Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription and Hyperglycemia Are Regulated by Glucocorticoids in Genetically Obesedb/db Transgenic Mice*

The molecular mechanisms underlying increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription and gluconeogenesis in type II diabetes are largely unknown. To examine the involvement of glucocorticoids and thecis-acting insulin response sequence (IRS, −416/−407) in the genetically obese db/db mouse model, we generated crosses between C57BL/KsJ-db/+ mice and transgenic mice that express −460 or −2000 base pairs of the rat PEPCK gene promoter containing an intact or mutated IRS, linked to a reporter gene. Transgenic mice expressing the intact PEPCK(460)-CRP (C-reactive protein) transgene bred to near homozygosity at thedb locus were obese, hyperinsulinemic, and developed fasting hyperglycemia (389 ± 26 mg/100 ml) between 4 and 10 weeks of age. Levels of CRP reporter gene expression were increased 2-fold despite severe hyperinsulinemia compared with non-diabetic non-obese transgenic mice. Reporter gene expression was also increased 2-fold in transgenic obese diabetic db/db mice bearing a mutation in the IRS, −2000(IRS)-hGx, compared with non-obese non-diabetic transgenic 2000(IRS)-hGx mice. Treatment of obese diabeticdb/db transgenic mice with the glucocorticoid receptor blocker RU 486 decreased plasma glucose by 50% and reduced PEPCK, GLUT2, glucose-6-phosphatase, tyrosine aminotransferase, CRP, and hGx reporter gene expression to levels similar to those of non-obese normoglycemic transgenic mice. Taken together, these results establish that −460 bp of 5′-flanking sequence is sufficient to mediate the induction of PEPCK gene transcription in genetically obesedb/db mice during the development of hyperglycemia. The results further demonstrate that the mechanism underlying increased expression of gluconeogenic enzymes in thedb/db mouse requires the action of glucocorticoids and occurs independently of factors acting through the PEPCK IRS (−416/−407) promoter binding site.

Non-insulin-dependent diabetes mellitus (NIDDM) 1 is a complex metabolic disease with environmental and genetic components (1,2). Hyperglycemia develops via mechanisms that are not understood completely; however, a classic defect involves the inability of insulin to inhibit hepatic glucose production. Most isotopic studies in man and rodents suggest that increased gluconeogenesis is a major source of increased glucose production in type II diabetes (NIDDM) (3,4); however, with rare exceptions, the primary mechanisms for increased gluconeogenesis at the molecular level are unknown (5)(6)(7). Gluconeogenesis is controlled from seconds to minutes by the delivery of substrates and by hormone-mediated changes in the activity of several key gluconeogenic enzymes. In the long term, usually involving hours to days, gluconeogenesis is regulated by the synthesis rate of gluconeogenic enzymes at the level of gene expression. In the liver, phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and is considered the major rate-controlling enzyme in the pathway of gluconeogenesis from pyruvate, lactate, and alanine (8). Normally, insulin rapidly and substantially inhibits transcription of the PEPCK gene in liver and in rat hepatoma cells. However, in several animal models of obesity and NIDDM, gluconeogenesis and PEPCK mRNA are increased by 2-3-fold over non-diabetic animals, despite circulating insulin levels that may be 4 -10-fold greater than nondiabetic controls (9 -11), suggesting a defect(s) in insulin regulation of gene expression.
In addition to abnormalities in insulin receptor signaling, defects (genetic or acquired) in regulatory proteins that control gene expression may play an important role in the pathogenesis and progression of type II diabetes. We found previously that deleting the glucocorticoid response unit (GRU; see Refs. 12 and 13), located between bases Ϫ455 and Ϫ349 upstream from the transcription start site of the PEPCK gene promoter in transgenic mice, prevented reporter gene induction in mice made diabetic with streptozotocin (14), whereas a mutation in the cAMP regulatory element, located between positions Ϫ93 and Ϫ86, had no effect on the diabetic response of the PEPCK promoter (21). The GRU (Fig. 1) is Х100 bp and consists of two glucocorticoid receptor binding sites GR1 and GR2, located between positions Ϫ395 and Ϫ349, flanking two accessory factor elements termed AF1 and AF2. AF1 (Ϫ455 to Ϫ431) is a * This work was supported in part by a career development award from the Juvenile Diabetes Foundation International and by National Institutes of Health Grant DK50272 (to J. E. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a Howard Hughes fellowship for summer undergraduate research.
ʈ Supported by a summer research fellowship from the Diabetes Association of Greater Cleveland.
§ To whom correspondence should be addressed: retinoic acid response element, whereas the AF2 site (Ϫ420 to Ϫ403) contains the insulin response sequence (IRS, Ϫ416 to Ϫ407) believed to mediate the negative effects of insulin on the PEPCK promoter (15). Although the protein(s) that mediate the effects of insulin have not been identified, it is suggested that insulin may inhibit PEPCK gene transcription by altering the binding and/or activation of a putative insulin response factor(s) that occupies the IRS (16,17). Thus, a defect in the expression, binding, and/or phosphorylation of nuclear proteins acting through the IRS might be expected to impair the ability of insulin to suppress PEPCK gene transcription, thereby leading to increased gluconeogenesis and NIDDM.
In an attempt to explore the role of glucocorticoids and the IRS binding site to increased PEPCK gene transcription in a model of type II diabetes, we generated a cross between transgenic mice expressing 2.0 or 0.46 kb of PEPCK regulatory sequences containing a mutated or intact IRS into the C57BL/KsJ-db/ϩ mouse. Our results indicate that increased PEPCK reporter gene transcription parallels the onset of hyperglycemia in the genetically obese db/db mouse, despite severe hyperinsulinemia. The overexpression of genes coding for gluconeogenic enzymes is driven by glucocorticoids and is independent of the cis-acting IRS sequence of the PEPCK promoter.

EXPERIMENTAL PROCEDURES
Materials-ATP, CTP, GTP, yeast tRNA, proteinase K, and restriction enzymes were purchased from Boehringer Mannheim. [␣-32 P]dCTP (3000 Ci/mmol), [␣-32 P]UTP (3000 Ci/mmol), and GeneScreen Plus were purchased from NEN Life Science Products. RNase-free DNase I (1000 units) was obtained from Promega. All other reagents were of the highest purity available. The following segments of DNA were used as hybridization probes. PEPCK corresponded to a 1.1-kb PstI-PstI fragment from the 3Ј-end of PEPCK cDNA. The C-reactive protein gene (CRP) corresponded to a 6.2-kb genomic fragment containing the entire rabbit CRP gene. Tyrosine aminotransferase (TAT) was obtained from American Type Culture Collection (Rockville, MD) and corresponded to a 0.43-kb BamHI-BamHI fragment of human cDNA. ␤-Actin corresponded to a 1.4-kb EcoRI-EcoRI fragment of mouse cDNA. hGx probe was a 640-bp SmaI-SmaI fragment from the genomic human growth hormone gene. GLUT2 was a 1.7-kb EcoRI-EcoRI fragment of the 3Ј-end of rat cDNA (provided by Christopher B. Newgard, Southwestern Medical Center, Dallas). Glucose-6-phosphatase (Glu-6-Pase) probe was a 1.1-kb XbaI-PstI fragment of human cDNA (provided by Dr. Janice Yang Chou, Genetics Branch, NICHD, National Institutes of Health). c-fos probe was a HindIII-BamHI 2.2-kb fragment from rat cDNA (provided by Tom Curran, Roche Pharmaceuticals, Nutley, NJ). 18 S ribosomal cDNA was a 5.8-kb HindIII-HindIII digest from rat cDNA. Anti-insulin receptor substrate 1 (IRS-1) and insulin receptor ␤ subunit (IR-␤) were obtained from Signal Transduction Laboratories (Lexington, KY). PI3-kinase (p85) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Animals-Two transgenic mouse lines were used to study the effects of obesity and diabetes on expression from the PEPCK promoter. The first utilized homozygous transgenic C57B6/SJL mice (a hybrid of C57BLK ϫ SJL) containing sequences from Ϫ460 to ϩ73 bp of the rat cytosolic PEPCK gene ligated to the rabbit CRP gene, PEPCK(460)-CRP, obtained from previously established transgenic lines (Dr. D. Samols, Department of Biochemistry, Case Western Reserve University). The production and characterization of PEPCK(460)-CRP trans-genic mice have been described in detail previously (18). Transgene expression in this model is directed by the Ϫ460 bp of the 5Ј-regulatory region of the PEPCK promoter and has been found to contain all of the necessary elements for developmental, hormonal (including diabetes), and dietary control in the liver in a manner similar to the endogenous PEPCK gene promoter (14, 19 -21). The second line of transgenic mice, termed PEPCK(IRS)-hGx, utilized a Ϫ2000-bp SacI-BglII 5Ј-flanking region of the rat cytosolic form of the PEPCK promoter containing a block mutation in the IRS (Ϫ416/Ϫ406). The larger Ϫ2000 promoter regulatory region was chosen to study regulation of reporter gene expression in adipose tissue that requires elements outside of Ϫ460 for tissue-specific expression (21)(22)(23). The PEPCK promoter was ligated to hGx. The hGx reporter gene contains a 2.2-kb BamHI-EcoRV sequence with a frameshift mutation in exon 5 of hGx, affecting the carboxylterminal 55 amino acids, resulting in a mutated hGx gene that does not produce active growth hormone (24, and present results). Transgenic mice were identified by Southern blot analysis, using a random primed 32 P-labeled 2.2-kb BamHI-EcoRV hGx gene fragment to probe samples of DNA isolated from mouse tails (25). Founder transgenic mice were bred to non-transgenic B6/SJL mice to generate male and female heterozygous mice. Heterozygous male and females from each line were bred to homozygosity and propagated up to 8 -10 generations. Three transgenic lines carrying an intact IRS, PEPCK(Ϫ2000)-hGx, and three lines carrying a mutation in the IRS of the PEPCK promoter, PEPCK-(IRS)-hGx, were studied. Basal reporter gene expression was similar in livers of mice carrying the intact PEPCK promoter compared with mice with a mutation in the IRS. hGx mRNA expression was characterized by Northern blot analysis in all six lines in response to feeding a high carbohydrate diet (decreased expression) or following a high protein diet (increased expression). There were no significant differences in response to dietary carbohydrate or protein in three lines of mice carrying the PEPCK(IRS)-hGx gene compared with induction and suppression of hGx in transgenic mice expressing the intact promoter. 2 Introduction of PEPCK Transgene into C57BL/KsJ-db/ϩ Mice-To study the effects of obesity and type II diabetes on promoter function, mice homozygous for the autosomal recessive db gene containing either intact or mutated promoter were produced using the breeding procedure outlined previously for outcrossing db/db mice (26). Because the recessive db mutation produces sterility in homozygotes, it was propagated by mating db/ϩ heterozygotes with homozygous transgenic mice. Based on the average basal level of expression and results from dietary manipulation, a representative transgenic line containing the intact promoter or a mutation in the IRS was chosen for breeding the PEPCK transgene onto the db/db background. Female C57BL/KsJ-m/Ϫdb/ϩ and db/db mice were obtained from Jackson Laboratories at 4 weeks of age and housed within the Case Western Reserve University animal resource facility. To produce homozygous db/db mice with the desired transgene, db/ϩ breeder females were mated with homozygous transgenic PEPCK(460)-CRP or PEPCK(IRS)-hGx males, and the F1 offspring were mated with known db/ϩ mice. Because the reporter gene is expressed in heterozygous transgenic mice, all F1 offspring expressed the reporter gene; however, only one out of four contained the db/ϩ locus. By test-crossing selected F1 offspring with breeder db/ϩ mice and observing the appearance of obesity and diabetes in the F2 generation, we identified transgenic mice carrying the db/ϩ locus. The compound db/ϩ transgenic offspring were back-crossed with known db/ϩ breeder mice up to five generations. Offspring were screened for the presence of the PEPCK transgene using DNA obtained from tail biopsies. DNA was extracted and Southern blotting performed using a random primed 32 P-labeled probe for identifying either CRP or hGX. Mice had free access to standard rat chow. Body weight was measured weekly beginning at 4 weeks of age, and fed and fasting plasma glucose were measured in venous blood from tail vein or the retro-orbital sinus as standard parameters for characterizing the appearance of the obesediabetic phenotype. Age-and sex-matched transgenic non-obese littermates (designated as ϩ/? at the db locus) were used as controls.
Treatment with RU 486 -RU 486 (also known as RU 38486 and mifepristone) was kindly supplied by Dr. D. Martini of Roussel Uclaf (Paris). RU 486 was dissolved at a concentration of 25 g/l in 100% ethanol and an equal volume of 0.9% NaCl. We studied a series of dosages of 10, 25, and 30 g/g body weight in 12-week-old male db/ϩ, db/db, and transgenic mice. Mice were given food and water ad libitum before receiving an intraperitoneal injection of the vehicle or RU 486 at 4:30 p.m. and a second injection at 8:00 a.m. Vehicle-and RU 486-

FIG. 1. Schematic representation of the GRU located between
؊455 and ؊349 bp upstream of the rat PEPCK gene promoter (12,13). The GRU consists of two AF binding sites, AF1 and AF2, necessary for the full response to glucocorticoids, and two adjacent GR binding sites, GR1 and GR2. The AF2 site contains an IRS, Ϫ416 to Ϫ407, which mediates a negative effect of insulin on PEPCK gene transcription (15). treated mice were killed by cervical dislocation at 12:30 p.m., and 0.5 ml of blood was withdrawn immediately from the abdominal aorta for insulin and corticosterone assay. Preliminary experiments showed that a dose of RU 486 at 25 g/g of body weight had no effect on food intake overnight in db/db mice and showed a maximal effect on lowering blood glucose (data not shown). The liver was removed and 200 mg frozen in liquid nitrogen for RNA isolation.
Isolation of Total RNA and Northern Blot Analysis-RNA was isolated from frozen mouse liver by the guanidine thiocynanate-phenol method of Chomczynski and Sacchi (27). For Northern blot analysis total RNA samples (20 g) were size fractionated in 0.9% agaroseformaldehyde gels and transferred to nitrocellulose filters. After washing with 2 ϫ SSC, the filter was dried and baked for 2 h at 80°C. Hybridization was carried out overnight at 65°C in Church buffer (1% bovine serum albumin, 1 mmol/liter EDTA, 0.7% SDS, 0.25 mol/liter Na 2 HPO4, pH 8.0). Membranes were hybridized with probes labeled with [␣-32 P]dCTP to a specific activity of Ϸ10 9 cpm/g DNA using a random primed labeling kit according to the manufacturer's instructions (Boehringer Mannheim). After hybridization, the filters were washed with 2 ϫ SSC; 0.1% SDS at room temperature; and 0.2 ϫ SSC, 0.1% SDS at 65°C for 30 min. Membranes were placed in contact with X-AR05 film (Eastman Kodak), and the image intensity of the autoradiogram was determined using a Sci-Scan 5000 laser densitometer (U. S. Biochemical Corp.). Levels of specific transcripts were estimated by quantitating probe-specific signals. Hybridization to ␤-actin or 18 S RNA was used to correct for differences in RNA content/loading.
Western Blot Analysis-Mice were anesthetized with chloralose (40 mg/kg), and the liver was removed, minced coarsely, and homogenized immediately in a 10 ϫ volume of solubilization buffer A (50 mM Hepes, pH 7.5, 137 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 2 mM Na 3 VO 4 , 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 2 g/ml aprotinin, 10 g/ml antipain, 5 g/ml leupeptin, 0.5 g/ml pepstatin, 1.5 mg/ml benzamidine, 34 g/ml phenylmethylsulfonyl fluoride) with a Polytron PTA 20S generator at maximum speed for 30 s. The homogenate was then centrifuged at 65,000 rpm at 4°C in a model Ti-70 rotor (Beckman Instruments, Inc.) for 60 min to remove insoluble material, and the supernatant was used for analysis. Protein was measured using the Bradford procedure (Pierce Biochemical). For IR-␤, IRS-1, and PI3-kinase, 40 g of protein was treated with Laemmli sample buffer containing 100 mM dithiothreitol, heated in a boiling water bath for 4 min, and subjected to SDS-polyacrylamide gel electrophoresis on a 7% Tris-acrylamide gel using a Bio-Rad mini-protein gel apparatus at 100 volts for 1 h. Proteins were electrotransferred from the gel to nitrocellulose at 90 V (constant) for 1 h using a mini-transfer apparatus. Nonspecific protein binding to the filter was blocked using 5% milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20. The polyvinylidene difluoride filter was incubated with antibodies to IR-␤ (1.5 g/ml), IRS-1 (1.5 g/ml), or PI3-kinase (0.75 g/ml) diluted in blocking buffer for 4 h at 22°C, followed by extensive washing with Tris-buffered saline (150 mM NaCl, 10 mM Tris ϩ Tween 20). At the end of the final wash, the blots were incubated with secondary antibody linked to horseradish peroxidase in 10 ml of blocking buffer for 1 h at 22°C and washed again before exposing the membranes to enhanced chemiluminesence reagent (Amersham). Autoradiography was carried out using Kodak X-AR x-ray film. The specific band intensities were quantitated by optical densitometry using a Digiscan scanner (U. S. Biochemical Corp.) for integrating the autoradiographic signals.
Plasma Hormones and Metabolites-Blood glucose levels were determined in samples using an Accu-Check II glucose monitor (Boehringer) and a glucose-oxidase assay kit (Sigma). The plasma immunoreactive insulin concentration was determined by radioimmunoassay using a rat insulin radioimmunoassay kit (Linco, Inc., St. Louis) using rat insulin as standard. The circulating level of rabbit CRP in transgenic mice was determined in blood samples collected by retro-orbital bleeding and a radial immunodiffusion assay in agarose, as described previously (18), using a goat anti-rabbit CRP antiserum specific for native rabbit CRP. This method is sensitive to levels as low as 1-2 g/ml and does not detect murine CRP. Plasma levels of corticosterone were assayed using using a rat corticosterone radioimmunoassay kit according to the manufacturer's instructions (ICN Biomedicals, Costa Mesa, CA). Glycerol was measured in deproteinized plasma samples by enzymatic determination using glycerol kinase-coupled with glycerophosphate dehydrogenase according to the conditions supplied by the manufacturer (Boehringer Mannheim).
Presentation of Data and Statistical Analysis-All data are presented as means Ϯ S.E. Specific mRNA levels were calculated by expressing the effects of perturbations as a percentage of readings in paired samples from non-treated or vehicle-treated control samples in the same Northern blot or transcription assay after correction for loading differences with signal from ␤-actin or ribosomal mRNA. Statistical analyses were performed using analysis of variance between treatments or groups. Differences were considered statistically significant at p Ͻ 0.05.

RESULTS
Obesity and Hyperglycemia in db/db Transgenic Mice-We introduced two separate PEPCK promoter-reporter genes from transgenic C57B6/SJL mice into the C57BL/KsJ-db strain, the background strain in which the db gene interacts to produce diabetes, obesity, and hyperinsulinemia. Data presented in Table I show the effects of the experimental cross on the development of obesity and diabetes in the PEPCK(460)-CRP, db/db mice. After five successive back-crosses between transgenic db/ϩ mice with breeder db/ϩ mice, more than 100 progeny were produced from which a group of 10 male transgenic db/db and 8 male transgenic ϩ/? mice were obtained for analysis. The transgenic PEPCK(460)-CRP, db/db mice demonstrated a developmental pattern of diabetes similar to that of C57BL/KsJ-db/db mice (9,28). At four weeks of age, PEPCK(460)-CRP, db/db mice were significantly heavier (p Ͻ 0.01) and had a 12-fold greater insulin level than their lean transgenic PEPCK(460)-CRP, ϩ/? littermates. However, plasma glucose and CRP (from the PEPCK promoter) were similar in transgenic PEPCK(460)-CRP, db/db and PEPCK(460)-CRP, ϩ/? mice, suggesting that higher plasma insulin levels in PEPCK(460)-CRP, db/db mice did not suppress PEPCK or CRP gene expression but were still capable of preventing hyperglycemia. At 10 weeks of age, hyperinsulinemia increased further by 1.75-fold (p Ͻ 0.01) in transgenic PEPCK(460)-CRP, db/db mice compared with 4-week-old PEPCK(460)-CRP, db/db mice. Fasting plasma glucose increased 2.8-fold (p Ͻ 0.01) above levels at 4 four weeks, and the levels of plasma CRP increased by 1.7-fold (p Ͻ 0.01), suggesting a transition to a state of hepatic insulin resistance and a failure of hyperinsulinemia to suppress expression from the PEPCK promoter.
The source of higher levels of plasma CRP noted in 10-weekold transgenic PEPCK(460)-CRP, db/db mice was identified by Northern blot analysis of RNA from the livers of the transgenic mice at 12 weeks of age (Fig. 2). The expression of both PEPCK and CRP mRNA was increased significantly by 2-fold (p Ͻ 0.01) in PEPCK(460)-CRP, db/db compared with PEPCK(460)-CRP, ϩ/? mice, thus confirming that the increased plasma levels of CRP most probably resulted from increased activity from the PEPCK gene promoter during the postweaning period, uninhibited by the excessive plasma insulin levels.
Effects of GR Antagonist RU 486 on Serum Parameters-Plasma corticosterone levels (Table II) in non-transgenic db/db mice were increased by 427% compared with db/ϩ mice (p Ͻ 0.01). To determine whether hypercorticism in the db/db mouse might underlie the mechanism for insulin resistance and increased PEPCK and CRP mRNA gene expression, we administered the synthetic glucocorticoid antagonist RU 486 to db/db and db/ϩ mice and later to PEPCK(460)-CRP, db/db mice. RU 486 was tested at doses 0.1, 0.25, and 0.5 mg/kg (data not shown) and found to have its maximal effect on blood glucose at a dose of 25 mg/kg of body weight. RU 486 treatment significantly increased the serum corticosterone levels in db/db and db/ϩ mice 2-11-fold over vehicle-treated controls, respectively (p Ͻ 0.01), indicative of GR blockade. RU 486 or vehicle treatment had no significant effects on plasma glucose in db/ϩ mice.
However, in hyperglycemic db/db mice, RU 486 decreased blood glucose levels by 49% (p Ͻ 0.01) with no change in plasma insulin levels. A similar result was obtained in PEPCK(460)-CRP, db/db mice (not shown). To determine whether RU 486 treatment resulted in a change in lipolysis, plasma glycerol levels were measured in db/ϩ and db/db mice. In the db/db mouse, plasma glycerol was elevated slightly by 25% compared with db/ϩ mice; however, there was no change after RU 486 treatment.
Effects of RU 486 on Liver Gene Expression in db/db Mice-To determine whether reducing hyperglycemia with RU 486 was associated with a coordinated change in expression of several other hepatic genes involved in glucose production, we measured hepatic mRNA for PEPCK, GLUT-2, Glu-6-Pase, TAT, and ␤-actin in db/ϩ and RU 486-treated db/db mice (Fig.  3). PhosphorImager quantitation of these results is presented  1 and 2), db/db vehicle-treated (lanes 3 and 4), and db/db mice hours after treatment with anti-glucocorticoid RU 486 (lanes 5 and 6) as outlined under "Experimental Procedures." Signals showing hybridization for PEPCK, GLUT2, CRP, Glu-6-Pase, TAT, and ␤-actin mRNAs are shown. in Fig. 4 and show that PEPCK mRNA concentration in the liver of db/db mice was increased 220 Ϯ 25% relative to db/ϩ control animals (p Ͻ 0.01), whereas RU 486 administration reduced the concentration of PEPCK mRNA in db/db to 125 Ϯ 22% of db/ϩ control mice. RU 486 had no effect on PEPCK gene expression in db/ϩ mice. The level of Glu-6-Pase mRNA expression in db/db mice was increased by 175 Ϯ 26% over db/ϩ controls, whereas RU 486 reduced the concentration of Glu-6-Pase mRNA to 89 Ϯ 23% of controls (p Ͻ 0.05). The level of GLUT2 mRNA in the liver of db/db mice was increased by 370 Ϯ 37% relative to db/ϩ controls (p Ͻ 0.05), and RU 486 reduced the concentration of GLUT2 mRNA expression to 120 Ϯ 17% of controls (p Ͻ 0.05). TAT mRNA concentration was increased in livers of db/db by 150 Ϯ 29% (p Ͻ 0.05) and was reduced to 110 Ϯ 18% of controls after RU 486 treatment.

Effect of a Mutation in the PEPCK IRS on Reporter Gene Expression in Transgenic Obese
Diabetic db/db Mice-In light of the increase in expression from the PEPCK promoter in PEPCK(460)-CRP db/db mice and reduction in transcription with RU 486 (Figs. 2 and 3), we hypothesized that increased PEPCK gene transcription in db/db might be caused by glucocorticoid-dependent interactions that stimulate transcription from the PEPCK promoter through factors binding at the IRS. The PEPCK(IRS)-hGx ϫ db/ϩ back-cross produced 86 offspring, of which 34 expressed the transgene, and 9 developed severe obesity and fasting hyperglycemia. At 12 weeks of age, the transgenic PEPCK(IRS)-hGx, db/db mice had 3.3-fold higher fasting glucose levels (p Ͻ 0.01) and were significantly heavier than their PEPCK(IRS)-hGx, ϩ/? littermates, and they exhibited similar fed and fasting hyperglycemia compared with db/db mice. In obese hyperglycemic transgenic PEPCK(IRS)-hGx, db/db mice treated with RU 486, blood glucose decreased by 53% from 342 to 159 mg/dl (p Ͻ 0.01). In the livers of obese diabetic transgenic PEPCK(IRS)-hGx, db/db mice, the level of hGx reporter gene expression increased 2-fold over normoglycemic PEPCK(IRS)-hGx, ϩ/? Mice (Fig. 5). PhosphorImager quantitation of the relative levels of PEPCK or reporter gene expression in obese hyperglycemic transgenic mice expressing either CRP or hGx is presented in Fig. 6; the relative increase in PEPCK or reporter gene expression is very similar in both lines of hyperglycemic transgenic mice and is reduced to levels similar to ϩ/? mice after RU 486 treatment. Levels of CRP mRNA in obese diabetic mice were increased 225 Ϯ 26% above normoglycemic transgenic PEPCK(460)-CRP, ϩ/? mice. CRP mRNA in RU 486-treated mice was significantly reduced to 135 Ϯ 15% above normoglycemic transgenic mice (p Ͻ 0.05). The levels of expression of hGx mRNA in obese diabetic transgenic mice were increased 201 Ϯ 18% above control PEPCK-(IRS)-hGx, ϩ/? mice (p Ͻ 0.05), and they were reduced to values similar to those of control non-obese non-diabetic mice after RU 486 treatment.
Effect of RU 486 on Levels of IR-␤, IRS-1, and PI3-Kinase (p85) Expression-To determine whether blocking the action of the GR affected the levels of insulin signaling intermediates IR-␤, IRS-1, and the 85-kDa regulatory subunit of PI3-kinase, Western blots were performed with liver cell extracts from vehicle-treated and RU 486-treated db/ϩ and db/db mice (Fig.  7). Compared with db/ϩ mice, the levels of IR-␤, IRS-1, and p85 in db/db mice were reduced by 42, 48, and 61, respectively (p Ͻ 0.01). Upon treatment with RU 486, there were no significant changes in the pattern of expression of any of these signaling proteins in either db/ϩ or db/db mice, suggesting that blocking FIG. 4. Densitometric quantitation of changes in liver PEPCK, CRP (from the PEPCK transgene), Glu-6-Pase, GLUT2, and TAT mRNA levels in C57BL/KsJ-db/db and transgenic db/db mice after treatment with RU 486. The relative levels of mRNA were expressed as a percentage of mRNA hybridization in liver from db/ϩ control mice detected on the same Northern blot after correction for ␤-actin to account for loading differences. Results are mean Ϯ 1 S.E. for C57BL6/KsJ, db/ϩ (n ϭ 12), C57BL6/KsJ, db/db (n ϭ 12), C57BL6/KsJ, db/db ϩ RU 486 (n ϭ 12). The plus signs (ϩ) indicate significance greater than db/ϩ controls, p Ͻ 0.05; asterisks (*) indicate significance reduced compared with db/db vehicle-treated, p Ͻ 0.05. the action of the GR did not correct the decrease in expression of insulin signaling proteins in db/db mice. DISCUSSION The present results suggest that elements located in the proximal Ϫ460 bp of the PEPCK promoter are of major importance in up-regulating PEPCK gene expression in the hyperinsulinemic obese diabetic db/db mouse. The elevated plasma CRP levels observed during the onset of hyperglycemia in transgenic PEPCK(460)-CRP, db/db mice and the fact that the half-life for clearance of circulating rabbit CRP in transgenic mice is 45 min (18) suggest a shift in hepatic metabolism during the postweaning stage and increases in transcription from the PEPCK gene promoter. Our studies suggest that interactions between the homozygous mutant leptin receptor db gene and transcriptional modifier gene(s) or protein(s) expressed in C57BL/KsJ background stimulate PEPCK gene transcription and increase hepatic gluconeogenesis and diabetes between 4 and 10 weeks of age. The genetic basis for susceptibility to NIDDM in the db/db mouse model bearing a mutation in the leptin receptor has been related to strainspecific differences in glucose metabolism and pancreatic ␤ cell function (29). However, it is difficult to distinguish between the pleiotropic effects of the mutation in the leptin receptor and the secondary effects of increased adiposity. To distinguish effects resulting from direct interactions between the NIDDM susceptibility genes in C57BL6/KsJ background and the db mutation from those resulting from obesity per se will require the identification of quantitative trait loci, i.e. genetic modifiers for NIDDM in this strain and subsequent outbreeding into other rodent models of genetic or diet-induced obesity.
Adrenalectomy has been shown to lessen or reverse many of the metabolic abnormalities found in genetic models of obesity and insulin resistance, including hyperglycemia in the C57BL/ KsJ-db/db mouse (30). We found that the steady-state levels of the mRNA coding for PEPCK, GLUT-2, Glu6-Pase, and TAT in the db/db mouse were elevated at 12 weeks of age and were reduced significantly after treatment with RU 486. Previous studies have shown that RU 486 administered chronically to genetically obese Zucker fatty fa/fa rats reduces hyperphagia and weight gain (31,32), whereas in rats made insulin-resist-ant with high fat feeding, RU 486 has been shown to reduce significantly whole-body and skeletal muscle insulin resistance without altering food intake, body weight, or insulin levels (33). Our results demonstrate that RU 486 can reduce hyperglycemia by decreasing glucocorticoid-regulated transcription of gluconeogenic enzymes independently of changes in insulin levels. Gluconeogenesis is also stimulated in part by increased levels of free fatty acids, which result in increased lipid oxidation, greater intramitochondrial concentrations of acetyl-CoA, and subsequent activation of pyruvate carboxylase (34,35). Thus, it is reasonable to assume that chronically elevated free fatty acids and glycerol, derived from adipose tissue, may also play an important role in promoting insulin resistance and increased gluconeogenesis. We found that the basal levels of plasma glycerol, a sensitive indicator of lipolysis, were slightly higher in the obese db/db mouse compared with db/ϩ controls, but RU 486 did not alter the circulating levels of plasma glycerol in lean or obese animals. These data suggest the reduction in blood glucose in the db/db mouse is most likely the direct effect of RU 486 on lowering hepatic enzyme gene expression rather than a decrease in lipolysis.
Because a mutation of the IRS in the PEPCK promoter has been shown to inhibit glucocorticoid-stimulated PEPCK gene transcription in vitro, we wanted to explore whether this regulatory element was involved in the induction of PEPCK gene expression in type II diabetes in the physiological context of the whole animal. It was hypothesized that mutating this element might actually reduce reporter gene expression during the onset of hyperglycemia because glucocorticoid induction of transcription from the PEPCK promoter is decreased by 50% in H4 cells transfected with a PEPCK-CAT (chloramphenicol acetyltransferase) promoter containing a mutation of the IRS (12). However, in transgenic PEPCK(IRS)-hGx, db/db mice, we found that reporter gene expression increased almost identically to the endogenous PEPCK gene, and RU 486 reduced expression from the promoter to levels similar to those found in lean ϩ/Ϫ mice. These results suggest that increased PEPCK gene transcription in db/db mice is driven by glucocorticoid-dependent interactions that are distinct from pathways terminating at the PEPCK IRS. We found previously that a deletion of the GRU (containing the IRS) prevented increased PEPCK gene expression in transgenic diabetic mice, whereas a combined block mutation in the cAMP-regulatory regions (Ϫ87/ Ϫ74) and P3 (Ϫ248/Ϫ230) of the PEPCK promoter had no effect on induction of reporter gene transcription in transgenic diabetic mice (14). Combined with our previous results, the present study suggests that increased PEPCK gene transcription in diabetes most likely results from activation of the GR involving the GR1 and GR2 sites but not the IRS of the PEPCK promoter.
Recently, the PI3-kinase inhibitor wortmannin has been shown to block the effects of insulin on PEPCK gene transcription in H4 cells (36,37), suggesting that decreased expression of these downstream insulin signaling intermediates is an important mechanism for the insulin resistance to gene expression (38,39). We found a significant decrease in the levels of IRS-1 and PI3-kinase (p85) protein (Fig. 6) which accounted for reduced tyrosine phosphorylation in IRS-1 and p85 after insulin stimulation in liver of db/db mice in vivo. 3 However, we found no change in the levels of IR-␤, IRS-1, or PI3-kinase (p85) protein in the liver of db/ϩ or db/db mouse after RU 486 treatment (Fig. 6). This suggests that the decrease in PEPCK gene transcription after RU 486 treatment may be caused by corrections in distal signaling pathways beyond IRS-1 and PI3-kinase which regulate transcription from the PEPCK promoter 3 T. Ishizuka and J. E. Friedman, unpublished observation. Proteins were extracted and resolved on a 6% SDS-polyacrylamide gel, transferred to nitrocellulose, and blotted with specific antisera. Scanning densitometry of autoradiograms was performed in three experiments performed on six mice from each group. The data are expressed as the mean Ϯ S.E. and are expressed relative to vehicle-treated db/ϩ control mice. Asterisks (*) indicate significant decrease compared with the db/ϩ vehicletreated group, p Ͻ 0.05. and reduce gluconeogenesis.
In most genetic models of obesity and type II diabetes, excess corticosterone is required to achieve hyperglycemia and increased gluconeogenesis; however Valera and Bosch (40) showed that transgenic mice overexpressing a PEPCK minigene driven by the PEPCK promoter developed hyperinsulinemia and chronic fasting hyperglycemia, suggesting that solely increasing PEPCK activity in the liver can increase gluconeogenesis, which results in hyperglycemia and a failure of hyperinsulinemia to down-regulate PEPCK gene expression in experimental animals. Although no evidence exists to suggest that the PEPCK promoter regulatory region is mutated in families with diabetes (41), it is possible that acquired or genetic defects in insulin or other signal transduction pathways could lead to increased PEPCK gene expression and gluconeogenesis because of a failure of insulin to modify one or more DNA-binding factors that regulate transcription from the PEPCK gene promoter. It is important to recognize, however, that gluconeogenesis is a coordinated pathway and that sites other than PEPCK may also contribute to stimulating increased hepatic glucose production, such as increased substrate delivery. However, inasmuch as the PEPCK gene is a major regulatory control point for gluconeogenesis and is highly resistant in hyperinsulinemic models of NIDDM, it represents a prime candidate for understanding the mechanisms whereby type II diabetes induces aberrant trans-regulation of otherwise normal genes coding for key enzymes of carbohydrate metabolism.