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Departments of Medicine (G.G.L., K.N.H., M.S., E.A.W., P.M.S.) and Physiology (D.H., S.M.B.), Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom
Address all correspondence and requests for reprints to: Gareth G. Lavery, Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom. E-mail: g.g.lavery{at}bham.ac.uk.
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Introduction |
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Target tissue exposure to GCs is determined by both circulating levels (regulated by the hypothalamic-pituitary-adrenal axis) and intracellular activation of GCs from inactive precursors. The local reactivation of GC hormone is modulated in a tissue-specific fashion by the expression and activity of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), a bidirectional enzyme catalyzing both oxidation and oxoreduction of GCs. 11ß-HSD1 is highly expressed in liver and adipose tissue, and in vivo acts predominantly as a nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-dependent oxoreductase converting hormonally inactive cortisone [or 11-dehydrocorticosterone (11-DHC) in rodents] to their active derivatives, cortisol (or corticosterone in rodents) (3).
An emerging and critical factor in regulating 11ß-HSD1 reductase activity is the provision of a high concentration of NADPH by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the endoplasmic reticulum (ER). H6PDH is a bifunctional enzyme that catalyzes the first two steps of an endoluminal pentose phosphate pathway, exploiting glucose-6-phosphate (G6P) as substrate but is distinct from its cytosolic homologue glucose-6-phosphate dehydrogenase in being localized exclusively to the ER lumen (4). Moreover, NADPH production by H6PDH may have an important function in maintaining reductive cofactors in the oxidizing environment of the ER (5, 6). In vitro studies have previously demonstrated close cooperativity between 11ß-HSD1 and H6PDH (7, 8, 9). We have shown that in H6PDH knockout (KO) mice the set point of 11ß-HSD1 activity is profoundly affected leading to a lack of reductase and a concomitant increase in dehydrogenase activity (10).
The autocrine generation of GCs by 11ß-HSD1 has emerged as an important factor in regulating hepatic glucose output by augmenting gluconeogenesis. In addition, within adipose tissue, the local generation of GCs increase adipocyte differentiation and adipogenesis (11). 11ß-HSD1 KO mice show improved glucose tolerance, enhanced insulin sensitivity, and reduced weight gain when fed a high-fat diet (12, 13). Similar data are reported as novel selective 11ß-HSD1 inhibitors are evaluated in animal models (14, 15, 16). Recently we identified a mechanism by which G6P availability can regulate 11ß-HSD1 activity (17). This raises the possibility that G6P metabolism through H6PDH is important, and therefore, its absence will have consequences for glucose homeostasis independent of its role in 11ß-HSD1 regulation.
On this basis we conducted a phenotypic analysis of the H6PDH KO mouse focusing on body weight, glucose homeostasis, and responses to metabolic stress such as fasting and a high-fat diet.
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Materials and Methods |
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Design
All animal experiments had the approval of the Institutional Animal Care and Use Committee and were performed according to procedures approved by that committee. Adult H6PDH KO mice were available in-house as previously described (10). All mice were housed in standard conditions on a 12-h light, 12-h dark cycle with access to standard rodent chow and water ad libitum. Age-matched male wild-type (WT) and KO mice were fed a high-fat diet (45% calories from fat; Research Diets Inc., NJ) or a regular rodent chow diet (10% calories from fat) for 16 wk from 6 wk of age. Mice were housed singly for the last days of the experiment and killed between 0800 and 1000 h. Body weight was monitored weekly for 10 wk and at the end of the experiment. Similarly, fasting glucose was determined every 2 wk for 10 wk and then a final determination at the end of the experiment by a rapid tail nick and quantified using a Onetouch Ultra glucometer (LifeScan Inc.; Johnson & Johnson, Milpitas, CA).
Intraperitoneal glucose tolerance tests
After 16 wk of high-fat or control diet, mice were fasted for 16 h and injected ip with 1.5 mg/g body weight of D-glucose. Blood samples were taken by tail nick at 0, 30, 60, 90, and 120 min after injection. Blood glucose was measured using a Onetouch Ultra glucometer.
Tissue glycogen content
Tissue glycogen concentrations were measured after alkali digestion of hepatic tissue (18). Briefly, samples of liver (25–50 mg) were digested in KOH [30% (wt/vol), 200 µl] at 70 C (1 h). After digestion, ice-cold absolute ethanol (
4 vol) was added to each sample and the glycogen allowed to precipitate overnight at 4 C. The precipitate was recovered and redissolved in water (200 µl) before reprecipitation in further ethanol (4 vol). This process was repeated a total of three times to remove any contaminating proteins and tritiated water. After the final precipitation, glycogen was resuspended in acetate buffer [50 mM (pH 4.8)] and an aliquot of glycogen digested using amyloglucosidase enzyme (100 U/ml final volume) and left overnight at room temperature. The concentration of glycogen in tissue was estimated as glucose released from glycogen as detailed in the glucose oxidase kit instructions. Aliquots of glycogen were counted by liquid scintillation counting for tritium incorporation to estimate the rate of synthesis of glycogen.
RNA isolation and real-time PCR
This was carried out according to procedures established previously in our group (9). Total RNA was extracted from liver pieces using TRI reagent (Sigma) and reverse transcribed using a high-capacity cDNA archive kit (Applied Biosystems, Cheshire, UK) following the manufacturer’s instructions. An ABI 7000 sequence detection system (Applied Biosystems) was used for real-time mRNA quantification. Reactions were performed with 25 ng cDNA as template and TaqMan universal PCR master mix. For phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) quantification, premade TaqMan gene expression assays were used (Applied Biosystems; PEPCK, Mm00440636_m1 and G6Pase, Mm00839363_m1). Gene expression levels were normalized for RNA loading using 18S (TaqMan assay reagent; Applied Biosystems) as an internal control. Arbitrary units for expression were calculated using 1000 x 2-
Ct, where Ct = [cycle threshold (Ct) value of the gene of interest] – (Ct value of 18S rRNA).
Histological analysis
Liver tissue was analyzed by hematoxylin and eosin staining in both KO and WT animals at 4, 12, and 24 wk of age after fixation in phosphate-buffered formalin and embedding in wax.
Estimation of glycogen synthesis rates
Glycogen synthesis rates in vivo were estimated using previously described methods (18, 19). Briefly, mice received a single dose of tritiated water (2 mCi per mouse injected ip) 2 h before the animals were killed. Animals had ad libitum access to both food and water. Animals were anesthetized by inhalation of isoflurane, and terminal blood samples were collected from the inferior vena cava (IVC) into heparinized syringes for estimation of the specific activity. Volumes of distribution for the tritiated water were estimated to be equivalent to body weight of the animal as previously outlined (20, 21). Samples of liver were then snap frozen in liquid nitrogen for estimation of glycogen concentration and the rate of synthesis of glycogen. In some experiments animals were either fasted overnight or fasted overnight followed by a period of refeeding (4 h).
Estimation of rates of gluconeogenesis
Gluconeogenesis was estimated from the rates of incorporation of U-14C-labeled lactate into plasma glucose and hepatic glycogen as previously outlined (22, 23). Briefly, mice fed ad libitum received a single dose of U-14C-labeled lactic acid in PBS (20 µCi per mouse) 15 min before the animals were killed. Mice were anesthetized by inhalation of isoflurane and terminal blood samples drawn from the IVC as outlined above. Samples of hepatic tissue were snap frozen in liquid nitrogen. Lactate incorporation was estimated from previously determined volumes of distribution (80% body weight) for lactate assuming a single compartment model of lactate clearance (22, 24). To discriminate between plasma 14C-labeled lactate and 14C-labeled glucose, unreacted lactate was removed from samples of plasma using ion-exchange resin as outlined below.
Extraction of unreacted lactic acid
Unreacted 14C-labeled lactate was removed from samples of plasma using Dowex 22Cl anion exchange resin (Aldrich, Poole, Dorset, UK) as outlined previously (25). Dowex resin was initially extensively washed with KOH (2 M) and then distilled water. Aliquots of plasma (50 µl) were diluted with water (final volume 200 µl), and a slurry of the pretreated resin was added (100 mg resin) and vortex mixed (30 sec). The supernatant was then recovered after centrifugation of the mixture through a centrifugal spin column. This method was validated using human plasma spiked with increasing concentrations of lactic acid (up to 20 mM) supplemented with radiolabeled lactic acid and further experiments confirmed that glucose was not recovered by the resin extraction.
Enzyme assays
Liver cytosolic PEPCK activity was assayed according to a previously described protocol (26). Briefly, 20% (wt/vol) homogenate was prepared at 4 C in buffer containing 0.25 M sucrose and 5 mM Tris-HCl (pH 7.4) (reagents from Sigma). The homogenate was centrifuged at 3500 x g for 15 min, with the supernatant further centrifuged at 11,000 x g for 40 min. The cytosolic supernatant was taken and subjected to a protein determination using an assay kit (Bio-Rad, Hercules, CA). To determine PEPCK activity, 0.2 mg of total protein was added to a 1-ml reaction media containing 50 mM Tris HCl, 50 mM NaHCO3, 1 mM MnCl2, 1 mM phosphoenolpyruvate, 2 U malate dehydrogenase, and 0.25 mM nicotinamide adenine dinucleotide (reduced). The reaction was commenced by adding 0.15 mM 2'-deoxyguanosine 5'-diphosphate, with the decrease in absorbance at 340 nm measured for 4 min.
11ß-HSD1 activity assays were carried out according to previously published protocols (9). Briefly, activity assays were performed by incubating 10–15 mg tissue with 1 ml of serum-free culture media with 100 nM 11-DHC (reductase) or corticosterone (dehydrogenase) and corresponding tritiated tracer for 2 h. After incubation, media were transferred to a glass tube and steroids were extracted with 5 ml dichloromethane. The aqueous phase was removed and dichloromethane phase concentrated to 100 µl, which was spotted on a thin-layer chromatography silica plate. Steroids were separated by thin-layer chromatography using a mobile phase of ethanol and chloroform (8:92) and quantitated using a Bioscan 2000 image analyzer (Lablogic, Sheffield, UK) or a phosphor imager (Fuji FLA-2000; Raytek Scientific Ltd., Sheffield, UK). Protein levels were assayed using a 96-well plate assay kit (Bio-Rad Laboratories). All experiments were carried out in triplicate.
Plasma metabolites
Plasma metabolite concentrations of glucose (Thermo-Electron), lactate (Randox), insulin (Crystal Chem), and corticosterone (Diagnostic Systems Laboratories, Webster, TX) were determined as outlined in the manufacturer’s instructions.
Corticosterone measurements were taken between 0800 and 0900 h in the fed state.
Statistical analysis
Statistical comparisons were performed using SPSS (version 12.0; SPSS Inc., Chicago, IL). Data are expressed as means ± SEM with statistical significance defined as P < 0.05. All data were tested for homogeneity of variance. Statistical analyses included repeated measures to compare longitudinal data for fasting glucose and body weight. Area under the curve followed by one-way ANOVA was used to determine the difference between glucose tolerance test profiles of WT and KO mice. One-way ANOVA was used to determine statistical significance between metabolite concentrations and both glycogen concentrations and rates of gluconeogenesis.
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Results |
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). The interconversion of 11-DHC and corticosterone was measured in explanted hepatic tissue maintained in media for 10 min postmortem. WT tissue maintained robust reductase activity (11-DHC to corticosterone) and little dehydrogenase activity (corticosterone to 11-DHC) (3 ± 0.2 and 0.03 ± 0.01 pmol of steroid per milligram tissue per hour, respectively, P < 0.01, Fig. 1B). KO tissue shows residual reductase activity and a significant increase in dehydrogenase activity (0.9 ± 0.07 and 0.3 ± 0.01 pmol of steroid per milligram tissue per hour, respectively, P < 0.01, Fig. 1B). Liver tissue sections from KO mice appeared histologically normal compared with age-matched WT mice at all ages studied, indicating that there are no gross changes or malformations (data not shown).
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Nonstressed fasting peripheral glucose levels revealed relative hypoglycemia in KO mice during the experimental period (P < 0.001), and the same was true when KO mice were fed a high-fat diet (P < 0.001, Fig. 2B).
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Blood drawn from the IVC, compared with peripheral tail nicking, indicated relative hypoglycemia in the fed and fasted refed state in KO mice, whereas their plasma glucose did not differ from WT in the fasted state (Table 1). Moreover, during the transition between fasting and refeeding, plasma glucose concentrations were not significantly different in KO mice (Table 1). Insulin values were similar in both genotypes, except when animals were fasted refed, revealing significantly lower plasma insulin levels in KO mice (P < 0.05).
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). These data represented a 3.4- and 2.2-fold induction of G6Pase mRNA in WT and KO, respectively, and a 4.5- and 4-fold induction of PEPCK in WT and KO, respectively, with fasting (Fig. 3B). Cytosolic PEPCK assays indicated that WT and KO had similar in vitro enzyme activity in hepatic tissues from both fed and fasted mice (Fed WT; 10 ± 0.8 vs. KO; 10.4 ± 1 nmol NADH per milligram per minute, P < 0.5, fasted WT; 14.5 ± 1.5 vs. 15.7 ± 0.7 nmol NADH per milligram per minute, P < 0.8; Fig. 3C). These data suggest no change in gluconeogenic response in KO mice, compared with WT, during the fasting period.
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). Refeeding reelevated glycogen to a similar level in both genotypes). Upon refeeding, WT mice overcompensate the fed state as expected until homeostasis is returned.
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), providing the basis for the excess storage of glycogen seen in KO liver. In the fasted and fasted refed state, glycogenesis proceeds at similar rates in both WT and KO mice.
Estimation of rates of gluconeogenesis
Gluconeogenesis was estimated in vivo from the incorporation of 14C-labeled lactate into plasma glucose and hepatic glycogen. Plasma rates of clearance estimated from a single compartment model revealed that, for both WT and KO mice, plasma lactate clearance was constant (T
= 48.0 ± 8.1 and 49.2 ± 4.0 min for WT and KO, respectively). After 15 min incubation, the fraction of unreacted lactate remaining in the plasma was also unchanged (WT = 26.2 ± 6.3%, KO = 26.2 ± 3.0% of initial injected dose). Incorporation of 14C-lactate into plasma glucose was not significantly different between WT and KO mice (Table 2); however, incorporation of 14C-glucose into hepatic glycogen was increased 50% in KO mice (P < 0.05; Table 2).
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Discussion |
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H6PDH KO mice had elevated basal circulating corticosterone levels, compared with WT, which was a consistent finding within this study. However, this was discordant with our previous data, which on further inspection revealed high corticosterone levels in WT mice and may have been due to the method of blood collection (10). In this study a more suitable method, namely a terminal bleed after isoflurane inhalation, was used. These data are more in keeping with the previously reported adrenal hyperplasia (10).
In hepatic explants we demonstrated a significant switch from 11ß-HSD1 reductase to dehydrogenase activity. Residual reductase activity was low, and an accurate interpretation of GC concentrations in hepatic tissue is difficult. Our previous data on urinary output of metabolized GCs clearly showed that most circulating GCs are inactivated to inert 11-DHC before excretion, suggesting both hepatic and peripheral 11ß-HSD1 dehydrogenase activity make important contributions to this process (10). Any residual reductase activity in H6PDH KO liver may reflect the available sources of NADPH in the ER during the assay. Experiments using hepatic microsomes report that ER membranes are impermeable to NADP/NADPH (8, 28). However, there is evidence to suggest NADPH regulation of 11ß-HSD1 reductase activity from the cytosolic pentose phosphate pathway in adipocytes. As this is a highly active pathway in hepatic tissue; it is this that we may have measured in H6PDH KO hepatic explants (29).
Adult H6PDH KO mice had attenuated weight gain and persistent fasting hypoglycemia as measured from tail vein nicking on a regular diet and high-fat diet. Glucose tolerance tests indicated improved glucose tolerance, most marked on a high-fat diet and indicated resistance to hyperglycemia. These data are consistent with insulin-sensitizing effects due to the observed decrease in GC regeneration. Liver-specific inactivation of the glucocorticoid receptor yields mice with poor induction of gluconeogenic G6Pase and PEPCK mRNAs on fasting, resulting in low plasma glucose and significant decreases in hepatic glycogen, underscoring the importance of GC regulation of gluconeogenesis (30). We showed similar levels of hepatic G6Pase and PEPCK in fed and fasted WT and KO mice and showed robust up-regulation of these GC-sensitive enzymes on fasting in the KO mice. In vitro PEPCK enzyme activity in fed WT and KO liver was similar, with activity significantly increasing when both genotypes were fasted, supporting the gene expression data and highlighting an inability to identify a defect in responses to fasting via GC regulation of gluconeogenesis. Thus, we postulate that in the H6PDH KO mouse, the increase in circulating corticosterone levels due to enhanced hypothalamic-pituitary-adrenal drive may partially rescue the ability to regulate gluconeogenesis. For the liver at least, the circulating concentrations of active GCs may be more important in hepatic glucose homeostasis than local reactivation via 11ß-HSD1. It is also possible that other pathways may promote the expression of these enzymes to combat the ensuing peripheral hypoglycemia.
To maintain euglycemia on fasting, hepatic glycogen stores are mobilized and glucose shuttled into the blood (31). We showed an increase in storage and synthesis rates of glycogen in fed KO, compared with WT mice. The result may be explained by decreased antagonism of the effects of insulin to drive glycogenesis in the postprandial state. Fasted H6PDH KO mice were able to fully mobilize stored glycogen to WT levels, suggesting that no defect was present in glycogenolysis, with synthesis rates suitably decreased. Refeeding reestablished hepatic glycogen storage and synthesis rates to similar levels for both WT and KO mice. Indeed, increased glycogen synthesis rates on refeeding in both genotypes resulted in increased glycogen stores, compared with the fed state, most likely due to overshoot storage during excessive feeding after the 16-h fast. However, WT glycogen storage resolves to its normal postprandial level, i.e. the fed state, whereas the KO hepatic glycogen store remains elevated.
Glycogen synthesis in the fasted-refed state revealed that increased rates of synthesis occurred on a background of relative hypoglycemia and relatively low plasma insulin concentrations (Fig. 4 and Table 1). Given that hepatic glucose uptake is driven by portal blood concentrations, we speculate that allosteric activation of glycogen synthase and inhibition of glycogen phosphorylase by G6P may be an important mechanism at work rather than, or at least in conjunction with, the direct effects of insulin. Indeed, acute inhibition of G6Pase leads to increased hepatic G6P levels and hepatic glycogen deposition without changes to gluconeogenesis rates (32). In addition, G6P also inhibits glycogenolysis through inactivation of glycogen phosphorylase (33). Our recent findings that G6P is a regulator of 11ß-HSD1 activity through H6PDH supports these notions and the implications for glucose homeostasis (17). The increased deposition of 14C-labeled lactate as glycogen may reflect the increased glycogen deposition observed in fed KO mice rather than changes to gluconeogenesis per se. The absence of changes to gluconeogenesis estimated from in vivo 14C-lactate incorporation into plasma glucose supports these observations and is further corroborated by the lack of changes to PEPCK in vitro activity measurements noted above.
We measured blood plasma derived from the IVC in the fed, fasted, and fasted refed cohorts as an adjunct to our data for fasting peripheral whole-blood glucose assays. These data indicated a relative hypoglycemia in the fed KO animals, compared with WT mice, and may reflect the increased storage of hepatic glucose as glycogen (and as yet undetermined peripheral glucose use). Interestingly, there was an absence of a fasting-related decrease in plasma glucose in the KO from the IVC sampling site. This is indicative of adequate buffering of plasma glucose concentrations by the combined effects of gluconeogenesis and the breakdown of an enlarged hepatic glycogen store in blood passing from the liver before peripheral perfusion. Moreover, the refeeding response documented for WT mice was severely blunted in KO mice, hinting at enhanced peripheral glucose disposal. This may also explain the lack of insulin response on refeeding, plasma glucose levels never rising sufficiently to trigger insulin secretion.
The discrepancy in fasting response noted for blood samples collected after an overnight fast from a peripheral tail nick and those collected from the IVC postmortem may simply reflect changes in methodology. However, given the central origins of IVC-drawn blood, we cannot dismiss the possibility that blood taken from tail-nick reflects enhanced extrahepatic glucose uptake in KO mice as a result of enhanced insulin sensitivity and changes to cellular G6P concentration.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online September 6, 2007
Abbreviations: Ct, Cycle threshold; 11-DHC, 11-dehydrocorticosterone; ER, endoplasmic reticulum; GC, glucocorticoid; G6P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; H6PDH, hexose-6-phosphate dehydrogenase; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; IVC, inferior vena cava; KO, knockout; NADP+, nicotinamide adenine dinucleotide phosphate (oxidized); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); PEPCK, phosphoenolpyruvate carboxykinase; WT, wild type.
Received July 13, 2007.
Accepted for publication August 27, 2007.
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References |
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in the control of SREBP activity and lipid synthesis in the liver. Biochem J 389:413–421[CrossRef][Medline]This article has been cited by other articles:
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  G. G. Lavery, E. A. Walker, N. Turan, D. Rogoff, J. W. Ryder, J. M. Shelton, J. A. Richardson, F. Falciani, P. C. White, P. M. Stewart, et al. Deletion of Hexose-6-phosphate Dehydrogenase Activates the Unfolded Protein Response Pathway and Induces Skeletal Myopathy J. Biol. Chem., March 28, 2008; 283(13): 8453 - 8461. [Abstract] [Full Text] [PDF]
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