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Endocrinology Vol. 147, No. 11 5061-5068
Copyright © 2006 by The Endocrine Society

Liver X Receptor Agonist T0901317 Inhibition of Glucocorticoid Receptor Expression in Hepatocytes May Contribute to the Amelioration of Diabetic Syndrome in db/db Mice

Yanjun Liu, Chaoying Yan, Ying Wang, Yuichi Nakagawa, Namiko Nerio, Adrian Anghel, Kabirullah Lutfy and Theodore C. Friedman

Division of Endocrinology (Y.L., Y.W., N.N., A.A., T.C.F.), Charles R. Drew University of Medicine and Sciences, University of California, Los Angeles, School of Medicine, Los Angeles, California 90059; Department of Pediatrics (C.Y.), First Hospital, JiLin University, ChangChun130021, China; Department of Pediatrics (Y.N.), Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan; and Department of Pharmaceutical Sciences (K.L.), College of Pharmacy, Western University of Health Sciences, Pomona, California 91766

Address all correspondence and requests for reprints to: Yanjun Liu, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine. Charles R. Drew University of Medicine and Sciences, University of California, Los Angeles, School of Medicine, 1731 East 120th Street, Los Angeles, California 90059. E-mail: dryanjunliu{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The glucocorticoid receptor (GR) is a crucial target gene for glucocorticoid-induced insulin resistance and hepatic gluconeogenesis linked to the development of type 2 diabetes. The liver X receptors (LXRs) are nuclear receptors that play an important role in the regulation of the metabolic gene linked to carbohydrate homeostasis. To assess the tissue-specific interaction of LXR with GR in the development of type 2 diabetes, we examined the possible effect of LXR agonist T0901317 on GR gene expression in vivo and in vitro in hepatocytes from db/db mice (a model of type 2 diabetes). Chronic ligand activation of LXR by a synthetic LXR T0901317 markedly decreased the expression of both GR mRNA and its protein in liver and improved the phenotype of type 2 diabetes in obese db/db mice. Suppression of hepatic GR expression was correlated with reduced levels of glucose and corresponded to the inhibition of phosphoenolpyruvate carboxykinase mRNA and 11ß-hydroxysteroid dehydrogenase type 1-mediated synthesis of active corticosterone from inactive 11-dehydrocorticosterone in liver. Treatment of db/db mouse primary hepatocytes with T0901317 resulted in dramatic suppression of GR mRNA and required ongoing protein synthesis. Addition of T0901317 to primary hepatocytes also suppressed the expression of both 11ß-hydroxysteroid dehydrogenase type 1 and phosphoenolpyruvate carboxykinase. These findings suggest that some of antidiabetic actions of LXR agonist T0901317 may be mediated, at least in part, through the suppression of hepatic GR gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLUCOCORTICOID RECEPTOR (GR) is a ligand-dependent transcriptional factor. At the cellular level, GR confers tissue-specific responsiveness to circulating corticosteroids and thus determines tissue sensitivity to glucocorticoids (1, 2). Increased glucocorticoid production induces obesity and type 2 diabetes (Cushing’s syndrome) via activation of intracellular GR, which mediates hypercortisolemia-related glucose intolerance and insulin resistance (3, 4). Importantly, activation of GR itself also promotes expression of phosphoenolpyruvate carboxykinase (PEPCK), a crucial enzyme in hepatic gluconeogenesis that is sufficient to cause hyperglycemia seen in diabetic animals (3, 4, 5). Moreover, increased skeletal muscle GR expression is thought to be an important component in the development of human insulin resistance and metabolic syndrome (6). Similarly, increased hepatic GR mRNA expression is positively correlated with the induction of insulin resistance, PEPCK mRNA expression, and hyperglycemia in obese Zucker rats and diabetic db/db mice (7, 8). In contrast, pharmacological antagonists of GR prevented the development of type 2 diabetes in these animal models of type 2 diabetes as well as in patients with Cushing’s syndrome (9, 10, 11, 12). Moreover, liver-specific GR knockout mice showed reduced expression of PEPCK mRNA and were resistant to streptozotocin-induced hyperglycemia (13). These studies implicate that tissue-specific alteration of GR may be crucial for the induction of insulin resistance and obesity and that GR may be a potential target for the treatment of type 2 diabetes.

The liver X receptors-{alpha} and -ß (LXRs) belong to a subclass of nuclear hormone receptors that are oxysterol ligand-activated transcription factors (14). The main target tissues for LXR{alpha} expression are liver and adipocytes, whereas LXRß is expressed ubiquitously. Activation of LXR have been shown to play a key role in the regulation of gene expression linked to carbohydrate homeostasis and to serve as the molecular target for the treatment of hyperglycemia and insulin resistance (15, 16, 17). In the liver, LXRs control lipid homeostasis through modulation of several genes involved in lipid and cholesterol metabolic pathways (18, 19, 20). Additionally, LXR agonists decrease the expression of critical enzymes involved in hepatic gluconeogenesis including suppression of PEPCK and glucose-6-phosphatase (G6P) (15, 21, 22). Importantly, suppression of PEPCK expression by LXR activation may also be involved in the regulation of GR signaling action because there is a positive relationship between PEPCK and GR expression in the control of hepatic gluconeogenesis linked to the development of type 2 diabetes (5, 7, 9). Moreover, LXRs could indirectly inhibit hepatic gluconeogenesis in normal mice by reducing the activity of 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) (23), a key enzyme in determining intracellular prereceptor glucocorticoid metabolism by regenerating active GR-ligand corticosterone (cortisol in human) from inactive 11-dehyderocorticosterone (cortisone) in hepatocytes and adipocytes (24, 25, 26). These observations raise the possibility that the beneficial metabolic effects of LXR activation on glucose homeostasis and insulin sensitivity may be associated with GR action. However, little is known about LXR activation-induced tissue-specific changes in GR expression because no direct information is currently available on the interactions between the LXRs and GR in the control of insulin resistance and obesity.

In this study, we examined the potential effects of chronic LXR agonist T0901317 treatment on the regulation of both GR mRNA and protein expression in vivo and in vitro in hepatocytes from db/db mice and their lean controls. We also investigated the possible role of T0901317 in prereceptor metabolism of corticosterone in the liver of db/db mice. Finally, we tested the direct effects of T0901317 on GR expression in primary cultures of hepatocytes from db/db mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatment
Male C57BL/KsJ-obese (db/db) mice and their lean littermates (db/+) were purchased at 12 wk of age from The Jackson Laboratory (Bar Harbor, ME) and housed in a room illuminated daily from 0700 to 1900 h (12-h light, 12-h dark cycle) with free access to water and standard laboratory chow. All animal experiments were approved by the Institutional Animal Care and Use Committee at Charles R. Drew University. Db/db mice and lean db/+ controls were divided into four groups (seven animals per group): db/+ mice treated with vehicle, db/+ mice treated with T0901317, db/db mice treated with vehicle, and db/db mice treated with T09091317. LXR agonist T0901317 (30 mg/kg body weight, dissolved in 0.125% carboxymethyl cellulose) or an equal volume of vehicle was injected ip twice daily for 1 or 3 wk (27, 28, 29). Food intake and body weight were measured before the initiation of the treatment and on the last day of treatment. Blood samples were collected into heparin tubes and then stored at –80 C until measurement of blood glucose, insulin, and corticosterone concentrations. Liver tissue was removed and stored in liquid nitrogen until extraction and analysis of protein and mRNA.

Insulin tolerance test
For the insulin tolerance test, animals were fasted for 8 h, and a basal blood sample was taken, followed by an injection of insulin (1 U/kg, ip; Novolin R; Eli Lilly, Indianapolis, IN). Blood samples were drawn at different times after insulin injection.

Analysis of blood parameters
Blood glucose levels were determined by the glucose oxidase method. Plasma corticosterone was determined by RIA using mouse corticosterone as a standard (ICN Biomedicals, Costa Mesa, CA). Serum insulin levels were measured by RIA using rat insulin as a standard (Crystal Chemicals, Chicago, IL).

Cell culture and drug treatment
Primary hepatocytes were isolated from db/db mice by a two-step collagenase perfusion procedure as previously described (26). The free hepatocytes were seeded onto collagen-coated dishes in William’s E medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine and 10 mM HEPES. Hepatocytes were plated in 60-mm dishes (coated with collagen; Sigma, St. Louis, MO) and incubated at 37 C for 4 h. Cells were then washed with PBS, and the medium was changed to William’s E medium without fetal bovine serum. After 12 h, cells were treated with LXR agonist T0901317 (10–9 to 10–5 M) in the presence or absence of 1.0 µg/ml cycloheximide for 12–72 h.

RNA isolation and analysis of semiquantitative RT-PCR
Total RNA was isolated from hepatocytes by using a single-step extraction method (RNA-zol B). RNA samples were quantified by spectrophotometry and integrity was assessed by 1.5% agarose gel electrophoresis and ethidium bromide staining. The mRNA levels of GR, PEPCK, G6P, and 11ß-HSD1 were measured using semiquantitative RT-PCR, with primers specific for mouse GR (forward: 5'-TGCTATGCTTTGCTCCTGATCTG-3', reverse: 5'-TGTCAGTTGATAAAACCGCTGC-3'), PEPCK (forward: 5'-AGCCTCGACAGCCTGCCCCAGG-3', reverse: 5'-CCAGTTGTTGACCAAAGGCTTTT-3'), G6P (forward: 5'-AAGACTCCCAGGACTGGTTCATCC-3', reverse: 5'-TAGCAGGTAGAATCCAAGCGCG-3'), 11ß-HSD1 (forward: 5'-TTGATGGCAGTTATGAAAAAT-3', reverse: 5'-TACAGAAGTATCAGGCAGGAC-3'), and 18S RNA (forward: 5'-GTAACCCGTTGAACCCCATT-3', reverse: CCATCCAATCGGTAGTAGCG-3'). The amplification conditions of GR, PEPCK, and 11ß-HSD1 were adjusted in preliminary studies to result in amplification within the linear range, as described previously (8). PCR products were visualized on 2% agarose gels by ethidium bromide staining, and gels were imaged under UV light. Target gene expression was measured by densitometry with the Eagle Eye II quantitation system (Stratagene, La Jolla, CA) and was normalized to the signals of 18S cDNA.

Immunoblotting analysis (Western blot)
Liver tissues were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 nmol/liter Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors) from various treatment groups by sonication. Homogenates were centrifuged at 4 C at 12,000 x g for 10 min, and the supernatants were collected. Protein concentrations of the supernatant were measured with the Bradford assay (protein assay kit; Bio-Rad Laboratories, Hercules, CA). In primary cultures of hepatocytes, cells were washed three times with ice-cold PBS and disrupted in ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 200 µM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µM pepstatin A, 40 mg/liter bestatin, 2 mg/liter aprotinin, 1% Triton X-100] (6, 30) to enable isolation of nuclear and cytosolic protein. Protein was boiled with sample buffer that contained 1% SDS and 10 mmol/liter Tris-HCl (pH 7.5), loaded, and separated on 8% SDS polyacrylamide gels and then electrophoretically transferred to Hybond-ECL nitrocellulose membranes. Subsequently membranes were blotted with blocking solution (5% skim milk in PBS containing 0.05% Tween 20) before GR protein was detected using a polyclonal anti-GR antibody at 1:200 dilution (Affinity BioReagents, Golden, CO). The bound primary antibody was visualized using ECL system (Amersham Biosciences, Piscataway, NJ). Densitometry was performed to determine the intensity of the GR protein signal relative to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Measurement of glucose uptake in liver
Glucose uptake was estimated in vivo by measuring the incorporation of [1,2-3H]deoxy-D-glucose (2-[3H]DG) as described previously (31). Briefly, after a 6-h fast, 2-[3H]DG (NEN Life Science Products, Boston, MA) (1.0 µCi/kg body weight) was administered as an iv bolus. Animals were killed 1 h after receiving the 2-[3H]DG. The livers were removed, cleaned, weighed, and solubilized with tissue solubilizer (Amersham, Arlington Heights, IL). Radioactivity was measured in the samples using a liquid scintillation counter.

Measurement of 11ß-HSD1 activity in liver homogenates
The 11ß-HSD1 activities were measured by a minor modification of previous reports (25, 26, 27). Briefly, liver tissue was homogenized in Krebs-Ringer buffer solution at 4 C in a Dounce tissue grinder. Protein concentrations were measured with the Bradford assay, and the supernatants were diluted to yield uniform protein concentrations. Liver homogenates were incubated with 2 µmol/liter 3H-corticosterone ({3H]B) (specific activity, 90 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA), 200 µM nicotinamide adenine dinucleotide phosphate (NADP) (for 11ß-dehydrogenase activity) or 2 µmol/liter 3H-11-dehydrocorticosterone ([3H]DHC) and 200 µM nicotinamide adenine dinucleotide phosphate reduced (NADPH) (for 11ß-reductase activity) using 0.25 mg protein/ml in Krebs-Ringer buffer solution [containing 0.2% BSA and glucose (pH 7.4)] at 37 C for 10 min in a shaking bath. Steroids were separated by thin-layer chromatography and analyzed by scintillation counting. The conversion of [3H]B to [3H]DHC or [3H]DHC to [3H]B was calculated from the radioactivity in each fraction.

Measurement of 11ß-HSD1 activity in hepatocytes
We have reported that 11ß-HSD1 functions mainly as a reductase to regenerate active corticosterone from inactive DHC in intact db/db mouse hepatocytes (26); thus, 11ß-HSD1 reductase activity was measured in primary cultures of db/db mouse hepatocytes by measuring the rate of conversion of [3H]DHC to [3H]B. Briefly, cells were incubated with 2 nmol/liter [3H]DHC with 18 nmol/liter unlabeled DHC at 10–30 min after the addition of steroids. After extraction from 0.5 ml culture media with ethyl acetate, steroids were separated by thin-layer chromatography and enzyme activity levels were estimated by radioactivity counting as described above. Protein concentrations were measured with the Bio-Rad protein assay kit. Enzyme activity was calculated as the percent conversion rate of [3H]DHC to [3H]B.

Statistical analysis
All data are expressed as the mean ± SEM. The normality of the distribution of date was established using Wilks-Shapiro test, and outcome measures between groups were compared by Student’s t test or ANOVA. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LXR agonist T0901317 attenuated the phenotype of type 2 diabetes in db/db mice
As shown in Table 1Go, chronic treatment of 12-wk-old male diabetic db/db mice with LXR agonist T0901317 lowered body weight (P < 0.05), blood glucose levels (P < 0.001), and plasma corticosterone levels (P < 0.005), with no significant changes in the levels of serum insulin, compared with those of vehicle-treated db/db controls. However, T0901317 treatment had no significant effects on body weight, blood glucose, insulin levels, or serum corticosterone levels in db/+ control mice (Table 1Go).


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  		TABLE 1. Body weight, blood glucose, serum insulin, and plasma corticosterone

 
LXR agonist T0901317 suppressed GR gene expression and the response to the reduction of hepatic PEPCK, G6P and 11ß-HSD1 expression
Quantitative RT-PCR analysis revealed that treatment of db/db mice with T0901317 significantly attenuated the increased hepatic GR mRNA expression observed in vehicle-treated db/db mice (Fig. 1AGo). This was confirmed by Western blot analysis, which revealed that hepatic GR protein expression in T0901317-treated db/db mice was decreased to 45% of vehicle-treated db/db mice (Fig. 1BGo). Treatment of db/db mice with T0901317 significantly decreased the mRNA expression levels of both PEPCK and G6P within liver, compared with those of vehicle-treated db/db mice (Fig. 2AGo). However, treatment of db/+ mice with T0901317 was without effect on hepatic PEPCK and G6P expression with no change in GR mRNA levels (Fig. 2AGo). Consistent with these observations, T0901317-treated db/db mice exhibited a 2.5-fold increased in 2-[3H]DG uptake in liver tissue, compared with vehicle-treated db/db mice (P = 0.00019) (Fig. 2BGo), but in db/+ mice, there was no effect. Moreover, an insulin tolerance test showed that treatment of db/db mice with T0901317 significantly improved the glucose-lowering effect of insulin in comparison with that observed in db/db mice treated with vehicle (Fig. 2CGo). In addition, a shorter treatment of db/db mice with T0901317 for 1 wk also reversed the phenotype of insulin resistance and hyperglycemia, which correlated with reduction in hepatic GR, PEPCK, and G6Pase expression (data not shown). These results indicate that T0901317 improved the phenotype of type 2 diabetes in db/db diabetic mice was associated with reduction of hepatic GR expression.


Figure 1
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  		FIG. 1. Hepatic GR mRNA (A) and protein (B) expression in db/+ mice treated with vehicle (db/+), db/db mice treated with vehicle (db/db), and db/db mice treated with T0901317 (db/db + T0) for 3 wk. A, Expression and relative quantification of GR mRNA levels expressed relative to the amount of 18S rRNA. B, Expression and relative quantification of GR protein levels expressed relative to the amount of GAPDH. Values are mean ± SEM of seven mice/group. #, P < 0.001 vs. db/+ controls; *, P < 0.005 vs. db/db mice; **, P < 0.01 vs. db/db mice.

 

Figure 2
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  		FIG. 2. A, Relative quantification of changes in liver GR, PEPCK, and G6P mRNA levels in db/+ mice treated with vehicle (db/+), db/+ mice treated with T0901317 (db/+ T0), db/db mice treated with vehicle (db/db), and db/db mice treated with T0901317 (db/db + T0). B, 2-[3H]DG uptake by liver of db/+ mice treated with vehicle (db/+), db/+ mice treated with T0901317 (db/+ T0), db/db mice treated with vehicle (db/db), and db/db mice treated with T0901317 (db/db + T0). C, Insulin tolerance test in db/+ mice treated vehicle (bullet, db/+), db/+ mice treated with T001317 ({diamondsuit}, db/+ T0), db/db mice treated with vehicle ({blacksquare}, db/db), and db/db mice treated with T0901317 ({blacktriangleup}, db/db + T0). Blood samples were obtained from the tail at the indicated time and glucose levels were measured. Data are mean ± SEM of six mice/group. **, P < 0.001 vs. db/+ controls; #, P < 0.01 vs. T0-treated db/db mice; ##, P < 0.001 vs. db/db controls; {dagger}, P < 0.01 vs. db/db controls; ¶, P < 0.005 vs. db/db controls; *, P < 0.001 vs. db/db controls.

 
Consistent with decreased GR and PEPCK expression, treatment of db/db mice with T0901317 for 3 wk significantly attenuated hepatic 11ß-HSD1 activity, compared with that of vehicle-treated db/db mice, although it did not restore enzyme levels to that of lean controls (Fig. 3AGo). Quantitative RT-PCR analysis showed T0901317 treatment in db/db mice resulted in a 2.2-fold decrease in hepatic 11ß-HSD1 mRNA expression, compared with controls (Fig. 3BGo).


Figure 3
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  		FIG. 3. Hepatic 11ß-HSD1 activity and mRNA expression in db/+ mice treated with vehicle (db/+), db/db mice treated with vehicle (db/db), and db/db mice treated with T0901317 (db/db + T0) for 3 wk. A, Enzyme activity was expressed as percentage of [3H]B converted to [3H] DHC. B, Expression and relative quantification of mRNA levels was done relative to the amount of 18S rRNA. Data are mean ± SEM of six mice/group. **, P < 0.001 vs. db/+ controls; #, P < 0.01 vs. db/db mice; ##, P < 0.01 vs. db/+ controls.

 
The influence of LXR agonist T0901317 on GR gene expression in primary cultures of hepatocytes
To confirm our observations in vivo, we treated db/db mouse primary hepatocytes with T0901317 and measured GR mRNA and protein expression. As shown in Fig. 4Go, treatment of primary cultures of hepatocytes with increasing doses of T0901317 led to a concentration-dependent decrease in GR mRNA expression (Fig. 4AGo). The decrease in GR mRNA levels was paralleled by Western blotting analysis showing that T0901317 treatment resulted in a 2- to 3-fold decrease in GR protein levels in comparison with control levels in primary hepatocytes (Fig. 4BGo). Moreover, treatment of primary hepatocytes with T0901317 showed a time-dependent decrease in GR expression (Fig. 5Go, A and B).


Figure 4
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  		FIG. 4. Effects of T0901317 on the expression of GR mRNA and protein levels in primary cultures of hepatocytes from db/db mice. Hepatocytes were incubated with T0901317 for 72 h. A, Expression and relative quantification of GR mRNA levels relative to the amount of 18S rRNA. B, Expression and relative quantification of GR protein levels relative to the amount of GAPDH. Data are the mean ± SEM from three separate culture preparations. *, P < 0.01 vs. controls; **, P < 0.005 vs. controls; #, P < 0.05 vs. controls; {dagger}, P < 0.01 vs. controls; {ddagger}, P < 0.001 vs. controls.

 

Figure 5
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  		FIG. 5. Effects of T0901317 on the expression of GR mRNA (A) and protein levels (B) in primary cultures of hepatocytes from db/db mice. Hepatocytes were incubated with T0901317 (10 µM) for different times. A, Expression and relative quantitation of GR mRNA levels expressed relative to the amount of 18S rRNA. B, GR protein levels were determined by Western blot analysis and expressed relative to the amount of GAPDH found in controls. Values are the mean ± SEM from three separate culture preparations. *, P < 0.05 vs. controls; {dagger}, P < 0.01 vs. controls; #, P < 0.001 vs. controls.

 
LXR agonist T0901317-induced suppression of GR mRNA expression required ongoing protein synthesis in primary hepatocytes
As shown in Fig. 6Go, treatment of hepatocytes with both T0901317 and the protein synthesis inhibitor cycloheximide (CHX) abolished the T0901317-mediated decreases in GR mRNA expression in comparison with hepatocytes with T0901317 alone (Fig. 6AGo), although CHX alone had little effect on control GR mRNA expression. These findings suggest that T0901317 reduction of GR expression is indeed dependent on ongoing protein synthesis. Similarly, CHX also reversed the effects of T0901317 on PEPCK mRNA expression in primary hepatocytes (Fig. 6BGo). However, CHX treatment alone increased PEPCK mRNA expression. These results indicate that T0901317 activation of LXR is an important metabolic signal leading to suppression of hepatic GR and PEPCK expression in vivo and in vitro.


Figure 6
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  		FIG. 6. Suppression of both PEPCK and GR mRNA expression by T0901317. Primary hepatocytes were incubated with T0901317 (1 µM) for 48 h in the absence or presence of 1.0 µg/ml CHX. The levels of GR (A) and PEPCK mRNA expression (B), quantified by quantitative RT-PCR, are expressed relative to the amount of 18S. {dagger}, P < 0.01 vs. controls; ¶, P < 0.01 vs. T0; ##, P < 0.001 vs. T0.

 
LXR agonist T0901317-induced suppression of GR mRNA expression is associated with the down-regulation of 11ß-HSD1 in primary hepatocytes
Parallel to its down-regulation on both GR and PEPCK expression, the level of 11ß-HSD1 activity in primary hepatocytes was decreased by 37% after treatment with T0901317 (Fig. 7AGo). The decrease in enzyme activity was consistent with results of quantitative RT-PCR analysis that showed significant decreases in 11ß-HSD1 mRNA levels in hepatocytes treated with T0901317 in comparison with control levels (Fig. 7BGo). Moreover, a positive correlation was observed between expression of both GR and PEPCK and 11ß-HSD1 activity in primary hepatocytes in response to treatment with T0901317.


Figure 7
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  		FIG. 7. Effects of T0901317 on 11ß-HSD1 activity (A) and mRNA levels (B) in primary cultures of hepatocytes from db/db mice. Hepatocytes were incubated in media with indicated concentrations of T0901317 for 48 h. Values are mean ± SEM from three separate culture preparations. A, 11ß-HSD1 activity was expressed as the percentage of [3H] DHC converted to [3H]B in medium from hepatocytes. B, The levels of 11ß-HSD1 mRNA expression, determined by quantitative RT-PCR, were expressed relative to the amount of mRNA found in controls. *, P < 0.001 vs. controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intracellular GR function determines tissue sensitivity to glucocorticoids and strongly affects the development of type 2 diabetes (1, 2, 3, 4, 5, 6). Activation of hepatic GR expression induces the tissues to respond to excess circulating corticosteroids and activates hepatic gluconeogenesis leading to the development of phenotype of type 2 diabetes in db/db mice and Zucker rats (7, 8). In contrast, reduction of GR expression in target tissues in rodents, either by gene disruption or pharmacological inhibition, decreased insulin resistance and hyperglycemia (9, 10, 11, 12). These studies indicate that alterations in intracellular GR function may contribute to the development of insulin resistance and hyperglycemia associated with type 2 diabetes and metabolic syndrome.

In the present study, we found that chronic treatment of db/db mice with the LXR agonist T0901317 reversed the induction of hepatic GR expression and attenuated the diabetic phenotype. Moreover, we also observed that the T0901317-mediated decrease in GR gene expression was associated with the suppression of PEPCK and G6P mRNA expression with induction of 2-[3H]DG uptake within liver, indicating that suppression of GR expression in the liver may be involved in the antidiabetic action of LXR in diabetic db/db mice. Supporting these conclusions are our cell culture findings that T0901317 reduced the both GR mRNA and protein levels in primary hepatocytes from db/db mice through a direct action. These findings suggest that LXR activation could inhibit GR gene expression and T0901317 exerts some antidiabetic action in diabetic db/db mice that may be mediated, at least in part, through inhibition of hepatic GR expression. Suppression of hepatic GR expression by LXR agonist T0901317 could attenuate the role of GR in the activation of both PEPCK and insulin action within hepatocytes, thereby reducing hepatic gluconeogenesis and circulating glucose levels, all of which may contribute to preventing the development of type 2 diabetes. Our present results are consistent with earlier reports of an inverse relation between LXR{alpha} and GR promoter activities in mouse hepatoma cell lines (32). Sequence analysis of the LXR gene has identified the multiple putative GR binding sites in the upstream flanking sequence of promoter region of mouse LXR{alpha} gene (32, 33). These studies could contribute to interpretation of our present study observed that LXR agonist T0901317 exerts a suppressive role in the control of hepatic GR expression linked to the development of type 2 diabetes.

The observed inhibitory effects of T0901317 on GR mRNA levels in hepatocytes paralleled the suppression of GR protein levels. Treatment of hepatocytes with the protein synthesis inhibitor CHX completely prevented the inhibitory effects of T0901317 on the levels of GR mRNA. These results indicate that T0901317 suppression of GR expression could be caused, at least in part, by decreased protein synthesis, consistent with predominantly transcriptional/posttranscriptional regulation of GR in hepatocytes by T0901317. Our findings are in agreement with previous studies showing that the protein synthesis was required for the regulation of selected metabolic genes by LXR agonists (23, 34). Similarly, our results showing that T0901317-induced suppression of PEPCK mRNA in primary hepatocytes was blocked by CHX, support a recent study suggesting that the indirect activation of LXR target gene-induced inhibition of PEPCK expression was dependent on ongoing protein synthesis in 3T3-L1 adipocytes (35).

The current study also showed that T0901317 treatment reversed hepatic PEPCK mRNA expression and exhibited antidiabetic actions in db/db mice support a previous study showing that the antidiabetic action of T0901317 in db/db mice and obese Zucker rats may be mediated by reduction of hepatic gluconeogenesis (22), although LXR activation did not affect gluconeogenesis in clamped ob/ob mice (36). Importantly, our findings suggest that T0901317-mediated reversal of PEPCK expression may be associated with inhibition of hepatic GR mRNA levels in db/db mice. This is supported by a recent study in which liver-specific inactivation of GR failed to induce hepatic PEPCK gene expression linked to the amelioration of hyperglycemia in diabetic model (13). In contrast, induction of hepatic GR expression was linked to increased PEPCK expression leading to hyperglycemia and insulin resistance (4, 5). Similarly, a recent report from our group showed that there is a positive interaction between PEPCK and GR expression in the control of glucose homeostasis in diabetic animals (8). These data indicate that hepatic GR may be an important factor for induction of PEPCK during the development of hyperglycemia in diabetic db/db mice.

In addition, our results also showed that chronic treatment of diabetic db/db mice with T0901317 reversed the increased hepatic 11ß-HSD1 activity with corresponding suppression in GR expression and improvement of the phenotype of type 2 diabetes in these diabetic mice. This is further confirmed by using isolated hepatocytes from db/db mice in which the inhibitory effects of T0901317 on GR gene expression in primary hepatocytes from db/db mice paralleled the suppression of 11ß-HSD1 by T0901317. These findings indicate that suppression of 11ß-HSD1 in hepatocytes may be involved in T0901317-induced inhibition of GR gene expression through reduction of intrahepatic synthesis of corticosterone to activate GR. This is supported by recent reports demonstrating that activation of 11ß-HSD1 expression results in the production of excess tissue glucocorticoids and GR occupancy, leading to the development of insulin resistance and metabolic syndrome (37). Similarly, we and others (8, 38) recently reported that the phenotype of type 2 diabetes in db/db mice and high-fat diet-induced obese animals was associated with induction of hepatic 11ß-HSD1. Moreover, a recent study (23) reported that LXR agonists could inhibit hepatic gluconeogenesis by reducing 11ß-HSD1 activity in normal mice, although no direct evidence exists about the LXR agonist-induced alteration of 11ß-HSD1 in diabetic rodents. Our results suggest that suppression of 11ß-HSD1 in hepatocytes may be an additional mechanism of T0901317-induced inhibition of GR gene expression in db/db mice.

In summary, we showed that the antidiabetic effects of LXR agonist T0901317 may be mediated, at least in part, through the suppression of hepatic GR expression. T0901317-induced suppression of hepatic GR expression may be an important metabolic signal that directly interferes with GR-mediated local glucocorticoid action linked to the pathogenesis of insulin resistance and gluconeogenesis. Thus, in addition to the novel roles of LXR agonists in suppression of GR gene expression in hepatocytes, the selective LXR agonist T0901317 could be considered as a potential pharmacotherapeutic agent for the treatment of type 2 diabetes.


   Acknowledgments
 
We thank Tripathi Rajavashisth, Ph.D. (Charles R. Drew University), for his helpful comments on this manuscript.


   Footnotes
 
Y.L. is supported by National Institute of Diabetes and Digestive and Kidney Disease KO1 Award 1KO1 DK073272-01A1. T.C.F. and Y.L. are supported by Center of Clinical Research Excellence Grant U54 RR14616 and Research Center in Minority Institutions Grant RR-03026 to Charles R. Drew University of Medicine and Sciences. T.C.F. is supported by National Institute of Health Grants DA-14659, DA-16867, and DA-15466.

The authors have nothing to disclose.

First Published Online July 27, 2006

Abbreviations: CHX, Cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6P, glucose-6-phosphatase; GR, glucocorticoid receptor; 2-[3H]DG, [1,2-3H]deoxy-D-glucose; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase; LXR, liver X receptor; PEPCK, phosphoenolpyruvate carboxykinase; SDS, sodium dodecyl sulfate.

Received February 23, 2006.

Accepted for publication July 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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