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ENDOCRINE AND DIABETES

Blood Glucose-Lowering Nuclear Receptor Agonists Only Partially Normalize Hepatic Gene Expression in db/db Mice

Clinical Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University Vienna, Vienna, Austria (M.L., W.W., T.M.S.); Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria (M.L., W.W., T.M.S.); Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna and Ludwig Boltzmann Institute for Clinical and Experimental Oncology, Vienna, Austria (M.B.); and Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland (M.R.)

Received for publication August 4, 2005
Accepted October 31, 2005.

	Abstract

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Agonists of the nuclear receptors peroxisome proliferator-activated receptor (PPAR) , PPAR, and liver X receptors (LXRs) reduce blood glucose in type 2 diabetic patients and comparable mouse models. Since the capacity of these drugs to normalize hepatic gene expression is not known, we compared groups of obese diabetic db/db mice treated with agonists for PPAR [rosiglitazone (Rosi); 10 mg/kg/day], PPAR [Wy 14643 (Wy; 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid); 30 mg/kg/day], and LXR [T0901317 (T09; N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide); 40 mg/kg/day] and from untreated nondiabetic litter mates (db/+) by oligonucleotide microarrays and quantitative reverse transcriptase-polymerase chain reaction. The 10-day treatment period of db/db mice with Rosi, Wy, and T09 altered expression of 300, 620, and 735 genes including agonist-specific target genes, respectively. However, from the 337 genes differentially regulated in untreated db/+ versus db/db animals, only 34 (10%), 51 (15%), and 82 (24%) were regulated in the direction of the db/+ group by Rosi, Wy, and T09, respectively. Gene expression normalization by drug treatment involved glucose homeostasis, lipid homeostasis, and local glucocorticoid activation. In addition, our data pointed to hitherto unknown interference of these nuclear receptors with growth hormone receptor gene expression and endoplasmic reticulum stress. However, many diabetes-associated gene alterations remained unaffected or were even aggravated by nuclear receptor agonist treatment. These results suggest that diabetes-induced gene expression is minimally reversed by potent blood glucose-lowering nuclear receptor agonists.

	Materials and Methods

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Animals. Male C57BL/KsJ-lepr^db/lepr^db diabetic (db/db) mice and their nondiabetic litter mates (db/+) were purchased from Charles River (Margate, Kent, UK) at 7 weeks of age and maintained under standard light (12 h light/dark) and temperature conditions (23°C). During 1 week of acclimatization, mice were provided with a low-fat standard rodent diet (<3% fat; N1324; Altromin, Detmold, Germany) and water ad libitum.

Treatment. For the experiment, the low-fat standard diet was mixed with vehicle alone (ethanol; untreated) or supplemented with 0.005% (w/w) PPAR agonist rosiglitazone (Rosi; Avandia; Glaxo-SmithKline, Welwyn Garden City, Hertfordshire, UK; corresponding to approximately 10 mg/kg/day; Hori et al., 2002), 0.02% PPAR agonist Wy 14643 (Wy; Sigma-Aldrich, St. Louis, MO; corresponding to approximately 30 mg/kg/day; Edvardsson et al., 1999), or 0.025% of the synthetic LXR agonist T0901317 (T09; generously provided by Amgen Biologicals, Thousand Oaks, CA; corresponding to approximately 40 mg/kg/day; Stulnig et al., 2002b), followed by extensive evaporation of ethanol. Four groups of db/db (untreated, Rosi, Wy, and T09; n = 5) and one group of db/+ mice (untreated; n = 8) were treated for 10 days.

Tissue and Blood Analyses. Mice were anesthetized with Isoflurane and sacrificed by neck dislocation after cardiac puncture. The liver was immediately cut into small homogenous regions, snap frozen in liquid nitrogen, and kept at -80°C until isolation of total RNA. Blood samples were drawn from the tail vein before starting the experimental diets. EDTA plasma separated from cardiac blood was stored in aliquots at -20°C until further analyses. Blood glucose was measured by an automated analyzer (ALCYON 300i; Abbott Laboratories, Abbott Park, IL) at the beginning and the end of the experiment. Plasma cholesterol and triglycerides were determined by the Alcyon 300i analyzer (Abbott Laboratories). Serum concentrations of nonesterified fatty acids were measured with the Wako FFA-kit (Wako Bioproducts, Richmond, VA), and insulin was measured by enzyme-linked immunosorbent assay (Linco Research, St. Charles, MO). Liver triglycerides were determined following lipid extraction as described previously (Haemmerle et al., 2002) but by using a commercially available enzymatic reagent (Rolf Greiner Biochemica, Flacht, Germany). The study protocol was approved by the local ethics committee for animal experiments and the Guidelines for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes were followed.

RNA Preparation. Total RNA was prepared by disrupting equivalent regions of approximately 50 mg of liver tissue from each animal per group in TRIzol reagent (Invitrogen, Carlsbad, CA) with a tissue homogenizer followed by RNA isolation according to the manufacturer's instructions. Total RNA samples were checked for integrity by agarose gel electrophoresis and the 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). For microarray cDNA synthesis, total RNA samples were repurified with RNeasy MinElute kit (QIAGEN, Valencia, CA).

Microarray Hybridization and Data Analysis. Ten micrograms of total RNA from each animal (n = 3 per group) was transcribed into first strand cDNA by Superscript II (Invitrogen) using T7-oligo(dT) primers followed by second strand synthesis and repurification according to the manufacturer's protocols (all Affymetrix, Santa Clara, CA). Following in vitro transcription, 15 µg of labeled and fragmented cRNA from each individual sample was hybridized to U74Av2 GeneChips (12k), which were scanned using the Gene-Array scanner and further analyzed with Microarray Suite version 5.0 software according to the manufacturer's protocols (all Affymetrix). Array quality criteria for all chips included control of expression report files for background, background noise, scale factor <2, internal control gene 3'/5' ratio, and hybridization control ratios, respectively. Normalization was performed by global scaling to an average intensity of 100 arbitrary units. Gene abundances were estimated by robust multiarray analysis (Irizarry et al., 2003) using the probe-level modeling (affyPLM) package from Bioconductor (http://www.bioconductor.org). This algorithm provides for calculation of means and S.E. of logarithmically transformed estimates, reflecting the inconsistency among the different probes for the same gene. We used a strategy similar to analysis of variance and computed a consensus estimate for the variability among all groups as a basis for Student's t test statistics. The multiple comparisons problem was addressed by estimating the false discovery rate (FDR) in a simple manner as the ratio of the expected number of false positives (resulting from permutation analyses with samples being randomly assigned to different groups) at a given p value threshold to the number of positives actually found (Irizarry et al., 2003; Storey and Tibshirani, 2003). Using this statistical approach, comparisons of gene expression with absolute -fold changes of at least 1.5-fold (increase or decrease) were selected at p < 0.01 as a default and p < 0.001 where indicated. Abbreviation, annotation, and analyses of genes meeting the selection criteria were done by combining available information from Affymetrix, Applied Biosystems (PANTHER), Jax, Genelynx, Kegg, Ensembl, Swiss-Prot, and PubMed in a File-maker Pro database.

Hierarchical Clustering Analysis. Genes altered by treatment with all nuclear receptor agonists by at least 1.5-fold (in either direction) and at p < 0.01 between mean gene expression and untreated db/db animals were selected for a cluster analysis. The genes were clustered using a distance measure defined as 1 minus the correlation between gene pair measures and using complete-linkage hierarchical clustering. The results are shown in Supplementary Fig. 2.

Quantitative Real-Time Polymerase Chain Reaction. One microgram of total RNA from each animal (n = 5 per group) was treated with DNase I and reverse transcribed into cDNA by Superscript II using random hexamer priming (all Invitrogen). Quantitative real-time polymerase chain reaction (QPCR) was performed for all samples per group using gene-specific 5-carboxyfluorescein-5-carboxytetramethylrhodamine-labeled commercial Assays-on-Demand (assay identification, Mm00839363_m1, G6pc; Mm00484574_m1, G6pt1; Mm00440636_m1, Pck1; Mm00662319_m1, fatty acid synthase; Mm00772290_m1, stearoyl-CoA desaturase 1 (Scd1); Mm008333-28_m1, glycerol-3-phosphate acyltransferase, mitochondrial; Mm-00483985_m1, Crat; Mm00451571_m1, Slc25a20; Mm00443579_m1, Acox1; Mm00470091_s1, Ehhadh; Mm00478137_m1, Pex11a; Mm0-0550050_m1, Hmgcs2; Mm00476182_m1, Hsd11b1; Mm004390-93_m1, Ghr; Mm00439561_m1, Igf1; Applied Biosystems, Foster City, CA) or self-designed primer/probe combinations (SREBP-1c; Stulnig et al., 2002a) normalized to 18S VIC-5-carboxytetramethylrhodamine labeled endogenous control (Applied Biosystems). Expression of specific mRNAs was quantitated in duplicates with a tolerated variance of 10% in an ABI PRISM 7000 Cycler (Applied Biosystems).

Statistics and Calculations. Data are given in means ± S.E.M. unless indicated otherwise. Study groups were compared with untreated db/db mice by univariate ANOVA using Dunnett's t test for post hoc analysis except for evaluation of gene expression profiles (see above). Data from quantitative polymerase chain reaction and microarrays as well as other data exhibiting inequality of variances between groups according to Levene's test were log-transformed before ANOVA. The effect of diabetes was evaluated by comparing db/+ to db/db mice resulting in its reciprocal (diabetes^-1) with facilitate direct comparisons with the normalizing effect of the compounds.

	Results

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Metabolic Data. This study was performed to elucidate to which extent blood glucose-lowering nuclear receptor agonists alter hepatic gene expression profiles of obese diabetic mice in convergence to nondiabetic animals. Gene expression profiles of db/db mice treated with agonists of PPAR (Rosi), PPAR (Wy), and LXR (T09) and untreated db/+ mice were compared with untreated diabetic mice. Treatment with each of the compounds resulted in reductions in blood glucose concentrations near to those of nondiabetic mice (Table 1). Plasma insulin concentrations were significantly increased only in the T09-treated group indicating that stimulation of insulin secretion as shown for cultured pancreatic islets and -cells (Efanov et al., 2004) could also occur in vivo. Body weight remained constant during the treatment except a borderline (p = 0.088) increase in the Rosi group. The Wy and particularly T09-treated animals showed significant hepatomegaly with increased liver/body weight ratio by 1.8- and 2.5-fold, respectively, due to the known development of hepatic steatosis by T09 as revealed by highly elevated hepatic triglyceride contents in T09-treated db/db mice (Table 1).

Gene Expression Profiling. Gene expression profiles were evaluated using 12k oligonucleotide microarrays for three individual mice of each group and robust multiarray analysis. p < 0.01 was predefined together with a -fold change of 1.5-/-1.5-fold as selection criteria for profile comparisons including detection of functional groups. Genes (337) were altered in untreated diabetic versus nondiabetic mice (Table 2). Drug treatment of db/db mice elicited significant changes in the expression of a comparable number of genes, namely 300, 620, and 735 genes for Rosi, Wy, and T09, respectively. Estimated FDRs were between 6 and 11% for p < 0.01, but approximately 80% of genes were altered at a p < 0.001 with FDR of 1 to 3%, respectively, indicating excellent confidence of data. Moreover, standardized logarithmic expression estimates from microarrays highly correlated with those from quantitative polymerase chain reaction (adjusted r² = 0.854, p < 0.0005) indicating valid relative quantitation by microarray hybridization (data not shown).

We used the reciprocal form of the diabetes effect (diabetes^-1), i.e., untreated nondiabetic versus diabetic mice, to facilitate direct comparison with treatment effects. Treatment-induced normalization of gene expression was defined as genes that were significantly changed by a compound in the same direction as diabetes^-1, i.e., in convergence to db/+ animals, without testing whether the gene actually reached the level of nondiabetic mice. From the genes (microarray probe sets) altered in db/db versus db/+ mice, only 34 (10% of all genes altered in diabetes^-1), 51 (15%), and 82 (24%) were normalized by Rosi, Wy, and T09, respectively (Fig. 1A). In total, only a set of 19 genes (6%) was normalized by all blood glucose-lowering drugs (listed in Supplementary Fig. 1A), indicating that these genes could be particularly important in mediating the glucose-lowering effects. These genes included some with clear implication in glucose metabolism and diabetes such as those involved in gluconeogenesis (e.g., glucose-6-phosphatase, fructose-1,6-bisphosphatase) and the glucocorticoid-activating enzyme 11-hydroxysteroid dehydrogenase type 1, whereas the functional implication of others for glucose lowering have to be assessed in detail. However, 56 (17%), 85 (25%), and 59 (18%) genes altered in db/db versus db/+ mice were regulated by Rosi, Wy, and T09, respectively, in the direction opposite to diabetes^-1 (Fig. 1B). The 30 genes (probe sets) regulated by all compounds in the direction opposite to diabetes^-1 (listed in Supplementary Fig. 1B), thus possibly giving insight into augmented adaptive processes, e.g., comprised genes involved in mitochondrial and peroxisomal -oxidation (Acaa1, Dci, Ehhadh), including the important peroxisomal biogenesis factor (Pex11a) as discussed in detail below. Treatment with nuclear receptor agonists regulated many genes not altered by diabetes^-1 itself and resulted in considerably overlaps in gene expression profiles in diabetic mice (Fig. 1C) similar to that shown for PPAR, LXR, and retinoid X receptor in nondiabetic mice (Anderson et al., 2004). The overlap of PPAR agonist treatment with other compounds is noteworthy because PPAR is only weakly expressed in liver; thus, direct cross-talk between nuclear receptors cannot be accounted for. Supplementary Fig. 2 provides a tree obtained by hierarchical clustering of genes altered in parallel by all nuclear receptors agonists to highlight the correlation of their regulation. A complete list of all genes altered in db/db versus db/+ mice (shown as diabetes^-1) or by treatment with any compound is given in Supplementary Fig. 3.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Overlap of diabetes and drug effects. Of the 337 genes (microarray probe sets) altered by diabetes, figures in overlapping wheels of A and B indicate the number of genes that were also regulated by PPAR, PPAR, and LXR agonists, respectively, in direction to normalize (A) or deteriorate (B) diabetes-associated changes (see Supplementary Figs. 1 and 2). C, total numbers of genes regulated by the compounds illustrating considerable overlap of drug effects. Numbers with connecting lines to the wheels indicate the total number of genes regulated by the respective nuclear receptor agonist. Note that only a minority of diabetes-associated gene regulations were normalized by the treatments and that many genes not associated with diabetes-associated alterations were regulated by the compounds.

Gluconeogenesis. Hepatic gluconeogenesis and glucose output are significantly enhanced in type 2 diabetes patients and comparable mouse models and contribute to high blood glucose levels. Treatment with each of the blood glucose-lowering nuclear receptor agonists resulted in normalization of gluconeogenetic key enzyme gene expression as shown for phosphoenol pyruvate carboxykinase 1 (phosphoenolpyruvate carboxykinase; gene Pck1; 1.2-fold; p < 0.05), fructose-1,6-bisphosphatase, and the catalytic and transport units of glucose-6-phosphatase (G6pc and G6pt1) (Fig. 2). Interestingly, the PPAR agonist treatment led to an increased expression of fructose-2,6-bisphosphatase, which could imply an additional regulation of fructose-1,6-bisphosphatase through competitive inhibition by fructose-2,6-bisphosphate. Expression of aldolase B, which catalyzes cleavage of fructose-1,6-bisphosphate and functions as a feed forward activator of pyruvate kinase, was normalized by both Wy (-1.8-fold; p < 0.002) and Rosi (-1.4-fold; p = 0.042). Glucokinase, whose expression was increased by 1.4-fold in diabetes^-1, was normalized in the Rosi group (1.4-fold; p = 0.003) and T09 group (1.8-fold; p < 0.001). Additional transfer of glucose 6-phosphate through the pentose phosphate shunt in the LXR-treated group is indicated by elevated expression of glucokinase, X-linked glucose-6-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase 2, ribose 5-phosphate isomerase A, and transketolase (Supplementary Fig. 3). In general, these data indicate that treatment of db/db mice with different blood glucose-lowering nuclear receptor agonists resulted in normalized expression of key genes involved in glucose homeostasis, even though some genes were regulated in an agonist-specific manner.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Expression of genes involved in gluconeogenesis. Diabetic db/db mice were treated for 10 days with Rosi, Wy, or T09, respectively, or left untreated, as were nondiabetic db/+ mice. Gene expression was evaluated by QPCR (default; n = 5) or by microarrays (indicated by black dotted line; n = 3) and is given in percentage of that in untreated db/db mice. G6pc, glucose-6-phosphatase, catalytic subunit; G6pt, glucose-6-phosphatase, transport subunit; phosphoenolpyruvate carboxykinase (Pck1), phosphoenolpyruvate carboxykinase 1, cytosolic; Fbp1, fructose bisphosphatase 1; Aldo2, aldolase 2, B isoform; ut, untreated. Significant differences compared with untreated db/db are indicated as follows: *, p < 0.05; , p < 0.01; and , p < 0.001.

Lipogenesis. Hepatic lipogenesis and fatty acid desaturation have been implicated in various pathological conditions including obesity and diabetes (Ntambi et al., 2002). Hepatic gene expression of lipogenic enzymes, e.g., Scd1, was generally increased in db/db versus db/+ animals (Fig. 3A). Treatment of db/db mice with any blood glucose-lowering compound led to a further strong increase of Scd1 gene expression (Fig. 3A). Scd1 expression is regulated by SREBP-1c-dependent and -independent mechanisms (Miyazaki et al., 2004). Since induction of Scd1 by drugs correlated with elevated SREBP-1c expression levels only in the T09 group (Fig. 3A), Scd1 up-regulation by PPAR agonists seems to occur predominantly by SREPB-1c-independent mechanisms. Parallel to Scd1 expression, fatty acid synthase and glycerol-3-phosphate acyltransferase were strongly induced by treatment with any agonist, whereas expression of hepatic lipase was decreased (Fig. 3A). Thereby, the nuclear receptor agonists aggravated diabetes-associated alterations in lipogenesis in parallel with their glucose-lowering effect (Way et al., 2001). Such regulation opposite to diabetes^-1 could on the one hand indicate enhancement of adaptive processes that were already induced by diabetes itself but also worsening of detrimental diabetes-associated alterations. Recent data suggest that up-regulation of lipogenesis provides an effective means to inhibit diabetes development by shifting the lipogenic burden from adipose tissue to the liver (Nadler and Attie, 2001). Altogether, alterations in hepatic lipid homeostasis provoked hepatic triglyceride accumulation, particularly in the T09-treated and, to a lesser extent, in Rositreated animals. Moreover, the LXR agonist increased blood plasma triglyceride concentrations, whereas Rosi and Wy lowered plasma triglyceride concentrations (Table 1; Oakes et al., 1994; Chisholm et al., 2003). In contrast to lipogenic enzymes, genes involved in cholesterogenesis such as 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgr) and synthase 1 (Hmgcs1), isopentenyl-diphosphate isomerase (Idi1), farnesyl diphosphate farnesyl transferase 1 (Fdft1), and NAD(P)-dependent steroid dehydrogenase-like (Nsdhl) were not altered by diabetes and were differently changed by nuclear receptor agonists (Supplementary Fig. 3; data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 3. Lipid homeostasis and ketogenesis. A, expression of genes involved in lipogenesis. Gene expression from mice is given as detailed in legend to Fig. 2. Gene expression was evaluated by QPCR (default; n = 5) or by microarrays (indicated by black dotted line; n = 3) and is given in percentage of that in untreated db/db mice. Fasn, fatty acid synthase; Gpam, glycerol-3-phosphate acyltransferase, mitochondrial; Dgat1, diacylglycerol O-acyltransferase 1; Lipc, hepatic lipase; ut, untreated; geometric mean is given for columns exceeding scale of y-axis. Significant differences compared with untreated db/db are indicated as follows: *, p < 0.05; , p < 0.01; and , p < 0.001. B, expression of genes involved in mitochondrial fatty acid import. Crat, carnitine acetyltransferase; Slc25a20, solute carrier family 25 (mitochondrial carnitine/acylcarnitine translocase), member 20; Cpt1a, carnitine palmitoyl-transferase 1, liver; Cpt2, carnitine palmitoyl-transferase 2. C, expression of genes involved in mitochondrial and peroxisomal -oxidation. Acox1, acyl-CoA oxidase 1, palmitoyl; Dci, dodecenoyl-CoA isomerase (3,2 trans-enoyl-CoA isomerase); Ehhadh, enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase; Acaa1, 3-ketoacyl-CoA thiolase B, acetyl-CoA acyltransferase 1; Pex11a, peroxisomal biogenesis factor 11a. D, expression of genes involved in ketogenesis. Hmgcs2, 3-hydroxy-3-methylglutaryl-CoA synthase 2; Hmgcl, 3-hydroxy-3-methylglutaryl-CoA lyase.

Mitochondrial and Peroxisomal -Oxidation. -Oxidation of free fatty acids provides energy and substrates for ketogenesis. Expression of genes implicated in mitochondrial fatty acid import including the carnitine acyltransferase Crat and the translocase Slc25a20 (Ramsay et al., 2001; Sekoguchi et al., 2003). Expression of both genes was significantly increased in diabetic versus nondiabetic mice and further elevated by treatment with blood glucose-lowering nuclear receptor agonists, indicating augmented mitochondrial fatty acid import (Fig. 3B). Expression of the long-chain-specific carnitine acyltransferases (Cpt1a, Cpt2) was increased by diabetes and further elevated in response to Wy treatment. Expression of enzymes involved in mitochondrial fatty acyl oxidation such as dodecenoyl-CoA isomerase (Dci) and t-acetyl-CoA dehydrogenases (Acads, Acadm, Acadl) was somewhat increased in diabetic animals and further elevated by nuclear receptor agonists (Fig. 3C; Supplementary Fig. 3). Peroxisomes oxidize fatty acids by different pathways with preference for long-chain and very-long-chain fatty acids (Van Veldhoven and Mannaerts, 1999). Expression of enzymes of the classic pathway, namely acyl-CoA oxidase 1 (Acox1), enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh), and acetyl-CoA acyltransferase 1 (Acaa1) was more pronounced in db/db versus db/+ animals (Lan et al., 2003) and further increased by nuclear receptor agonist treatment, particularly Wy (Fig. 3C), in parallel with peroxisomal biogenesis factor 11a (Pex11a; Fig. 3C). Notably, hepatic triglyceride accumulation particularly in T09-treated animals revealed that increased -oxidation did not sufficiently counteract elevated lipogenesis to prevent hepatic steatosis. Increased ketogenesis in diabetes was indicated by elevated expression of key genes 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) and 3-hydroxy-3-methylglutaryl-CoA lyase (Hmgcl; Fig. 3D). In particular, PPAR agonists further increased Hmgcl and Wy also increased Hmgcs2 expression, suggesting increased ketogenesis. In conclusion, treatment with different blood glucose-lowering nuclear receptor agonists resulted in increased expression of most genes contributing to mitochondrial and peroxisomal fatty acid oxidation and ketogenesis, thereby aggravating the diabetes-associated shift to fatty acid metabolism.

Glucocorticoid Activation. Type 2 diabetes has been shown to be associated with alterations in local activation of inactive glucocorticoid precursors (cortisone, 11-dehydro corticosterone) to active glucocorticoids (corticosterone, cortisol) by 11-hydroxysteroid dehydrogenase type 1 (Hsd11b1). Hsd11b1 is particularly expressed in human and rodent liver and adipose tissue (Bujalska et al., 2002), and increased expression has been linked to development of diabetes and the metabolic syndrome (Aoki et al., 2001; Masuzaki et al., 2001). Treatment with agonists of the PPAR and LXR family has been shown recently to affect expression of Hsd11b1 in adipose tissue and liver (Stulnig et al., 2002a; Cao et al., 2003). Treatment of either nuclear receptor agonists diminished Hsd11b1 expression even below the level of nondiabetic mice (Fig. 4). Since oxo-reductase activity of Hsd11b1 depends on NADPH, mutations in hexose-6-phosphate dehydrogenase (H6pd) synergize with weak Hsd11b1 mutations in glucocorticoid activation (Draper et al., 2003). However, H6pd gene expression was not changed in this study (Fig. 4). Notably, Hsd11b1 was one of the only 19 transcripts that were normalized by all blood glucose-lowering nuclear receptor agonists (Supplementary Fig. 1A). These data emphasize the importance of 11-hydroxysteroid dehydrogenase 1 for the development of insulin resistance and suggest the use of specific inhibitors of this enzyme in diabetes treatment.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Glucocorticoid metabolism. Gene expression from mice is given as detailed in legend to Fig. 2. Gene expression was evaluated by QPCR (default; n = 5) or by microarrays (indicated by black dotted line; n = 3) and is given in percentage of that in untreated db/db mice. Hsd11b1, hydroxysteroid 11- dehydrogenase 1; H6pd, hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase); ut, untreated. Significant differences compared with untreated db/db are indicated as follows: , p < 0.001.

Growth Hormone Signaling. Growth hormone (GH) antagonizes insulin signaling and elicits insulin resistance resulting in increased hepatic glucose production. In contrast to unchanged expression in diabetic versus nondiabetic mice, treatment with blood nuclear receptor agonists down-regulated growth hormone receptor gene (Ghr) expression (Fig. 5), whose abundance is related to GH tissue sensitivity (Dominici and Turyn, 2002; Iida et al., 2004), as well as expression of insulin-like growth factor-1, a prototype GH-responsive gene in the liver. These data indicate interference with GH action by nuclear receptor agonist treatment. Since inhibition of GH action improves insulin sensitivity (Yakar et al., 2004), this could be an additional mechanism of how PPAR and LXR agonists ameliorate glucose homeostasis in diabetes.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Growth hormone signaling. Gene expression from mice is given as detailed in legend to Fig. 2. Gene expression was evaluated by QPCR (default; n = 5) or by microarrays (indicated by black dotted line; n = 3) and is given in percentage of that in untreated db/db mice. Ghr, growth hormone receptor; Igf-1, insulin-like growth factor 1; ut, untreated. Significant differences compared with untreated db/db are indicated as follows: , p < 0.01; and , p < 0.001.

Endoplasmic Reticulum Stress. Endoplasmic reticulum stress is a major contributor for the development of obesity and insulin resistance as shown very recently (Özcan et al., 2004). Obesity triggers an unfolded protein response (UPR) in liver that is controlled by X-box-binding protein-1 (XBP-1) (Yoshida et al., 2001). Notably, XBP-1^+/- mice are prone to insulin resistance due to impaired insulin signal transduction during endoplasmic reticulum stress (Özcan et al., 2004). Glucose-regulated/binding immunoglobulin protein Grp78 (Hspa5), an endoplasmic reticulum chaperone, and the protein kinase inhibitor p58^ipk (Dnajc3) are up-regulated during UPR, and the latter has been shown to be an XBP-1 target gene, as is thioredoxin domain-containing protein-7 (Txndc7) (Lee et al., 2003). XBP-1 expression was significantly lowered by all blood glucose-lowering nuclear receptor agonists (Fig. 6) in parallel with Grp78, DNajc3, and Txndc7, pointing to reduced XBP-1 activity and amelioration of endoplasmic reticulum stress. Reduced expression of Grp78 in untreated db/db animals compared with lean mice could be secondary to the nearly 2-fold reduced expression of XBP-1 in untreated db/db versus db/+ mice. It is intriguing to speculate that the reduced expression of XBP-1 in db/db animals indicates a reduced capacity to deal with endoplasmic reticulum stress similar to that seen in XBP-1^+/- mice, whereas further reduction of the expression of UPR genes by nuclear receptor agonist treatment of diabetic mice reflects a decline of endoplasmic reticulum stress. Thus, reduction of endoplasmic reticulum stress could be a novel mechanism of how PPAR and LXR agonists lower blood glucose concentrations in diabetic mice prone to UPR by lowered XBP-1 expression.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Expression of genes involved in endoplasmic reticulum stress. Gene expression from mice is given as detailed in legend to Fig. 2. Gene expression was evaluated by QPCR (default; n = 5) or by microarrays (indicated by black dotted line; n = 3) and is given in percentage of that in untreated db/db mice. Grp78 (Hspa5), heat shock 70-kDa protein 5 (glucose-regulated protein); Dnajc3, DnaJ (Hsp40) homolog, subfamily C, member 3; Txndc7, thioredoxin domain containing protein-7; ut, untreated; *, p < 0.05; , p < 0.01; , p < 0.001.

	Discussion

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In conclusion, this study revealed that apart from pointing to novel modes of action, currently available blood glucoselowering nuclear receptor agonists by far do not normalize diabetes-associated molecular alterations. Despite our advances during recent years, there is still a need for developing novel drugs for effective treatment of type 2 diabetes at the molecular level.

	Acknowledgements

 
We thank Sylvia Molzer for technical assistance and Jelena Todoric for liver triglyceride determination.

	Footnotes

 
This work was supported by the Center for Molecular Medicine, a basic research institute within the companies of the Austrian Academy of Sciences (to T.M.S. and W.W.), and by the Joseph Skoda Award of the Austrian Society for Internal Medicine (to T.M.S.).

doi:10.1124/jpet.105.093831.

ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; LXR, liver X receptor; SREBP, sterol regulatory element-binding protein; Rosi, rosiglitazone; Wy, Wy 14643, 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid; T09, T0901317, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide; FDR, false discovery rate; QPCR, quantitative real-time reverse-transcriptase polymerase chain reaction; Scd1, stearoyl-CoA desaturase 1; ANOVA, analysis of variance; GH, growth hormone; UPR, unfolded protein response; XBP-1, X-box-binding protein-1.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Thomas M. Stulnig, Clinical Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. E-mail: thomas.stulnig{at}meduniwien.ac.at

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