Distinct Mechanisms of Glucose Lowering by Specific Agonists for Peroxisomal Proliferator Activated Receptor γ and Retinoic Acid X Receptors
2005; Elsevier BV; Volume: 280; Issue: 46 Linguagem: Inglês
10.1074/jbc.m505853200
ISSN1083-351X
AutoresXiangquan Li, Polly A. Hansen, Xi Li, Roshantha A.S. Chandraratna, Charles Burant,
Tópico(s)Adipose Tissue and Metabolism
ResumoAgonists for the nuclear receptor peroxisomal proliferator-activated receptor-γ (PPARγ) and its heterodimeric partner, retinoid X receptor (RXR), are effective agents for the treatment of type 2 diabetes. To gain insight into the antidiabetic action of these compounds, we treated female Zucker diabetic rats (ZFF) with AGN194204, which we show to be a homodimer-specific RXR agonist, or the PPARγ agonist, troglitazone. Hyperinsulinemic-euglycemic clamps in ZFF showed that troglitazone and AGN194204 reduced basal endogenous glucose production (EGP) ∼30% and doubled the insulin suppression of EGP. AGN194204 had no effect on peripheral glucose utilization, whereas troglitazone increased insulin-stimulated glucose utilization by 50%, glucose uptake into skeletal muscle by 85%, and de novo skeletal muscle glycogen synthesis by 300%. Troglitazone increased skeletal muscle Irs-1 and phospho-Akt levels following in vivo insulin treatment, whereas AGN194204 increased hepatic Irs-2 and insulin stimulated phospho-Akt in liver. Gene profiles of AGN194204-treated mouse liver analyzed by Ingenuity Pathway Analysis identified increases in fatty acid synthetic genes, including Srebp-1 and fatty acid synthase, a pathway previously shown to be induced by RXR agonists. A network of down-regulated genes containing Foxa2, Foxa3, and G-protein subunits was identified, and decreases in these mRNA levels were confirmed by quantitative reverse transcription-PCR. Treatment of HepG2 cells with AGN194204 resulted in inhibition of glucagon-stimulated cAMP accumulation suggesting the G-protein down-regulation may provide an additional mechanism for hepatic insulin sensitization by RXR. These studies demonstrate distinct molecular events lead to insulin sensitization by high affinity RXR and PPARγ agonists. Agonists for the nuclear receptor peroxisomal proliferator-activated receptor-γ (PPARγ) and its heterodimeric partner, retinoid X receptor (RXR), are effective agents for the treatment of type 2 diabetes. To gain insight into the antidiabetic action of these compounds, we treated female Zucker diabetic rats (ZFF) with AGN194204, which we show to be a homodimer-specific RXR agonist, or the PPARγ agonist, troglitazone. Hyperinsulinemic-euglycemic clamps in ZFF showed that troglitazone and AGN194204 reduced basal endogenous glucose production (EGP) ∼30% and doubled the insulin suppression of EGP. AGN194204 had no effect on peripheral glucose utilization, whereas troglitazone increased insulin-stimulated glucose utilization by 50%, glucose uptake into skeletal muscle by 85%, and de novo skeletal muscle glycogen synthesis by 300%. Troglitazone increased skeletal muscle Irs-1 and phospho-Akt levels following in vivo insulin treatment, whereas AGN194204 increased hepatic Irs-2 and insulin stimulated phospho-Akt in liver. Gene profiles of AGN194204-treated mouse liver analyzed by Ingenuity Pathway Analysis identified increases in fatty acid synthetic genes, including Srebp-1 and fatty acid synthase, a pathway previously shown to be induced by RXR agonists. A network of down-regulated genes containing Foxa2, Foxa3, and G-protein subunits was identified, and decreases in these mRNA levels were confirmed by quantitative reverse transcription-PCR. Treatment of HepG2 cells with AGN194204 resulted in inhibition of glucagon-stimulated cAMP accumulation suggesting the G-protein down-regulation may provide an additional mechanism for hepatic insulin sensitization by RXR. These studies demonstrate distinct molecular events lead to insulin sensitization by high affinity RXR and PPARγ agonists. Thiazolidinediones (TZDs) 2The abbreviations used are: TZD, thiazolidinedione; RXR, retinoid acid X receptor; PPAR, peroxisomal proliferator activated receptor; FAS, fatty acid synthase; ACO, acyl-CoA oxidase; PPRE, peroxisomal proliferator response element; GIR, glucose infusion rate; HGO, hepatic glucose output; Rd, whole body glucose disposal; FABP, fatty acid-binding protein; PEPCK, phosphoenolpyruvate carboxykinase; SREBP-1, sterol regulatory element-binding protein-1; FATP, fatty acid transport protein; ZFF, Zucker diabetic rat; CMV, cytomegalovirus; EGP, endogenous glucose production. and other compounds that bind and enhance the transcriptional activity of peroxisomal proliferator-activated receptor γ (PPARγ) have proven to be effective treatments for insulin-resistant diabetes (1.Vasudevan A.R. Balasubramanyam A. Diabetes Technol. Ther. 2004; 6: 850-863Crossref PubMed Scopus (91) Google Scholar, 2.Alarcon de la Lastra C. Sanchez-Fidalgo S. Villegas I. Motilva V. Curr. Pharm. Des. 2004; 10: 3505-3524Crossref PubMed Scopus (54) Google Scholar). The increase in insulin sensitivity that occurs after treatment with TZDs likely involves actions in adipose tissue, muscle, and liver (3.Tonelli J. Li W. Kishore P. Pajvani U.B. Kwon E. Weaver C. Scherer P.E. Hawkins M. Diabetes. 2004; 53: 1621-1629Crossref PubMed Scopus (219) Google Scholar, 4.Miyazaki Y. Mahankali A. Wajcberg E. Bajaj M. Mandarino L.J. DeFronzo R.A. J. Clin. Endocrinol. Metab. 2004; 89: 4312-4319Crossref PubMed Scopus (202) Google Scholar, 5.Stumvoll M. Expert Opin. Investig. Drugs. 2003; 12: 1179-1187Crossref PubMed Scopus (63) Google Scholar, 6.Burant C.F. Sreenan S. Hirano K. Tai T.A. Lohmiller J. Lukens J. Davidson N.O. Ross S. Graves R.A. J. Clin. Invest. 1997; 100: 2900-2908Crossref PubMed Scopus (323) Google Scholar), although the complete set of genes that are modulated to result in improved insulin action by TZDs remains unknown. The heterodimeric partner of PPARγ receptor is the retinoid X receptor (RXR) (7.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). In overexpression studies, binding of ligand to the RXR receptor has been reported to result in the recruitment of coactivators to the RXR/PPARγ heterodimer (8.Martin G. Schoonjans K. Lefebvre A.M. Staels B. Auwerx J. J. Biol. Chem. 1997; 272: 28210-28217Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar) and increased transcription from idealized peroxisomal proliferator response elements (PPREs). Some RXR activators can increase transcription of genes in vitro that are also increased by PPARγ ligands (8.Martin G. Schoonjans K. Lefebvre A.M. Staels B. Auwerx J. J. Biol. Chem. 1997; 272: 28210-28217Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 9.Schulman I.G. Shao G. Heyman R.A. Mol. Cell. Biol. 1998; 18: 3483-3494Crossref PubMed Google Scholar). Like TZDs, RXR agonists can differentiate 3T3-L1 adipocytes (10.Canan Koch S.S. Dardashti L.J. Cesario R.M. Croston G.E. Boehm M.F. Heyman R.A. Nadzan A.M. J. Med. Chem. 1999; 42: 742-750Crossref PubMed Scopus (66) Google Scholar), and administration of specific, high affinity RXR agonists to hyperglycemic ob/ob (11.Lenhard J.M. Lancaster M.E. Paulik M.A. Weiel J.E. Binz J.G. Sundseth S.S. Gaskill B.A. Lightfoot R.M. Brown H.R. Diabetologia. 1999; 42: 545-554Crossref PubMed Scopus (84) Google Scholar) and db/db (12.Mukherjee R. Davies P.J. Crombie D.L. Bischoff E.D. Cesario R.M. Jow L. Hamann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. Paterniti Jr., J.R. Heyman R.A. Nature. 1997; 386: 407-410Crossref PubMed Scopus (576) Google Scholar) mice is effective in lowering glucose levels. These results have led to the hypothesis that the antihyperglycemic effect of RXR agonists is due to transactivation of the PPARγ/RXR heterodimer. In contrast, other studies suggest that in vivo, specific RXR and PPARγ ligands regulate the expression of different genes in adipose tissue and liver, suggesting distinct mechanisms for glucose lowering by these ligands (13.Davies P.J. Berry S.A. Shipley G.L. Eckel R.H. Hennuyer N. Crombie D.L. Ogilvie K.M. Peinado-Onsurbe J. Fievet C. Leibowitz M.D. Heyman R.A. Auwerx J. Mol. Pharmacol. 2001; 59: 170-176Crossref PubMed Scopus (81) Google Scholar). In addition, a recent report showed a different effect of the rexinoid LG268 and the PPARγ activator rosiglitazone on signal pathways in skeletal muscle of diabetic (db/db) mice (14.Shen Q. Cline G.W. Shulman G.I. Leibowitz M.D. Davies P.J. J. Biol. Chem. 2004; 279: 19721-19731Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Although both result in a significant increases in insulin-stimulated glucose transport activity in skeletal muscle, LG268 increased Irs-1 and Akt phosphorylation while rosiglitazone increased the levels of CAP expression and insulin-stimulated c-Cbl phosphorylation without having an effect on the Irs-1/Akt pathway, suggesting distinct sensitizing pathways. In preliminary studies, we used microarrays to evaluate gene expression in insulin-responsive tissue of diabetic ZFF rats following treatment with the high affinity RXR ligand AGN194204 or troglitazone, a prototypical PPARγ agonist. We found distinct populations of genes were regulated by each compound. 3C. F. Burant, unpublished observation. In light of this finding, we sought to systematically evaluate the ability of these compounds to increase transcription in vitro and determine their pharmacological activities in vivo. We also utilized a new gene array analysis tool to gain insight into the pharmacological effects of AGN194204. We found that the AGN194204 does not transactivate PPARγ in vitro and lowers glucose in vivo only by suppressing hepatic glucose production without affecting peripheral insulin sensitivity. We also utilized a novel gene expression analysis tool to identify the potential pathways in the liver that contribute to the ability of RXR agonists to lower liver glucose production. Transient Transfection Studies—CV-1 cells were grown in 6-well plates with minimal essential medium containing 10% fetal bovine serum and l-glutamine (2 mm). Cells were transfected with plasmids pGL3Luc containing the PPRE fragment of the fatty acid transport protein (FATP) or the acyl-CoA oxidase (ACO) PPRE (0.5 μg) with or without expression constructs pcDNA3 containing RXRα and/or PPARγ (0.5 μg) using Fugene6 transfection reagent (Roche Applied Science). CMV-β-galactosidase DNA (5 ng) was co-transfected for normalization of transfection efficiency. Twenty-four hours after transfection, cells were treated with different concentrations of troglitazone, AGN194204, AGN195203, or LG100268. After 24 h, cells were extracted and the luciferase activity and β-galactosidase activity were measured using the Dual-light assay system (Tropix, Inc., Foster City, CA). Data are expressed as the ratio of Luc/β-galactosidase activity. Animals and Biochemical Measurements—Female Zucker diabetic fatty rats (ZDF/Gmi-fa/fa; "ZFF" rats) were purchased from Genetic Models, Inc. (Indianapolis, IN) at 5–6 weeks of age and were fed with a semipurified high fat diet (48% fat, 16% protein, diet 13004) prepared by Research Diets Inc. (New Brunswick, NJ). All studies were approved by the Animal Care and Use Committees at Park-Davis, Inc. and at the University of Michigan, Ann Arbor. After the animals became hyperglycemic (fed blood glucose > 250 mg/dl, 3–4 weeks on the diet) they were treated orally with vehicle (carboxymethylcellulose), troglitazone (400 mg/kg), or AGN194204 (1 mg/kg) (15.Beard R.L. Colon D.F. Song T.K. Davies P.J. Kochhar D.M. Chandraratna R.A. J. Med. Chem. 1996; 39: 3556-3563Crossref PubMed Scopus (66) Google Scholar) at 10 a.m. for 7 days. On the morning of the 8th day, the animals were anesthetized and blood was collected by cardiac puncture. Whole blood glucose was determined using a Hemocue (Angelholm, Sweden), serum triglycerides were determined using a colorimetric assay (Wako Chemicals, Richmond, VA), and serum insulin concentrations were measured by radioimmunoassay (Linco, St. Louis, MO) using rat insulin standards. In some experiments, control C57Bl/6 or db/db mice on a C57Bl/6 background (Jackson Laboratories, Bar Harbor, ME) were treated with vehicle or with AGN194204 or AGN195203 (both at 1 mg/kg) for 1, 3, or 7 days. The livers were removed and subjected to either gene expression profiling or Northern and Western blotting as described below. Euglycemic, Hyperinsulinemic Clamp in ZFF Rats—After ∼24 days on the diet, indwelling catheters were surgically implanted in the jugular vein and carotid artery of rats. Starting 3–5 days after surgery, rats were treated with AGN194204 or troglitazone as described above. The clamp procedure was initiated in the morning of day 8, following overnight food restriction (6–7 g after 6:00 p.m.). One hour before starting the glucose and insulin infusions (t =–60 min), a prime-continuous infusion (15 μCi of bolus, 0.15 μ Ci/min) of high-performance liquid chromatography-purified [3H]glucose (PerkinElmer Life Sciences) was initiated. At t =–30 min, four arterial plasma samples were obtained at 10-min intervals for determination of plasma glucose specific activity in the basal state. At t = 0 min, a prime-continuous infusion of porcine insulin (72 milliunits · kg–1 · min–1; Eli Lilly & Co., Indianapolis, IN) was initiated. Plasma glucose concentration, determined every 5–10 min, was maintained at 100–110 mg/dl by a variable infusion of 45% dextrose. Steady state was generally achieved within 120–150 min, at which time four arterial plasma samples were obtained at 10-min intervals for determination of plasma glucose specific activity. Plasma [3H]glucose specific activity was measured after barium hydroxide-zinc sulfate precipitation. Aliquots of the supernatant were evaporated to dryness to eliminate tritiated water prior to counting. In a subset of clamp animals, in vivo glucose uptake in skeletal muscle and adipose tissue was determined using the 2-deoxyglucose bolus technique (16.James D.E. Jenkins A.B. Kraegen E.W. Am. J. Physiol. 1985; 248: E567-E574PubMed Google Scholar) at ∼150 min after initiation of the insulin infusion. Tissue accumulation of 2-deoxyglucose was assessed by counting neutralized perchloric acid extracts before (free and phosphorylated) and after (free) barium hydroxide-zinc sulfate precipitation. Assessment of Liver Insulin Signaling following in Vivo Insulin Administration—Following an overnight fast, three ZFF rats from each treatment group were anesthetized with sodium pentobarbital. A bolus of saline (1.0 ml) was administered via a catheter placed in the jugular vein. The lateral head of the right gastrocnemius muscle was rapidly excised and clamp frozen (basal). A bolus of insulin (10 units/kg body weight in 1.0 ml of saline) was injected via the catheter. The liver and the lateral head of the left gastrocnemius were clamp frozen at 60 and 120 s, respectively, following insulin administration. Liver and muscle lysates were prepared according to the method of Saad et al. (17.Saad M.J. Folli F. Araki E. Hashimoto N. Csermely P. Kahn C.R. Mol. Endocrinol. 1994; 8: 545-557Crossref PubMed Scopus (63) Google Scholar). Lysates were subjected to Western blot analysis with commercially available antibodies against the Irs-1 or Irs-2 proteins, Akt (purchased from Upstate Biotechnology, Waltham, MA), p85 subunit of phosphoinositide 3′-kinase (BD Transduction Laboratories, San Diego, CA), or phospho-Akt (Ser-473) (New England Biolabs, Beverly, MA) and developed by enhanced chemiluminescence (ECL). GeneChip Analysis—GeneChip analysis was performed according to the standard Affymetrix (Santa Clara, CA) protocol using liver RNA from four C57Bl/6J mice treated for 7 days with AGN194204. Following probe preparation according to the manufacturer's instructions, the fragmented cRNAs were hybridized on Affymetrix GeneChips using quantitative controls, processed, and scanned according to the manufacturer. A total of 1,466 genes prefiltered to have an average difference score of >100 and showed a mean change (both decrease and increase) in expression level by at least 1.5-fold between treated and control samples were used for the network generation and pathway analysis. Gen-Bank™ accession numbers were imported into the Ingenuity Pathway Analysis Application Tool (Ingenuity Systems). Of these, 741 genes were mapped to the Ingenuity Knowledge Base, which were assembled into biological networks and were ranked by score. The score is the likelihood of a set of genes being found in the networks due to random chance (supplemental Table S1). A score of 3 indicates that there is a 1/1000 chance that the focus genes are in a network due to random chance. Therefore, scores of 3 or higher have a 99.9% confidence of not being generated by random chance alone. This score was used as the cut-off for identifying gene networks significantly affected by treatment with AGN194204. Northern Blot Analysis—Total RNA from liver was subjected to Northern analysis with the indicated cDNA probes, obtained either from the American Type Tissue Collection (Manassas, VA) or generated by PCR amplification and confirmed by direct sequencing. The blots were washed and subjected to autoradiography and quantified by phosphorimaging. Western Blotting—Membrane fractions for sterol regulatory element-binding protein-1 (SREBP-1) determination from mouse or rat liver were prepared as previously described (18.Sheng Z. Otani H. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 935-938Crossref PubMed Scopus (279) Google Scholar). Twenty micrograms of protein was separated on 4–20% gradient SDS-PAGE gel and transferred to nitrocellulose membranes. Blots were incubated with polyclonal antibody SREBP-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and developed with ECL reagent using goat anti-rabbit antisera. For FAS protein determination, liver tissues were homogenized in extraction buffer containing 1% Triton X-100, 1% Nonidet P-40, 10% glycerol, 50 mm HEPES (pH 7.9), 100 mm sodium pyrophosphate, 100 mm sodium fluoride, 10 mm EDTA, 5 mm sodium vanadate, and 0.5 mm phenylmethylsulfonyl fluoride and centrifuged at 55,000 × g for 1 h .25 μg of protein from the supernatant fraction was subjected to Western blot analysis with a monoclonal antibody against FAS (BD Transduction Laboratories, San Diego, CA). Real-time Reverse-transcription PCR—Total RNA from rat livers (1 μg) was used for reverse transcription reaction in accordance to the manufacturer's instructions (Promega, Madison WI). Real-time PCR was performed on a DNA Engine Opticon PCR cycler (MJ Research, Waltham, MA), by using the following protocol: one cycle of 15 min at 95 °C, 40 cycles of 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C, and plate read, 1 cycle of 10 min at 72 °C, and 1 cycle of melting curve from 65 °C to 95 °C. For FoxA2, the anneal temperature is 56 °C. The following synthetic oligonucleotide primers were used in real-time PCR: rGnas (forward, 5′-ACGCCTCCCCGAGACGTGCGC-3′; reverse, 5′-GGACGGAGTCACCCATTAGTG-3′), rGnai2 (forward, 5′-AGCCCCCTGACCATCTGTTTC-3′; reverse, 5′-CTGCCCCTCAGAAGAGGCCAC-3′), rGnb2 (forward, 5-ATCAGATGATGCCACATGTCG-3′; reverse, 5′-ACAGCCATCCCATCATCTGTG-3′), rFoxA2 (forward, 5′-ATCAACAACCTCATGTCCT-3′; reverse, 5′-CGAGTTCATAATAGGCCTG-3′), rFoxA3 (forward, 5′-CACCCTATTTCACTGGCCTGG-3′; reverse, 5′-GAACCGGTCATCTGTCACAGC-3′), rGlut2 (forward, 5′-ACATCCTACTTGGCCTATCTG-3′; reverse, 5′-CCAACGCCGATGGTTGCATAC-3′), rGcgr (forward, 5′-AGGGTCTGCTGGTGGCTGTTC-3′; reverse, 5′-GTCTTCGCAGAGGGCTCACAG-3′), and universal 18 S (forward, 5′-ACTCAACACGGGAAACCTCACC-3′; reverse, 5′-CCAGACAAATCGCTCCACCAAC-3′). Results of the real-time PCR data were calculated from Ct values where Ct was defined as the threshold cycle of PCR that the amplified product was first detected. The PCR reaction and protocol for the 18 S ribosome were the same as described above. ΔCt was the difference in the Ct values derived from the specific gene being assayed and the 18 S control, whereas ΔCt represented the difference between the paired tissue samples, as calculated by the formula ΔCt =ΔCt of wild type tissue–ΔCt of other tissue. The n-fold differential expression in a specific gene of a sample compared with the wild type counterpart is expressed as 2Ct. Relative RNA equivalents for each sample were obtained by normalizing to 18 S levels. Each of the 3 samples per group was run in duplicate to determine sample reproducibility, and the average relative RNA equivalents per sample pair was used for further analysis. cAMP Assay in HepG2 Cells—HepG2 cells were grown in 6-well dishes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and treated with 10 μm troglitazone or 500 nm AGN194204 for 48 h. Cells were starved for 2 h and then incubated with 100 nm glucagon for 30 min, extracted, and assayed for cAMP according to the manufacture's instructions (R&D Systems, Minneapolis, MN). Statistical Analysis—Data were analyzed by Student's t test or analysis of variance with Turkey's post-hoc testing as appropriate using SAS. Differences were determined to be significant at p < 0.05. RXR Agonists Do Not Transactivate PPARγ—The RXR agonists used in this study, AGN194204 and AGN195203, were selected as potent activators of RXR subtype homodimers (15.Beard R.L. Colon D.F. Song T.K. Davies P.J. Kochhar D.M. Chandraratna R.A. J. Med. Chem. 1996; 39: 3556-3563Crossref PubMed Scopus (66) Google Scholar). The compounds show no activity toward retinoic acid receptor subtypes (not shown). We determined the ability of these RXR compounds to drive transcription in the context of PPARγ/RXR heterodimers. CV-1 cells, which contain endogenous PPARγ and RXR receptors, were transfected with luciferase reporter constructs containing the PPRE from either the ACO or FATP promoter. The PPARγ agonist troglitazone increased transcription nearly 3-fold, whereas treatment with AGN194204 and AG195203 caused non-significant increases in transcription from the ACO promoter (Fig. 1A). When AGN194204 and troglitazone were added together, the TZD-induced transcriptional activity was consistently blunted (Fig. 1B). AGN194204 treatment also significantly blunted transcription induced by the PPARγ ligands rosiglitazone and non-thiazolidinedione GI62570 (not shown). Overexpression of RXRα, or RXRα and PPARγ, into CV-1 cells resulted in an increase of ACO-luc activity in response to RXR agonist treatments (Fig. 1B). Because the ACO promoter can be activated in the absence of PPAR (19.Krey G. Mahfoudi A. Wahli W. Mol. Endocrinol. 1995; 9: 219-231Crossref PubMed Scopus (63) Google Scholar), these results suggest that the RXR homodimer is binding to the PPRE of the ACO promoter and activating transcription. We also evaluated transcriptional activity using the FATP PPRE, which binds the PPARγ/RXR heterodimer in preference to RXR or PPARγ homodimers (20.Corsetti J.P. Sparks J.D. Peterson R.G. Smith R.L. Sparks C.E. Atherosclerosis. 2000; 148: 231-241Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In this context, treatment with troglitazone increased luciferase expression to nearly 3-fold, whereas neither AGN194204 nor LG100268 (another RXR agonist (21.Liu Y.L. Sennitt M.V. Hislop D.C. Crombie D.L. Heyman R.A. Cawthorne M.A. Int. J. Obes. Relat. Metab. Disord. 2000; 24: 997-1004Crossref PubMed Scopus (38) Google Scholar)) induced luciferase reporter expression above baseline (Fig. 1C). Thus, in contrast to previous reports (8.Martin G. Schoonjans K. Lefebvre A.M. Staels B. Auwerx J. J. Biol. Chem. 1997; 272: 28210-28217Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 9.Schulman I.G. Shao G. Heyman R.A. Mol. Cell. Biol. 1998; 18: 3483-3494Crossref PubMed Google Scholar, 11.Lenhard J.M. Lancaster M.E. Paulik M.A. Weiel J.E. Binz J.G. Sundseth S.S. Gaskill B.A. Lightfoot R.M. Brown H.R. Diabetologia. 1999; 42: 545-554Crossref PubMed Scopus (84) Google Scholar), the agents used in the present study show no ability to transactivate PPARγ/RXR. We also found that rats treated with AGN194204 showed no induction of PPARγ target genes in adipocytes following treatment with the RXR agonist (data not shown) suggesting minimal transactivation of PPARγ in vivo. Metabolic Effects of AGN194204 and Troglitazone in ZFF Rats—Previous studies have suggested a peripheral insulin-sensitizing effect of RXR agonists. To investigate the effect of AGN194204 and AGN195203, we studied the physiological effect of these agents in ZFF. ZFF rats become diabetic after ∼3 weeks feeding of high fat diet (20.Corsetti J.P. Sparks J.D. Peterson R.G. Smith R.L. Sparks C.E. Atherosclerosis. 2000; 148: 231-241Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Treatment of 9- to 10-week-old diabetic ZFF rats (ZFF-diabetic) with AGN194204 (1 mg/kg), AGN195203 (not shown), or troglitazone (400 mg/kg) orally for 7 days lowered blood glucose and insulin to control levels (Fig. 2A). In contrast to troglitazone, AGN194204 treatment resulted in markedly elevated triglyceride (Fig. 2A) and free fatty acid levels (not shown). ZFF rats treated with AGN195203 showed the same glucose lowering and triglyceride elevating effects (data not shown). Thus, like other RXR agonists, AGN194204 and AGN195203 are effective antihyperglycemic agents but also increase of serum lipid levels. Site of Action of AGN194204 and Troglitazone by Hyperinsulinemic-Euglycemic Clamp—We next performed hyperinsulinemic-euglycemic clamps with tracer [3H]glucose infusion in a separate group of ZFF rats to determine the degree of insulin sensitization in the liver and peripheral tissue afforded by each agent. ZFF rats fed high fat diet (ZFF-diabetic) for 5 weeks were ∼20 g heavier, and had significantly higher fasting serum glucose and insulin concentrations than those rats fed a standard chow diet (TABLE ONE). Treatment of the diabetic rats with AGN194204 or with troglitazone resulted in an additional increase in body weight (5.8% and 11.3%, respectively).TABLE ONEFasting plasma glucose and insulin concentrations in ZFF rats following 7 days of treatment with RXR or PPARγ agonistsBody weightPlasma glucosePlasma insulingmg/dlng/mlObese (9.Schulman I.G. Shao G. Heyman R.A. Mol. Cell. Biol. 1998; 18: 3483-3494Crossref PubMed Google Scholar)252.9 ± 6.6138.3 ± 3.76.3 ± 0.6Diabetic (9.Schulman I.G. Shao G. Heyman R.A. Mol. Cell. Biol. 1998; 18: 3483-3494Crossref PubMed Google Scholar)273.7 ± 5.6179.3 ± 9.717.3 ± 9.74204 (5.Stumvoll M. Expert Opin. Investig. Drugs. 2003; 12: 1179-1187Crossref PubMed Scopus (63) Google Scholar)290.2 ± 7.0165.0 ± 5.25.4 ± 0.6Troglitazone (6.Burant C.F. Sreenan S. Hirano K. Tai T.A. Lohmiller J. Lukens J. Davidson N.O. Ross S. Graves R.A. J. Clin. Invest. 1997; 100: 2900-2908Crossref PubMed Scopus (323) Google Scholar)305.4 ± 9.0134.9 ± 5.15.6 ± 0.6 Open table in a new tab Steady-state plasma glucose concentrations during the insulin clamp were not significantly different among groups (TABLE TWO). Steady-state plasma insulin concentrations, however, were 2-fold higher in the untreated diabetic rats compared with the obese controls, despite identical insulin infusion rates (72 milliunits· kg–1 ·min–1), suggesting that insulin clearance was impaired in the diabetic rats. Plasma insulin concentrations in the diabetics treated with RXR agonists or troglitazone were decreased 25.5% and 21.4%, respectively, but still slightly higher compared with control group.TABLE TWOSteady-state glucose infusion rates and plasma glucose and insulin concentrations during the hyperinsulinemic-euglycemic clamp in ZFF ratsGlucose infusion ratePlasma glucosePlasma insulinmg·kg-1·min-1mg/dlng/mlObese (8.Martin G. Schoonjans K. Lefebvre A.M. Staels B. Auwerx J. J. Biol. Chem. 1997; 272: 28210-28217Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar)14.1 ± 0.7102.0 ± 2.2509 ± 59Diabetic (8.Martin G. Schoonjans K. Lefebvre A.M. Staels B. Auwerx J. J. Biol. Chem. 1997; 272: 28210-28217Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar)8.5 ± 1.8110.0 ± 2.41030 ± 1264204 (5.Stumvoll M. Expert Opin. Investig. Drugs. 2003; 12: 1179-1187Crossref PubMed Scopus (63) Google Scholar)11.8 ± 1.4108.9 ± 1.7767 ± 38Troglitazone (5.Stumvoll M. Expert Opin. Investig. Drugs. 2003; 12: 1179-1187Crossref PubMed Scopus (63) Google Scholar)21.7 ± 1.1112.4 ± 0.9809 ± 75 Open table in a new tab Basal endogenous glucose production (EGP), likely reflecting primarily hepatic glucose production, was increased ∼25% in ZFF-diabetic rats compared with obese controls (Fig. 2B). Suppression of EGP by insulin during the hyperinsulinemic phase of the clamp was impaired in the diabetic rats, such that glucose production during steady-state in the diabetics was 2-fold higher than that in obese controls, even in the face of significantly higher steady-state insulin concentrations. Treatment of ZFF-diabetic with AGN194204 or troglitazone restored basal EGP to control levels and completely normalized insulin suppression of EGP during the clamp. The rate of glucose infusion for maintaining steady-state plasma glucose levels in ZFF-diabetic rats during the insulin clamp was 40% less than that required in obese controls (TABLE TWO). Insulin-stimulated whole body glucose disposal (Rd), however, was only slightly lower in the diabetics than in the obese controls (Fig. 2C). These findings indicate that the primary effect of the high fat diet in the ZFF female rat is on glucose production by the liver, with little change in insulin action in the already highly insulin-resistant peripheral tissues. Troglitazone treatment increased the rate of insulin-stimulated whole body glucose disposal by ∼50% (versus ZFF-diabetic). Interestingly, ZFF-diabetic rats treated with AGN194204 showed no improvement and even a slight decline in Rd. Thus, although the PPARγ agonist troglitazone improved both peripheral and hepatic insulin actions, the RXR agonists selectively improved glucose metabolism in the liver, with no significant improvements in insulin sensitivity in peripheral tis
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