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Inactivation of the Glucocorticoid Receptor in Hepatocytes Leads to Fasting Hypoglycemia and Ameliorates Hyperglycemia in Streptozotocin-Induced Diabetes Mellitus

Molecular Biology of the Cell I (C.O., F.T., C.K., W.S., G.S.), Deutsches Krebsforschungszentrum, and Division of Metabolic and Endocrine Diseases (D.K., A.S.), University Children’s Hospital, 69120 Heidelberg, Germany; Molecular Genetics and Neurophysiology (F.T.), FRE2401, Collège de France, 75231 Paris Cedex 05, France; and Center for Neurobiology and Behaviour (C.K.), Columbia University, New York, New York 10033

Address all correspondence and requests for reprints to: Günther Schütz, Division Molecular Biology of the Cell I, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: g.schuetz{at}dkfz.de.

	ABSTRACT

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Hepatic glucose production by gluconeogenesis is the main source of glucose during fasting and contributes significantly to hyperglycemia in diabetes mellitus. Accordingly, glucose metabolism is tightly controlled by a variety of hormones including insulin, epinephrine, glucagon, and glucocorticoids (GCs) acting on various cell types. GC effects are mediated by the GC receptor (GR), a ligand-dependent transcription factor, which in the liver and kidney controls gluconeogenesis by induction of gluconeogenic enzymes. To specifically study the contribution of GC on liver carbohydrate metabolism, we generated mice with an inactivation of the GR gene exclusively in hepatocytes using the Cre/loxP technology. Half of the mutant mice die within the first 2 d after birth most likely due to hypoglycemia. Adult mice have normal blood sugar under basal conditions but show hypoglycemia after prolonged starvation due to reduced expression of genes involved in gluconeogenesis. We further demonstrate that absence of GR in hepatocytes limits the development of hyperglycemia in streptozotocin-induced diabetes mellitus probably due to impaired induction of gluconeogenesis. These findings show the essential role of GR function in liver glucose metabolism during fasting and in diabetic mice and indicate that liver-specific GC antagonists could be beneficial in control of diabetic hyperglycemia.

	INTRODUCTION

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IN MAMMALS A SOPHISTICATED hormonal control by insulin, glucagon, glucocorticoids (GCs), and epinephrine maintains blood glucose within narrow limits, orchestrating the uptake and the release of glucose by peripheral organs and the production of glucose by the liver and kidney. Insulin suppresses glucose production by inhibiting hepatic glycogenolysis and gluconeogenesis and stimulates glucose uptake, storage, and utilization by tissues such as muscle and fat. GCs, glucagon, and epinephrine raise blood glucose by inhibiting glucose uptake in the periphery and by stimulating hepatic glucose release. Their effects in peripheral tissues are mainly due to antagonizing insulin action (1). They impair glucose uptake by skeletal muscle and adipose tissues and increase lipolysis, leading to an augmentation of circulating free fatty acids (2). Finally, by acting directly on pancreatic ß-cells, GCs reduce insulin release (3). A dysregulation of these functions may lead to pathological states. GC deficiency in humans is characterized by impaired gluconeogenesis leading to hypoglycemia (1). GC excess is associated with glucose intolerance and the appearance of insulin resistance and is a risk factor for the development of type 2 diabetes (1, 4).

GC effects are mediated by ligand-dependent activation of the GC receptor (GR) that is expressed in almost every cell type. The activated GR acts as a transcription factor and controls the level of expression of target genes but also modulates intracellular signaling pathways (5, 6, 7, 8, 9, 10).

Hepatic gluconeogenesis is the main source of hepatic glucose production in states of prolonged fasting and contributes significantly to the development of diabetes mellitus (11). The key gluconeogenic enzymes involved are tightly controlled by GCs and glucagon on the transcriptional level (11, 12). GCs exert their activity by the binding of GR dimers to glucocorticoid response elements (GREs), such as in the phosphoenol pyruvate carboxykinase (PEPCK) promoter (12), tyrosine aminotransferase (TAT) (13, 14, 15), and glucose-6-phosphatase (G6Pase) enhancers (16, 17). Glucagon, on the other hand, signals through the cAMP/PKA pathway that leads to activation of cAMP response element (CRE) binding protein (CREB). CREB can directly stimulate the transcription of genes for gluconeogenic enzymes by binding to CREs or acts through peroxisome proliferator-activated receptor coactivator 1, a GR coactivator, and thereby potentiates GR activity (18).

To specifically study the hepatic effects of GC on carbohydrate metabolism, we generated mice with an inactivation of the GR gene exclusively in hepatocytes using the Cre/loxP recombination system (19). This approach allowed us to distinguish the contribution of GC signaling in hepatocytes vs. in other cell types and to bypass the perinatal lethality we observed in GR mutant animals (6, 20). We show that disruption of the GR in hepatocytes leads to hypoglycemia during prolonged starvation due to a reduced expression of genes involved in gluconeogenesis. Furthermore, we demonstrate that absence of GR in hepatocytes restricts gluconeogenesis and the development of hyperglycemia in streptozotocin-induced diabetes mellitus.

	RESULTS

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Loss of GR in Liver Parenchymal Cells Impairs Survival in the Perinatal Period
Ubiquitous inactivation of the GR in mice leads to lethality shortly after birth (6, 20). Mice with a hepatocyte-specific inactivation of the GR generated with the help of the Cre/loxP system have been described (19). To generate these mice, homozygous GR^loxP/loxP (referred to as GR^loxP) or control females, which show normal expression of GR, were crossed with GR^loxP/loxP;AlfpCre males (referred to as GR^AlfpCre or mutants). Genotype analysis of 397 mice at weaning revealed frequencies of 67% control (267 animals) and 33% mutant mice (130 animals), suggesting that the absence of GR in hepatocytes leads to death of about half of the mutant animals. The increased mortality of mutant pups occurred exclusively during the first 48 h. After this period, no difference of mortality between wild-type and mutant pups was observed. Before death, offspring became lethargic and pale. Blood glucose levels were reduced in 1-d-old mutant mice compared with control animals (40.0 ± 7 mg dl^–1 vs. 62.8 ± 6 mg dl^–1; P < 0.05). By inspection, mice lacking GR in hepatocytes were not distinguishable from control animals until puberty when they displayed a growth deficiency [Tronche et al., (20A )]. The amount of fat of body carcass was 23% higher in adult mutant animals when compared with wild-type animals (19.1% ± 1.3 vs. 15.6% ± 0.5), and the dried mass, excluding fat, was reduced by 8% (27.2% ± 0.4 vs. 29,5% ± 0.3). The size of the liver was proportionally reduced in mutant animals (Table 1). In sections stained with hematoxylin-eosin and periodic acid Schiff, no differences between livers from control and mutant animals were observed (Fig. 1, A and B, and data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 1. Absence of GR in GRAlfpCre Mice Does Not Affect Liver Histology and Basal Functions No differences were observed between sections from 1.5- to 2-month-old GRAlfpCre mice (A) and from control littermates (B) (stained by eosin and hematoxylin; magnification, x200). C, Northern blot analysis of total liver RNA from fed animals showed only limited changes in expression of GR target genes under basal conditions. The mRNA levels in control liver were arbitrarily set to 100, S26 was used for normalization of other mRNAs levels. P < 0.05.

Consistent with the apparently unaltered glucose metabolism in GR^AlfpCre mice, the expression levels of mRNAs encoding several gluconeogenic enzymes, including PEPCK, which is rate-limiting in gluconeogenesis, and TAT, were not affected under basal conditions (Fig. 1C). G6Pase mRNA levels had a tendency to be increased in GR^AlfpCre mice, but the difference did not reach significance (Fig. 1C). All of these genes are induced by GC via the binding of GR to GR binding sites present in their promoters or enhancers (14, 15, 21). As expected, the induction observed in control mice after injection of dexamethasone, a potent glucocorticoid agonist, is absent in GR^AlfpCre mice (Fig. 2).

View larger version (65K): [in this window] [in a new window]   Fig. 2. Induction of Gluconeogenic Enzymes by a GC Agonist Is Defective in GRAlfpCre Animals Induction of PEPCK, G6Pase, and TAT gene expression was clearly observed in the livers of 6- to 8-wk-old control animals 2 h after ip injection of dexamethasone (0.1 mg/g) but not in mutant mice. A, Northern blot analysis. B, Quantification of the signals. The mRNA levels in control liver were arbitrarily set to 100. The signals were quantified using PhosphorImager, and loading was controlled using S26 antisense probe. and , P values <0.05 and <0.01, respectively.

GR^AlfpCre Mice Develop Hypoglycemia upon Prolonged Fasting
Because we observed normal serum glucose levels in unstressed GR^AlfpCre mice we evaluated the role of GR in hepatocytes by prolonged starvation. During fasting, glucose levels are maintained during the first 24 h mainly by breakdown of glycogen in liver and kidneys. After prolonged starvation, liver gluconeogenesis becomes the major source of serum glucose (11). We therefore studied the consequences of prolonged starvation (48 h). During fasting, GR^AlfpCre mice showed a faster decline in plasma glucose levels than their wild-type littermates that were able to sustain serum glucose levels at nearly normal levels during the time of starvation (Fig. 3A). As expected, GC levels were markedly increased in response to fasting (Fig. 3B). This elevation was higher in mutant animals, possibly reflecting an attempt to sustain gluconeogenesis in these animals. Glycogen stored in the liver of GR^AlfpCre mice showed a normal mobilization during fasting, probably explaining the only moderate difference in glucose levels between mutant and control groups after 24 h of starvation (Fig. 3C). The lower serum glucose levels in mutant animals is due to impaired gluconeogenesis (Fig. 3D) as reflected in the impaired induction of PEPCK mRNA. Lipid metabolism was not affected by the inactivation of GR in hepatocytes. After fasting, free fatty acids plasma levels were similar in both genotypes (mutant: 0.47 mmol/liter ± 0.02 vs. control: 0.51 ± 0.04). In addition, fatty acid oxidation determined by measuring levels of ketone bodies in plasma was not markedly different between genotypes (acetoacetate: 18.3 µmol/liter ± 1.7 in mutants vs. 16.0 ± 1.3; butyric acid: 1264 µmol/liter ± 201 in mutants vs. 748 ± 302; this increase does not reach significance).

View larger version (24K): [in this window] [in a new window]   Fig. 3. GRAlfpCre Mice Develop Hypoglycemia during Fasting A, Blood glucose levels: at time zero (beginning of the light cycle), food was withdrawn and glucose measured over 2 d as indicated. The experiment was performed with five GRAlfpCre (black bar) and five control (white bar) males, 2 to 3 months old. The glucose values are expressed as percentage of untreated wild-type mice. B, Plasma corticosterone: basal values are similar in mutant and control animals, but fasting led to a more marked increase in mutant animals. C, Liver glycogen: levels were unaffected in fed GRAlfpCre mice compared with control animals (n = 8 per group). After 48 h fasting liver glycogen was lower in mutant animals (n = 5 per group). D, Induction of gluconeogenic enzymes is defective in GRAlfpCre animals: induction of PEPCK mRNA expression in the livers of fed and fasted mice after 48 h starvation was studied by Northern blot analysis. Loading was controlled using S26 antisense probe. Quantification of mRNA expression in mutant and control livers of fed and fasted animals is shown on the right. , P <0.05; , P <0.01.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Absence of GR in Hepatocytes Reduces Hyperglycemia in Experimentally Induced Diabetes A, At d 0, 1.5- to 3-month-old male mice received an ip injection of streptozotocin (0.2 mg kg–1), to induce diabetes, or saline. Blood was collected by tail phlebotomy, and plasma glucose levels were measured in GRAlfpCre and control males. B, PEPCK mRNA expression after streptozotocin treatment is impaired in liver-specific GR mutant mice. Mice were killed 4 d after streptozotocin injection. Total cellular RNA was isolated from livers of each mutant and control animal under normal and diabetic conditions. The relative levels of PEPCK mRNAs were determined as described in Material and Methods and expressed as percentage of mRNA hybridization in liver from control animals injected with a saline solution. Results are means ± SEM of five to eight animals per group. , Significant difference (P < 0.05) between mutant and control groups calculated by unpaired Student’s t test.

	DISCUSSION

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Mice with a germline inactivation of the GR die shortly after birth due to lung atelectasis (20). We observed here that about 50% of newborn GR^AlfpCre mice die within the first 2 d of life possibly due to hypoglycemia. The partial penetrance of the lethal phenotype might be due to individual variations in the timing of recombination. In the perinatal period, expression of gluconeogenic enyzmes such as PEPCK is activated by glucagon via cAMP and GCs by activation of a variety of transcription factors, including GR, CREB/CRE modulator, hepatocyte nuclear factor (HNF)4, HNF3, HNF1, CCAAT enhancer binding protein (C/EBP), and C/EBPß (21, 22, 23, 24, 25). Targeted disruption of one of the members of these clusters leads to defective expression of PEPCK. C/EBP knockout mice and a large fraction of C/EBPß-deficient mice die shortly after birth due to defective hepatic glucose production (22, 26). Our results show that gluconeogenesis after inactivation of GR in the GR^AlfpCre mice is diminished and that other transcription factors can compensate only partially for the loss of GR that leads to the death of half of the mutants shortly after birth. Interestingly, mice with an ubiquitous inactivation of PEPCK also die within this period, whereas liver-specific inactivation of the PEPCK gene is compatible with survival (27), indicating that the lethality observed in the GR^AlfpCre cannot be explained solely by the defective induction of PEPCK. This suggests that GR exerts more pleiotropic actions on neonatal gluconeogenesis in the liver.

In contrast to newborns, in adult mice the loss of hepatic GR did not have a major impact on blood glucose level in the fed or overnight fasted animals. Mutant animals showed normal glycogen content and unaltered expression of gluconeogenic enzymes in the liver, indicating a sufficient compensation by GC-independent mechanisms. Indeed, a compensatory increase in glucagon and a concomitant decrease in insulin was noted. These changes are apparently sufficient to maintain glucose levels within normal range at basal conditions. However, after prolonged fasting, a metabolic challenge, in which gluconeogenesis becomes the major source of hepatic glucose production (11, 28), the mutant animals showed marked hypogylcemia associated with a reduced induction of the rate limiting enzymes of gluconeogenesis such as PEPCK. The low glucose levels we observed could also be due to changes in lipid metabolism in the liver of mutant animals. Our results are in accordance with mice deficient in hepatic PEPCK (27), liver-specific C/EBP (26), and C/EBPß knockout mice (29), which show no impairment of glucose homeostasis under unstressed conditions but display fasting hypoglycemia associated with a reduced induction of PEPCK.

The development of hyperglycemia in diabetes mellitus is still not fully understood. Insulin deficiency or resistance tips the hormonal balance toward glucose production by increasing hormones such as glucagon and glucocorticoids. Thereby, glucose uptake in the periphery is lowered, and peripheral proteolysis and lipolysis is increased. On the other hand, hepatic glucose production is stimulated by gluconeogenic precursors in serum and by increased expression of gluconeogenic enzymes such as PEPCK in the liver. Induced production of GCs has been considered to be an important factor in diabetic hyperglycemia for at least 60 yr (30). Adrenalectomy (30), RU486 administration (31), or ß-hydroxysteroid dehydrogenase-1 inactivation [which converts inactive into active GC in target tissues (32)] limits hepatic PEPCK induction and hyperglycemia in diabetes mellitus. Targeted deletion of the glucocorticoid-responsive unit in the PEPCK promotor also limits the induction of a reporter gene in diabetes mellitus (33). However, in these transgenic models the isolated hepatic effect of GCs and the physiological consequences in terms of hyperglycemia could not be determined. The GR^AlfpCre mutants give the opportunity to exclusively study the hepatocyte-specific contribution of GR in diabetes mellitus because GR actions in other organs, such as inhibition of glucose uptake and proteolysis, are intact. In streptozotocin-induced insulin deficiency, GR^AlfpCre mice developed less severe hyperglycemia and completely failed to induce PEPCK mRNA in the liver. The amelioration of hyperglycemia in liver-specific mutant mice indicates that hepatic GR is a crucial factor for PEPCK induction during insulin deficiency and thus reveals the important contribution of GR signaling in the liver in the development of diabetic hyperglycemia. This finding is intriguing because liver-specific antagonists of GR might be of therapeutic value of hyperglycemia in diabetes mellitus.

	MATERIALS AND METHODS

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Animals
Animals were housed and bred according to international standard conditions with a 12-h dark, 12-h light cycle. Mice were genotyped by hybridization of a P³²-labeled probe for Cre recombinase to tail DNA immobilized on nylon filters (dot blot) and by PCR for the GR^loxP-allele (34). Mice used in this study were 1.5 to 6 months old. To determine the fat content of animal carcasses, 3-month-old female mice were decapitated and the gastrointestinal tract was removed. The carcasses were autoclaved at 122 C for 20 min and homogenized with a Polytron after the addition of an equal weight of water. The fat content was determined by methanol/chloroform extraction, and the dried mass was measured after the homogenate had been dried for 5 d at 70 C. Dexamethasone phosphate disodium salt (Sigma Chemical Co., St. Louis, MO) was injected ip at 0.1 mg/g, dissolved in PBS. For induction of diabetes mellitus, mice housed individually were fasted overnight and injected ip with streptozotocin (Sigma, 0.2 mg/g) in 0.05 M citrate, pH 5. Two hours after the injection mice were fed ad libitum. Induction of diabetes was determined by measuring insulin levels in plasma by RIA (Crystal Chem, Downers Grove, IL).

Northern Blot Analysis
Preparation of total RNA from liver and Northern blot analysis were performed as previously described (35). Briefly, 30 µg total RNA from the liver of a single animal were separated on a 1% agarose/formaldehyde gel, transferred to a nylon membrane (Hybond +, Amersham Pharmacia Biotech, Arlington Heights, IL), and sequentially hybridized at 65 C with radiolabeled cDNA probes (23). For each single measure, the signal was quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and normalized to the quantitated signal obtained using a probe hybridizing to S26 mRNA. S26 ribosomal protein RNA was used for normalization (36). For each probe, normalized signals were averaged. The significance of observed differences were determined using Student’s t test.

Determination of Serum Metabolites and Hormones
To determine glucose values, metabolites, and amino acid levels, 40–60 µl of blood were collected by phlebotomy from mice housed individually. For determination of glucagon and insulin, mice were killed by C0₂ inhalation, and blood was collected from the inferior cava vein. Glucose was measured using an automated analyzer (Accutrend GC, Roche Clinical Laboratories, Indianapolis, IN). The amounts of total protein, albumin, triglycerides, free fatty acids, ketone bodies, and cholesterol were determined in plasma, using microassay procedures (Wako Chemicals GmbH, Neuss, Germany) (37).

To measure blood amino acid levels, a drop of blood was spotted onto filter paper and dried (S&S 903, Schleicher & Schuell, Dassel, Germany). Amino acid levels were determined by mass spectrometry (PE Sciex, Canada), as previously described with minor modifications (38, 39). Internal deuterated standards (Cambridge Isotope Laboratories, Kit NSK-A, Cambridge, UK) were used for quantification. Corticosterone, glucagon, and insulin levels were measured by RIA (ICN Biomedicals, Cleveland, OH; and Crystal Chem). Hepatic glycogen content was measured as previously described (40). Significance was calculated by Student’s t test. P values < 0.05 were considered significant.

	FOOTNOTES

 
This work was supported by the "Deutsche Forschungsgemeinschaft" through SFB 405, SFB 488, FOR 302, Graduierten-Kolleg (GRK) 791/1.02, GRK 484, and Sachbeihilfe Schu 51/7-2; by the "Fonds der Chemischen Industrie"; the European Community through Grant QLG1-CT-2001-01574; the BMBF through NGFN Grant FZK 01GS01117; the Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren (HGF) through the "Strategiefonds DNA-CHIPS"; the Alexander von Humboldt-Stiftung through the Max-Planck-Forschungspreis für Internationale Kooperation 1998; and by the Volkswagen-Stiftung through Grant I/76 234. F.T. is supported by the Centre National de la Recherche Scientifique.

C.O and F.T. contributed equally to this work and should both be considered as first authors.

Abbreviations: C/EBP, CCAAT enhancer binding protein; CRE, cAMP response element; CREB, CRE binding protein; GC, glucocorticoid; G6Pase, glucose-6-phosphatase; GR, GC receptor; GRE, GC response element; HNF, hepatocyte nuclear factor; PEPCK, phosphoenol pyruvate carboxykinase; TAT, tyrosine aminotransferase.

Received for publication July 17, 2003. Accepted for publication March 10, 2004.

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