.09) toward such an interaction for TAT. There was a significant genotype effect on GCK expression (P = 0.02).


Figure 5
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  		FIG. 5. Gene expression of PEPCK, TAT, Glu-6-Pase, and GCK in livers from fed and 24-h fasted animals (WT: 13 females, 10 males, age 12.4 ± 3.6 wk, 13 fed, 10 fasted; mutant: 12 females, nine males, age 13.2 ± 3.9 wk, 12 fed, nine fasted). A, gene expression is displayed in arbitrary units (1000 x 2{Delta}Ct), where {Delta}Ct = (average Ct of the gene of interest) – (average Ct of 18S rRNA) for each enzyme in fed (black bars) and fasted (shaded bars) animals. WT animals respond to fasting with a significant increment in gene expression of gluconeogenic enzymes and with suppression of the glycolytic enzyme GCK (+, P < 0.05; *, P = 0.01; **, P < 0.001). These responses are blunted in mutant animals (P > 0.1). GCK is markedly increased in mutant mice (+, P = 0.02). B, PEPCK enzymatic activity. As with gene expression, WT animals respond to fasting with an increment in enzyme activity, whereas mutant animals do not (+, P = 0.04). A.U., Arbitrary unit; NADH, reduced nicotinamide adenine dinucleotide.

 
We also investigated PEPCK enzymatic activity in fed and fasted WT and H6PD mutant mice. WT mice increased enzymatic activity significantly when fasted (fed 16.3 ± 1.8 nmol/min·mg protein, fasted 28 ± 3.6; P = 0.04), whereas H6PD mutant animals did not (fed 24.3 ± 2.2, fasted 27.3 ± 0.3; P = 0.1) (Fig. 5BGo). There was a trend toward significance for a genotype/feeding status interaction by ANOVA (P = 0.09).

Changes in gene expression in response to corticosterone in primary hepatocyte cultures.
To see whether attenuated responses to glucocorticoids could explain the observed abnormalities in regulation of hepatic enzymes in response to fasting, we incubated primary hepatocytes with corticosterone in 24-h cultures. In hepatocytes from WT animals, corticosterone induced the expression of PEPCK (P = 0.01) and TAT (P = 0.04) (Fig. 6AGo). These responses were blunted in hepatocytes from mutant animals. GCK was up-regulated significantly in both groups (WT P = 0.0005; H6PD P = 0.004). Genotype effects by ANOVA for expression of PEPCK, TAT, and GCK were P = 0.03, 0.04, and 0.03, respectively. Although there was a trend toward increased expression of Glu-6-Pase with corticosterone, this did not reach significance (P = 0.1). There were no significant differences between WT and mutant hepatocytes in their responses to dexamethasone, a potent synthetic glucocorticoid that does not require activation by 11ß-HSD1 (Fig. 6BGo).


Figure 6
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  		FIG. 6. Modulation by glucocorticoids of gene expression of the same enzymes as in Fig. 5Go, in cultured hepatocytes from WT (four females, four males, age 11.5 ± 2 wk) and mutant (five females, three males, age 10.8 ± 0.6) animals. Corticosterone and dexamethasone comparisons are displayed separately for clarity, but the identical controls are used in both panels. A, After stimulation with corticosterone, WT hepatocytes increased the expression of the gluconeogenic enzymes PEPCK (*, P = 0.01) and TAT (+, P = 0.04), whereas H6PD mutant hepatocytes did not respond adequately (P > 0.3). Contrary to observations in vivo, GCK was up-regulated both in WT and mutant hepatocytes (**, P = 0.0005; *, P = 0.004). B, There were no differences in responses to dexamethasone between the groups (*, P = 0.01). A.U., Arbitary units.

 
Glucose uptake in muscle ex vivo.
To see whether increased glucose use or insulin sensitivity might contribute to fasting hypoglycemia in H6PD mutant mice, we examined these parameters in muscle explants. Mutant animals had significantly greater glucose incorporation in their EDL muscles than WT mice (genotype effect by ANOVA, P = 0.0002) under both basal and insulin-stimulated conditions (P = 0.02 and 0.004, respectively). There were no significant differences in glucose uptake between WT and mutant soleus muscles (Fig. 7Go).


Figure 7
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  		FIG. 7. Glucose incorporation in the EDL and soleus muscles from male WT (n = 6; age: 15.3 ± 3.3 wk) and mutant (n = 6; age: 18.2 ± 5.3 wk) animals, with and without insulin stimulation. Mutant animals have greater glucose incorporation in EDL muscles under both basal (+, P = 0.02) and insulin-stimulated conditions (*, P = 0.004), confirming higher insulin sensitivity in this model. Other P values: EDL basal vs. insulin, WT, 0.0004 (**), mutant, 0.01 (*); solens basal vs. insulin, WT, 0.0008 (**), mutant, 0.01 (*).

 
HPA axis
It was important to determine the extent to which the known abnormalities in corticosteroid metabolism in mutant mice (16) affected the HPA axis, given that adrenal insufficiency can cause hypoglycemia. Mutant mice had higher ACTH levels than WT (P = 0.01 by ANOVA). When analyzed separately, the difference was significant at diurnal nadir (0800 h; P = 0.006), but not at diurnal peak (1800 h; P = 0.23) (Fig. 8Go).


Figure 8
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  		FIG. 8. Top, Corticosterone at 1800 and 0800 h (WT: 11 females, two males, age 15.4 ± 3.8 wk; mutant: eight females, four males, age 13.1 ± 4 wk), and after ACTH stimulation tests in eight each of the WT and mutant animals. Bottom, ACTH and DOC levels in WT and mutant animals. H6PD animals have higher corticosterone levels both at diurnal nadir (WT 71 ± 19 ng/ml, H6PD 269 ± 62; *, P = 0.004) and at diurnal peak (WT 404 ± 76, H6PD 670 ± 56; +, P = 0.02). ACTH was significantly increased in H6PD mutant mice at 0800 h (WT 291 ± 39 pg/ml, H6PD 615 ± 145; *, P = 0.006). DOC was higher in mutant animals (WT 93 ± 23 ng/dl, H6PD 227 ± 25; +, P = 0.04).

 
Paralleling the differences in ACTH, mutant mice had higher corticosterone levels than WT (genotype effect by ANOVA, P = 0.002) at both diurnal nadir and diurnal peak (P = 0.004 at 0800 h and 0.02 at 1800 h). Corticosterone levels after ACTH stimulation did not differ between the groups (P = 0.17) (Fig. 8Go).

We measured DOC levels at 0800 h in 23 WT and 23 mutant animals but pooled the WT samples into four sets and mutant samples into three sets to meet sample volume requirements. DOC levels were higher in mutant animals compared with WT animals (P = 0.04) (Fig. 8Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that the directionality of the reactions catalyzed by 11ß-HSD1 depends on the availability of NADPH provided by H6PD, as evidenced by abnormal glucocorticoid metabolism in mice with a targeted mutation of H6PD. In this report we further characterized the effects of targeted disruption of the H6PD gene on glucose metabolism and the HPA axis.

Glucose homeostasis
Glucose levels depend on the balance between glucose output and glucose use. In fasting, the maintenance of normal glucose levels depends on glycogen breakdown and on de novo synthesis from other precursors, such amino acids, lactate, and glycerol (1, 2). H6PD mutant mice have altered glucose metabolism, with lower fasting glucose levels.

Glucose output is stimulated by glucagon, catecholamines, and glucocorticoids, and inhibited by insulin (1, 3, 25). In H6PD mutant mice, effective intracellular corticosterone levels are apparently low despite high circulating levels. Besides their direct actions, glucocorticoids exert a permissive effect on activation of gluconeogenic and glycogenolytic pathways by glucagon and epinephrine (26). Glucocorticoids also antagonize insulin action at the transcriptional level, thus inhibiting glucose transport and reducing cellular glucose uptake (3, 4, 27, 28, 29). Therefore, attenuated glucocorticoid actions are associated with impaired glucagon stimulatory effects and increased insulin sensitivity (30, 31). These are indeed observed in H6PD mutant mice, evidenced by decreased glucagon-stimulated glucose output by cultured hepatocytes, by reduced insulin resistance, and by increased insulin-stimulated glucose uptake in muscle explants.

Our experiments allowed us to assess abnormalities in both glycogen use and gluconeogenesis. Glycogen metabolism is clearly impaired in H6PD mutant animals. Fed H6PD mutant mice have reduced hepatic glycogen content, which is consistent with the reduced hepatic GS activity in these animals when fed. Similar results are observed in adrenalectomized mice and rats (32, 33, 34, 35), in which decreased hepatic glycogen synthesis in response to glucose is thought to result from impaired inactivation of phosphorylase and decreased glycogen-associated synthase phosphatase.

WT animals respond to fasting with a significant reduction in glycogen content, whereas fed and fasted H6PD mutant animals have similar hepatic glycogen content. Thus, they cannot mobilize glycogen adequately when fasting. This aspect of glycogen metabolism is not identical to that seen in adrenalectomized mice, suggesting that it is not solely the result of attenuated glucocorticoid effects. In fasted adrenalectomized mice, even though glycogenolysis is impaired (22, 32, 33, 36), there is an even more significant reduction in GS activity, leading to a net decrease in glycogen content compared with intact mice (24, 37, 38, 39). In contrast, fasted H6PD mutant mice have increased GS activity and higher hepatic glycogen content compared with WT mice. The reason for this difference is not immediately apparent. The higher expression of GCK in H6PD mutant mice may increase the cytoplasmic G6P concentration, which in turn increases GS activity both directly and indirectly (40). Alternatively, Glu-6-Pase is sensitive to lipid peroxidation (41, 42), so perhaps altered redox potential inside the ER (due to an abnormal NADPH to /NADP+ ratio) affects Glu-6-Pase activity, leading to higher glycogen deposition when animals are fasting. Further experiments are needed to test these hypotheses.

It is noteworthy that GS activity is higher in fasting than fed mice in both WT and H6PD mutant animals, as has been reported previously (22, 24, 43, 44). Higher GS activity prevents hepatocytes from completely depleting glycogen stores during fasting, and also enables the liver to rapidly reaccumulate glycogen during refeeding (43, 44).

In contrast to their markedly abnormal regulation of glycogen metabolism, direct measurement of gluconeogenesis in intact livers of fasted mutant animals using stable isotope techniques reveals normal levels of gluconeogenesis from both pyruvate and glycerol. However, it might be expected that gluconeogenesis would be increased to defend against hypoglycemia in an animal that is unable to mobilize glycogen well, suggesting that regulation of gluconeogenesis is somewhat abnormal as well in H6PD mutant mice. It should be noted that under the conditions used for the gluconeogenesis experiments in intact livers, glycogenolysis represents a negligible portion of glucose output (Fig. 3Go), and this may explain why glucose output from mutant livers was unaffected in these experiments, despite the abnormalities in glycogen use previously discussed.

WT animals respond to fasting by inducing enzymes involved in gluconeogenesis and by down-regulating GCK, a key enzyme for glucose use. Both responses were indeed blunted in mutant animals. Our in vitro experiments implicate attenuated corticosterone effects as a primary cause of impaired regulation of gluconeogenesis. WT hepatocytes responded to corticosterone by increasing expression of enzymes involved in gluconeogenesis. In contrast, H6PD mutant hepatocytes did not increase their expression of gluconeogenic enzymes in response to corticosterone, but their responses to dexamethasone were similar to those observed in WT hepatocytes. This presumably occurs because H6PD deficiency reduces intraluminal NADPH concentrations, causing 11ß-HSD1 to function as a dehydrogenase in the liver, thus inactivating corticosterone, which, therefore, cannot reach glucocorticoid receptors in adequate concentrations. Dexamethasone, a synthetic glucocorticoid, is not a substrate for 11ß-HSD1 and is not inactivated (45).

It should be noted that in cultured WT hepatocytes (and to a lesser degree. in mutant hepatocytes), GCK expression is increased by glucocorticoids, whereas its expression in vivo is normally suppressed by fasting. Similar results have been previously reported (46), suggesting that glucocorticoids are not the predominant regulators of expression of this enzyme in vivo. Whereas glucocorticoids exert a permissive action on GCK induction in vivo (47), expression of this enzyme is regulated mainly by the balance between stimulation by insulin and repression by glucagon via cAMP (48).

In addition to abnormalities in glucose output, glucose use is increased in H6PD mutant mice, as demonstrated by higher glucose uptake in muscle, both basally and after insulin stimulation. Changes in glucose uptake were observed only in the EDL muscle, which is normally rich in relatively insulin-insensitive, type IIB (fast-twitch, glycolytic) muscle fibers; no changes were seen in the soleus muscle, which is enriched in more insulin-sensitive, type I (slow-twitch) fibers (20, 30, 31). These abnormalities are corroborated by the increased insulin sensitivity observed in vivo. The mechanisms underlying increased glucose uptake in mutant EDL muscles remain to be elucidated.

HPA axis
Corticosterone levels were higher in H6PD mutant animals at diurnal peak and particularly at diurnal nadir compared with WT animals, but responses to a pharmacological dose of ACTH were unchanged. These results are similar to those seen in mice with a mutation in 11ß-HSD1 itself (5). Because 11ß-HSD1 is expressed in the hypothalamus and pituitary (49), it is plausible that deficient 11ß-HSD1 reductase activity decreases intracellular glucocorticoid concentrations within the hypothalamus and/or pituitary, leading to relative resistance to feedback inhibition by corticosterone, with increased ACTH secretion, and a consequent increment in corticosterone secretion. This activation of the HPA axis is supported by the increased levels of ACTH and DOC found in mutant animals compared with WT. Further studies are needed to explore the effect of the excess of DOC, a weak mineralocorticoid, in the cardiovascular system of these mice.

It was previously reported (16) that the H6PD mutant mouse had decreased circulating corticosterone levels at diurnal nadir relative to WT; this is not supported by the current data. Further inspection of the previous data reveals unusually high corticosterone levels in WT mice. In the previous experiment, blood was collected by cardiac puncture rather than retro-orbital sinus bleeding of anesthetized mice [the latter is known to be a relatively low-stress method of blood collection in mice (50)], suggesting that technical differences in blood collection technique may have excessively stressed the animals and confounded the results.

In summary, targeted inactivation of the H6PD gene in mice causes abnormalities of glucose homeostasis and regulation of the HPA axis. The fasting hypoglycemia seen in H6PD mutant mice apparently results from abnormal hepatic glycogen storage and use, thus affecting hepatocyte glucose output, combined with increased glucose use and increased insulin sensitivity in muscle. Gluconeogenesis is abnormally regulated and is unable to defend fasting animals from hypoglycemia; this abnormal regulation is caused by abnormal corticosterone metabolism within hepatocytes (Fig. 9Go).


Figure 9
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  		FIG. 9. Schematic of hypothesized alterations in glucose homeostasis in fasted H6PD mutant mice. ->, stimulation; {dashv}, inhibition. Red arrows, words, or boxes denote processes, substances, or levels of enzymatic expression, respectively, which are reduced in mutant mice, whereas green arrows, words, or boxes indicate factors that are increased. Blue circled numbers refer to this figure legend. H6PD is inactivated by targeted mutation (1 ). This decreases conversion of NADP+ to NADPH within the lumen of the ER (2 ). The altered ratio of NADP+ to NADPH changes the equilibrium of the interconversion of 11-dehydrocorticosterone (abbreviated A) and corticosterone (B) catalyzed by 11ß-HSD1(3 ), such that intracellular levels of corticosterone are decreased. Likewise, the altered NADP+ to NADPH ratio might change the intraluminal redox state, indirectly decreasing Glu-6-Pase activity (4 ). Decreased levels of corticosterone are available to occupy glucocorticoid receptors (GRs) (5 ). Although most glucocorticoid effects are mediated through nuclear glucocorticoid receptors at the level of gene expression (the nucleus in the schematic is denoted by the circle with the double helix), for simplicity the schematic summarizes the effects of glucocorticoids on cytoplasmic enzyme activity without reference to the nucleus, and the glucocorticoid receptor is otherwise not displayed. Decreased intracellular corticosterone levels result in augmented insulin effects, due to less antagonism by glucocorticoids, and attenuated glucagon actions, due to lack of the permissive effects of glucocorticoids. These changes affect glucose metabolism in several ways. GCK expression is not appropriately repressed during fasting (6 ), thus glucose use is not repressed, and G6P (Glu-6-P) levels are inappropriately high. GS activity is increased (7 ) both by the elevated levels of the substrate, G6P, and by the disturbed balance of glucagon and insulin actions, as previously described. Glucose production from glycogenolysis (GLy) in response to fasting is decreased (8 ). Expression of gluconeogenic (GNG) enzymes in response to fasting is blunted in H6PD mutant mice, both as a direct consequence of glucocorticoid deficiency, and also due to augmented suppression by insulin and attenuated stimulation by glucagon (9 ). The net results are decreased hepatocyte glucose output in response to glucagon and increased sensitivity to insulin in suppressing glucose output. In addition, there is increased glucose uptake by muscle both basally and in response to insulin (10 ). Higher glucose uptake combined with decreased hepatic glucose output results in fasting hypoglycemia. 6P-GLN, 6-Phosphogluconolactone.

 


   Acknowledgments
 
We thank Heather Powell, Charles Storey, and Angela Milde for technical assistance, and Keith Parker for providing hexose-6-phosphate dehydrogenase mutant mice and critically reading the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grants R01DK68101 (to P.C.W.) and U24DK076169.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 26, 2007

Abbreviations: DOC, Deoxycorticosterone; EDL, extensor digitorum longus; ER, endoplasmic reticulum; GCK, glucokinase; Glu-6-Pase, glucose 6-phosphatase; G6P, glucose-6-phosphate; GS, glycogen synthase; H6PD, hexose-6-phosphate dehydrogenase; HOMA, homeostasis assessment model; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; HPA, hypothalamic-pituitary-adrenal; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NMR, nuclear magnetic resonance; PEPCK, phosphoenolpyruvate carboxykinase; TAT, tyrosine aminotransferase; WT, wild type.

Received May 4, 2007.

Accepted for publication July 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pilkis SJ, Granner DK 1992 Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Physiol 54:885–909[CrossRef][Medline]
  2. Nordlie RC, Foster JD, Lange AJ 1999 Regulation of glucose production by the liver. Annu Rev Nutr 19:379–406[CrossRef][Medline]
  3. Lowenberg M, Tuynman J, Scheffer M, Verhaar A, Vermeulen L, van Deventer S, Hommes D, Peppelenbosch M 2006 Kinome analysis reveals nongenomic glucocorticoid receptor-dependent inhibition of insulin signaling. Endocrinology 147:3555–3562[Abstract/Free Full Text]
  4. Ruzzin J, Wagman AS, Jensen J 2005 Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor. Diabetologia 48:2119–2130[CrossRef][Medline]
  5. Harris HJ, Kotelevtsev Y, Mullins JJ, Seckl JR, Holmes MC 2001 Intracellular regeneration of glucocorticoids by 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 plays a key role in regulation of the hypothalamic-pituitary-adrenal axis: analysis of 11ß-HSD-1-deficient mice. Endocrinology 142:114–120[Abstract/Free Full Text]
  6. Seckl JR, Walker BR 2001 Minireview: 11ß-hydroxysteroid dehydrogenase type 1–a tissue-specific amplifier of glucocorticoid action. Endocrinology 142:1371–1376[Abstract/Free Full Text]
  7. Walker EA, Clark AM, Hewison M, Ride JP, Stewart PM 2001 Functional expression, characterization, and purification of the catalytic domain of human 11-ß-hydroxysteroid dehydrogenase type 1. J Biol Chem 276:21343–21350[Abstract/Free Full Text]
  8. White PC, Mune T, Agarwal AK 1997 11ß-Hydroxysteroid dehydrogenase and the syndrome of apparent mineralocorticoid excess. Endocr Rev 18:135–156[Abstract/Free Full Text]
  9. 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]
  10. Bujalska IJ, Draper N, Michailidou Z, Tomlinson JW, White PC, Chapman KE, Walker EA, Stewart PM 2005 Hexose-6-phosphate dehydrogenase confers oxo-reductase activity upon 11 ß-hydroxysteroid dehydrogenase type 1. J Mol Endocrinol 34:675–684[Abstract/Free Full Text]
  11. Ricketts ML, Shoesmith KJ, Hewison M, Strain A, Eggo MC, Stewart PM 1998 Regulation of 11ß-hydroxysteroid dehydrogenase type 1 in primary cultures of rat and human hepatocytes. J Endocrinol 156:159–168[Abstract]
  12. Odermatt A, Atanasov AG, Balazs Z, Schweizer RA, 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]
  13. Hewitt KN, Walker EA, Stewart PM 2005 Minireview: hexose-6-phosphate dehydrogenase and redox control of 11ß-hydroxysteroid dehydrogenase type 1 activity. Endocrinology 146:2539–2543[CrossRef][Medline]
  14. 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 oxoreductase. FEBS Lett 571:129–133[CrossRef][Medline]
  15. 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]
  16. Lavery GG, Walker EA, Draper N, Jeyasuria P, Marcos J, Shackleton 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]
  17. Inoue Y, Inoue J, Lambert G, Yim SH, Gonzalez FJ 2004 Disruption of hepatic C/EBPalpha results in impaired glucose tolerance and age-dependent hepatosteatosis. J Biol Chem 279:44740–44748[Abstract/Free Full Text]
  18. Rao R, Hao CM, Redha R, Wasserman DH, McGuinness OP, Breyer MD 2007 Glycogen synthase kinase 3 inhibition improves insulin-stimulated glucose metabolism but not hypertension in high-fat-fed C57BL/6J mice. Diabetologia 50:452–460[CrossRef][Medline]
  19. Petrescu I, Bojan O, Saied M, Barzu O, Schmidt F, Kuhnle HF 1979 Determination of phosphoenolpyruvate carboxykinase activity with deoxyguanosine 5'-diphosphate as nucleotide substrate. Anal Biochem 96:279–281[CrossRef][Medline]
  20. Ryder JW, Long YC, Nilsson E, Mahlapuu M, Zierath JR 2005 Effects of calcineurin activation on insulin-, AICAR- and contraction-induced glucose transport in skeletal muscle. J Physiol 567:379–386[Abstract/Free Full Text]
  21. Burgess SC, Hausler N, Merritt M, Jeffrey FM, Storey C, Milde A, Koshy S, Lindner J, Magnuson MA, Malloy CR, Sherry AD 2004 Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase. J Biol Chem 279:48941–48949[Abstract/Free Full Text]
  22. Hutson NJ, Brumley FT, Assimacopoulos FD, Harper SC, Exton JH 1976 Studies on the {alpha}-adrenergic activation of hepatic glucose output. I. Studies on the {alpha}-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells. J Biol Chem 251:5200–5208[Abstract/Free Full Text]
  23. Nakatani T, Tsuboyama-Kasaoka N, Takahashi M, Miura S, Ezaki O 2002 Mechanism for peroxisome proliferator-activated receptor-{alpha} activator-induced up-regulation of UCP2 mRNA in rodent hepatocytes. J Biol Chem 277:9562–9569[Abstract/Free Full Text]
  24. Margolis RN, Curnow RT 1983 The role of insulin and glucocorticoids in the regulation of hepatic glycogen metabolism: effect of fasting, refeeding, and adrenalectomy. Endocrinology 113:2113–2119[Abstract/Free Full Text]
  25. Bollen M, Keppens S, Stalmans W 1998 Specific features of glycogen metabolism in the liver. Biochem J 336(Pt 1):19–31
  26. Runge D, Schmidt H, Christ B, Jungermann K 1991 Mechanism of the permissive action of dexamethasone on the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes. Eur J Biochem 198:641–649[Medline]
  27. Goodman PA, Medina-Martinez O, Fernandez-Mejia C 1996 Identification of the human insulin negative regulatory element as a negative glucocorticoid response element. Mol Cell Endocrinol 120:139–146[CrossRef][Medline]
  28. Saad MJ, Folli F, Kahn JA, Kahn CR 1993 Modulation of insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats. J Clin Invest 92:2065–2072[Medline]
  29. Caro JF, Amatruda JM 1982 Glucocorticoid-induced insulin resistance: the importance of postbinding events in the regulation of insulin binding, action, and degradation in freshly isolated and primary cultures of rat hepatocytes. J Clin Invest 69:866–875[Medline]
  30. Henriksen EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, Holloszy JO 1990 Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol 259(4 Pt 1):E593–E598
  31. Daugaard JR, Richter EA 2001 Relationship between muscle fibre composition, glucose transporter protein 4 and exercise training: possible consequences in non-insulin-dependent diabetes mellitus. Acta Physiol Scand 171:267–276[CrossRef][Medline]
  32. Exton JH, Friedmann N, Wong EH, Brineaux JP, Corbin JD, Park CR 1972 Interaction of glucocorticoids with glucagon and epinephrine in the control of gluconeogenesis and glycogenolysis in liver and of lipolysis in adipose tissue. J Biol Chem 247:3579–3588[Abstract/Free Full Text]
  33. Chan TM, Blackmore PF, Steiner KE, Exton JH 1979 Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Reciprocal change in alpha- and ß-adrenergic activation of hepatic glycogen phosphorylase and calcium mobilization in adrenalectomized rats. J Biol Chem 254:2428–2433[Free Full Text]
  34. Allende G, Chavira R, Quintanar-Stephano A 2004 The role of corticosterone in the metabolic recovery after intrasplenic adrenal autotransplantation in rats. Endocr J 51:325–332[CrossRef][Medline]
  35. Reese JB, Mayer SE, Brunton LL 1984 Effects of adrenalectomy on activation of glycogen phosphorylase in rat myocardium. Am J Physiol 247(3 Pt 1):E389–E397
  36. Chan TM, Steiner KE, Exton JH 1979 Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats. J Biol Chem 254:11374–11378[Abstract/Free Full Text]
  37. Doherty MJ, Cadefau J, Stalmans W, Bollen M, Cohen PT 1998 Loss of the hepatic glycogen-binding subunit (GL) of protein phosphatase 1 underlies deficient glycogen synthesis in insulin-dependent diabetic rats and in adrenalectomized starved rats. Biochem J 333(Pt 2):253–257
  38. Exton JH, Miller TB, Harper SC, Park CR 1976 Carbohydrate metabolism in perfused livers of adrenalectomized and steroid-replaced rats. Am J Physiol 230:163–170[Abstract/Free Full Text]
  39. Bollen M, Gevers G, Stalmans W 1983 The activity of glycogen synthase phosphatase limits hepatic glycogen deposition in the adrenalectomized starved rat. Biochem J 214:539–545[Medline]
  40. Harndahl L, Schmoll D, Herling AW, Agius L 2006 The role of glucose 6-phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism. FEBS J 273:336–346[CrossRef][Medline]
  41. Gonzalez Padron A, de Toranzo EG, Castro JA 1996 Depression of liver microsomal glucose 6-phosphatase activity in carbon tetrachloride-poisoned rats. Potential synergistic effects of lipid peroxidation and of covalent binding of haloalkane-derived free radicals to cellular components in the process. Free Radic Biol Med 21:81–87[CrossRef][Medline]
  42. Jung UJ, Lee MK, Park YB, Jeon SM, Choi MS 2006 Antihyperglycemic and antioxidant properties of caffeic acid in db/db mice. J Pharmacol Exp Ther 318:476–483[Abstract/Free Full Text]
  43. Chen C, Williams PF, Cooney GJ, Caterson ID, Turtle JR 1992 The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice. Horm Metab Res 24:161–166[Medline]
  44. Hue L, Bontemps F, Hers H 1975 The effects of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations. Biochem J 152:105–114[Medline]
  45. Best R, Nelson SM, Walker BR 1997 Dexamethasone and 11-dehydrodexamethasone as tools to investigate the isozymes of 11ß-hydroxysteroid dehydrogenase in vitro and in vivo. J Endocrinol 153:41–48[Abstract/Free Full Text]
  46. Spence JT, Pitot HC 1979 Hormonal regulation of glucokinase in primary cultures of adult rat hepatocytes. J Biol Chem 254:12331–12336[Free Full Text]
  47. Sibrowski W, Seitz HJ 1980 Hepatic glucokinase turnover in intact and adrenalectomized rats in vivo. Eur J Biochem 113:121–129[Medline]
  48. Iynedjian PB, Jotterand D, Nouspikel T, Asfari M, Pilot PR 1989 Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system. J Biol Chem 264:21824–21829[Abstract/Free Full Text]
  49. Lakshmi V, Sakai RR, McEwen BS, Monder C 1991 Regional distribution of 11 ß-hydroxysteroid dehydrogenase in rat brain. Endocrinology 128:1741–1748[Abstract/Free Full Text]
  50. Grouzmann E, Cavadas C, Grand D, Moratel M, Aubert JF, Brunner HR, Mazzolai L 2003 Blood sampling methodology is crucial for precise measurement of plasma catecholamines concentrations in mice. Pflugers Arch 447:254–258[CrossRef][Medline]



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