Muraglitazar, a Novel Dual (/) Peroxisome ProliferatoreCActivated Receptor Activator, Improves Diabetes and Other Metabolic Abnormalities an
http://www.100md.com
糖尿病学杂志 2006年第1期
1 Department of Metabolic Diseases Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
2 Department of Metabolic Diseases Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
3 Department of Applied Genomics, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
ACO, acyl coenzyme-A oxidase; FFA, free fatty acid; HMW, high molecular weight; LMW, low molecular weight; MMW, medium molecular weight; PPAR, peroxisome proliferatoreCactivated receptor; WAT, white adipose tissue
ABSTRACT
Muraglitazar, a novel dual (/) peroxisome proliferatoreCactivated receptor (PPAR) activator, was investigated for its antidiabetic properties and its effects on metabolic abnormalities in genetically obese diabetic db/db mice. In db/db mice and normal mice, muraglitazar treatment modulates the expression of PPAR target genes in white adipose tissue and liver. In young hyperglycemic db/db mice, muraglitazar treatment (0.03eC50 mg · kgeC1 · dayeC1 for 2 weeks) results in dose-dependent reductions of glucose, insulin, triglycerides, free fatty acids, and cholesterol. In older hyperglycemic db/db mice, longer-term muraglitazar treatment (30 mg · kgeC1 · dayeC1 for 4 weeks) prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment (10 mg · kgeC1 · dayeC1 for 2 weeks) improves oral glucose tolerance and reduces plasma glucose and insulin levels. In addition, treatment increases insulin content in the pancreas. Finally, muraglitazar treatment increases abnormally low plasma adiponectin levels, increases higheCmolecular weight adiponectin complex levels, reduces elevated plasma corticosterone levels, and lowers elevated liver lipid content in db/db mice. The overall conclusions are that in db/db mice, the novel dual (/) PPAR activator muraglitazar 1) exerts potent and efficacious antidiabetic effects, 2) preserves pancreatic insulin content, and 3) improves metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and elevated corticosterone levels.
Peroxisome proliferatoreCactivated receptor (PPAR) and PPAR are ligand-activated nuclear hormone receptors that regulate the transcription of genes involved in carbohydrate and lipid metabolism pathways (1eC4). Activation of PPAR, which is predominantly expressed in adipose tissue, results in insulin-sensitizing antidiabetic effects (5,6). Activation of PPAR, which is highly expressed in the liver, results in the lowering of triglycerides and the elevation of plasma HDL cholesterol levels (7,8). In addition, both PPAR and PPAR selective activators have been demonstrated to suppress vessel wall inflammatory activity and reduce atherosclerosis in experimental animal models through complementary mechanisms (9,10). Since type 2 diabetic patients often develop dyslipidemia and other metabolic abnormalities, eventually resulting in atherosclerotic coronary heart disease, an agent that simultaneously activates both PPAR and PPAR has the potential to be useful for the treatment of these patients (11,12).
The discovery and preliminary biological and pharmacokinetic properties of muraglitazar (BMS-298585), a novel oxybenzylglycine dual (/) PPAR activator, have been recently described (13). Muraglitazar binds with high affinity to both human PPAR and PPAR ligand binding domain protein (IC50 for binding = 0.19 and 0.25 e蘭ol/l, respectively) and potently transactivates full-length human PPAR- or PPAR-mediated reporter gene activity (EC50 for transactivation = 0.11 and 0.32 e蘭ol/l, respectively). We assessed the effects of muraglitazar treatment on diabetes and other metabolic abnormalities in genetically obese, diabetic, and hyperlipidemic db/db mice. Untreated db/db mice exhibit progressive deterioration of glycemic control and develop insulin deficiency and loss of pancreatic insulin content (14eC17). In these mice, the clinically used PPAR selective activators (e.g., rosiglitazone and piogitazone) have been reported to show antidiabetic effects, and the PPAR selective activators (e.g., gemfibrozil) have been reported to lower plasma triglyceride levels and also show some improvement in insulin sensitivity (14eC17). Furthermore, we assessed the effects of muraglitazar treatment on diet-induced hyperglycemia and hyperlipidemia in C57BL/6J mice (diet-induced obese [DIO]) (18)
RESEARCH DESIGN AND METHODS
Compounds.
Muraglitazar and rosiglitazone were synthesized by BMS Medicinal Chemistry. Fenofibric acid was purchased from Sigma (St. Louis, MO).
Mice.
db/db mice (C57BL/6ks lepreC/eC) and age-matched lean normal C57BL/6J or Swiss Webster mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under controlled temperature (23°C) and lighting (12 h of light between 6 A.M. and 6 P.M.) with free access to water and standard mouse diet (18% protein rodent diet no. 2018; Harlan). The db/db mice were prebled, and those within a narrow range of fasted glucose levels were selected for studies to minimize variability between control and drug-treated groups. C57BL/6J mice on experimental diet were fed research diet no. 12327, which contains 40% sucrose/40% fat by calorie (Research Diets, New Brunswick, NJ) for 12 weeks before the start of the experiment and were maintained on this diet for the duration of the experiment. Mice were dosed daily by oral gavage in a vehicle composed of 20% polyethylene glycol (vol/vol), 5% N-methyl pyrrolidone, and 75% 10 mmol/l phosphate buffer, pH 7.4. Bristol-Myers Squibb study guidelines were strictly followed in the investigations.
Gene expression profiling.
Lean normal mice or db/db mice that were treated with vehicle or compounds, respectively, were killed, and their inguinal white adipose tissue (WAT) and liver were harvested. Total RNA was isolated from WAT or liver samples using RNeasy (Qiagen, Valencia, CA). For Northern blot analysis, 15 e蘥 RNA was subjected to MOPS-formaldehyde gel electrophoresis. The gels were blotted to nylon membranes and hybridized to 32P-cDNA probes according to standard procedures. The radioactivity in the hybridized bands was counted on an Instant Imager (Packard Instruments, Meridian, CT). Alternatively, SYBR-Green PCR analysis was carried out (Applied Biosystems, Foster City, CA). Oligonucleotide primers were designed using Primer Express, and RT-PCRs were carried out (primer sequences and protocol available upon request). The mRNA levels of target genes were normalized to control glyceraldehyde-3-phosphate dehydrogenase mRNA levels. WAT RNA samples from vehicle- and compound-treated mice were also analyzed by Affymetrix microarray for changes in gene expression pattern (protocol available upon request).
Triglyceride/VLDL secretion assay.
C57BL/6J mice that were treated with vehicle or compounds for 7 days were fasted overnight and intravenously injected with Triton-WR1339 (250 mg/kg) 1 h after the final dosing. The injection of Triton prevents the degradation of triglyceride-rich VLDL (triglyceride/VLDL) particles in plasma, resulting in an accumulation of triglycerides. The secretion rate (typically 0.16eC0.20 mg · mineC1 · 100 g body wteC1, linear for 5 h after Triton administration) was determined by calculating the amount of triglycerides accumulated 2.5 h after the Triton injection/100 g body wt. Triglyceride levels were determined using a Roche Cobas blood chemistry analyzer.
Acyl coenzyme-A oxidase activity.
The db/db mice that were treated with vehicle or compounds for 14 days were killed, and their liver was harvested. Liver acyl coenzyme-A oxidase activity (ACO) activity [(slope of the rate of A502 increase after addition of substrate eC the slope of the background rate)/mg protein] was measured according to a published method (19).
Plasma chemistry analysis.
About 50 e蘬 tail vein blood from overnight-fasted or ad libitumeCfed mice was collected in EDTA-coated tubes. Plasma glucose, triglyceride, free fatty acid (FFA), cholesterol, and HDL cholesterol levels were determined using a Roche Cobas blood chemistry analyzer; insulin, adiponectin, and corticosterone levels were determined by mouse enzyme-linked immunosorbent assay kits (Linco Research, St. Charles, MO). Corticosterone levels were assessed at 10 A.M. during regular 12-h diurnal light cycle. ED50 to normalization is calculated as the midpoint of the dose-response activity curve using a four-parameter-fit equation.
HigheC, mediumeC, and loweCmolecular weight adiponectin complexes.
The higheCmolecular weight (HMW), mediumeCmolecular weight (MMW), and loweCmolecular weight (LMW) adiponectin complexes in db/db mouse plasma were detected according to the method described by Waki et al. (20). A total of 0.5 e蘬 db/db mouse plasma samples were diluted (1:12) and incubated for 1 h at room temperature in reducing sample buffer (3% SDS, 50 mmol/l Tris-HCl, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, and 10 mmol/l dithiothreitol) or nonreducing sample buffer (3% SDS, 50 mmol/l Tris-HCl, pH 6.8, and 10% glycerol) and subjected to SDS-PAGE under reducing/heat-denaturing conditions (samples were heated at 95°C for 10 min) or nonreducing/nonheat-denaturing conditions, according to the standard Laemmli’s method with Criterion precast Tris-HCl 4eC15% gel (Bio-Rad, Hercules, CA). For immunoblotting, proteins separated by SDS-PAGE were transferred to nitrocellulose membranes, blocked with StartingBlock (Tris-buffered saline) Blocking Buffer (Pierce, Rockford, IL), and then incubated with 1:5,000 diluted anti-mouse adiponectin globular domain monoclonal antibody (Chemicon, Temecula, CA) in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature. After washing, the membranes were incubated with goat anti-mouse IRDye 800 (1:10,000) (Rockland, Gilbersville, PA) for 1 h at room temperature and then washed thoroughly. The membrane was scanned with the Odyssey Imaging System (Li-Cor, Lincoln, NE).
Oral glucose tolerance test.
The db/db mice, which were on a 2-week dosing regimen, were fasted overnight on day 13. On day 14, an oral dose of vehicle alone or compound was given in the morning, and blood samples were collected from the tail vein for determination of baseline values (t = 0 min). The mice were then gavaged with an oral bolus of glucose (2 g/kg), and additional blood samples were collected at regular intervals (t = 15, 30, 60, and 90 min) for glucose and insulin measurement. Homeostasis model assessment index values were calculated using the following equation: (the product of the fasting insulin levels [mU/l] x fasting glucose levels [mmol/l]/22.5).
Pancreatic insulin content.
Pancreata were harvested from overnight-fasted vehicle- and drug-treated mice, placed in liquid N2, then stored at eC20°C. Pancreata were homogenized in acid-ethanol (75% ethanol, 23.5% water, and 1.5% c-HCl in 1.8 ml volume) with a polytron homogenizer. The homogenates were stored at 4°C for 28 h and then centrifuged at 1500g for 30 min at 4°C. The supernatants were diluted (1:20,000), and insulin levels were determined by enzyme-linked immunosorbent assay (21).
Liver lipid analysis.
Liver triglyceride levels were determined using a Wako Kit (no. 997-69801). Frozen liver pieces were homogenized in saline and brought to a concentration of 0.05 mg/1 ml. Twenty microliters of the sample were solubilized with 20 e蘬 deoxycholate (1.6% wt/vol in water), and 1 ml of the Wako reagent was added. The mixture was incubated at 37°C for 15 min, and the absorbance was read at 505 nm.
Statistical analysis.
Unpaired, two-tailed Student’s t tests were performed for comparisons between compound-treated and vehicle control groups. Differences were considered significant at P < 0. 05.
RESULTS
Muraglitazar modulates PPAR target gene expression in mice.
As previously described, muraglitazar potently stimulates full-length human PPAR- and PPAR-mediated reporter gene expression (EC50 for PPAR and PPAR transactivation = 0.11 and 0.32 e蘭ol/l, respectively; 13) The ability of muraglitazar to transactivate full-length mouse PPAR or PPAR receptor has not been determined. However, in a chimeric Gal4/mouse PPAR-mediated reporter gene assay, muraglitazar shows mouse PPAR agonist activity at levels comparable with its human PPAR activity (EC50 for mouse PPAR = 0.09 e蘭ol/l for muraglitazar and 0.08 e蘭ol/l for the PPAR selective activator rosiglitazone; the PPAR selective activator fenofibric acid was inactive) and mouse PPAR agonist activity that is weaker than its human PPAR activity (observed EC50 for mouse PPAR = 23.8 e蘭ol/l for muraglitazar and 16.3 e蘭ol/l for fenofibric acid; rosiglitazone was inactive). The disparity between the mouse and human PPAR activity is likely due to mouse/rodent-specific differences in the interactions of muraglitazar with several amino acid residues that are altered between mouse and human PPAR ligand binding domains (22).
The effects of muraglitazar treatment on the expression of PPAR target genes in WAT and liver were determined in db/db and normal mice. In db/db mice, muraglitazar treatment (10 mg · kgeC1 · dayeC1 for 2 weeks) increases mRNA levels of fatty acid binding protein aP2, GLUT4 glucose transporter, and lipoprotein lipase in WAT and stimulates both mRNA and activity levels of ACO and suppresses apolipoprotein CIII mRNA levels in liver (Fig. 1A and B). Microarray analysis of WAT RNA from muraglitazar- or rosiglitazone (10 mg · kgeC1 · dayeC1 for 7 days)-treated db/db mice shows that expression levels of genes that are implicated in 1) adipocyte differentiation, 2) insulin signaling and glucose metabolism, 3) fatty acid transport, 4) fatty acid oxidation, 5) triglyceride synthesis, and 6) energy expenditure are modulated by both muraglitazar and rosiglitazone treatment (Table 1). Both muraglitazar and rosiglitazone treatment (10 mg · kgeC1 · dayeC1 for 3 days) stimulate aP2 and lipoprotein lipase mRNA levels and suppress 11-hydroxy steroid desaturase 1 mRNA levels in normal mouse WAT (Fig. 1C). Muraglitazar, but not rosiglitazone, stimulates ACO mRNA levels in normal mouse liver (Fig. 1D). Finally, in normal mice, muraglitazar treatment (3, 10, and 30 mg · kgeC1 · dayeC1 for 7 days) dose dependently inhibits triglyceride/VLDL secretion from the liver without promoting liver weight increase (Fig. 1E and F). Fenofibrate treatment (30, 50, and 100 mg · kgeC1 · dayeC1) also inhibits triglyceride/VLDL secretion (Fig. 1E and F). However, this effect is accompanied by dose-dependent increases in liver weight, which is a known fibrate-induced phenomenon in rodents (23). Rosiglitazone treatment (3, 10, and 30 mg · kgeC1 · dayeC1), by contrast, does not inhibit triglyceride/VLDL secretion (Fig. 1E and F). The gene expression data thus demonstrate that muraglitazar treatment results in modulation of PPAR target gene expression in WAT and liver. The PPAR agonist activity of muraglitazar may have contributed to the differences in the expression levels of various PPAR target genes in WAT and liver as well as inhibition of VLDL secretion in muraglitazar-treated compared with rosiglitazone-treated mice.
Muraglitazar treatment ameliorates diabetes and hyperlipidemia and increases pancreatic insulin content in db/db mice.
Muraglitazar was investigated in three separate studies for 1) dose-dependent lowering of fasted and fed glucose, insulin, FFA, triglyceride, and cholesterol levels in young hyperglycemic db/db mice (8-week-old males; 0.03eC50 mg · kgeC1 · dayeC1 orally for 2 weeks), 2) effect on time-dependent deterioration of glycemic control and plasma insulin levels in older db/db mice (12-week-old females; 30 mg · kgeC1 · dayeC1 for 4 weeks), and 3) improvements in hyperglycemia and glucose tolerance and effect on pancreatic insulin content in severely hyperglycemic db/db mice (10-week-old females with fasting plasma glucose >500 mg/dl; 10 mg · kgeC1 · dayeC1 for 2 weeks; rosiglitazone at 10 mg · kgeC1 · dayeC1 was used as a positive control in the study).
In study 1, muraglitazar treatment results in dose-dependent lowering of both fasted (day 7 data shown, similar data were also obtained after 14 days) and fed (on day 15) plasma glucose, FFA, insulin, triglyceride, and cholesterol levels (Fig. 2AeCE). Amelioration of hyperglycemia in the presence of reduced plasma insulin levels suggests that insulin sensitivity has been improved in muraglitazar-treated young db/db mice. As previously observed with PPAR activators in rodents, the cholesterol-lowering effect of muraglitazar is restricted to a reduction of the HDL cholesterol fraction (data not shown) (24). The ED50 to normalization of glucose and triglyceride levels in fasted animals on day 14 are 0.1 and 0.2 mg · kgeC1 · dayeC1, respectively, and in fed animals on day 15 are 0.5 and 1.3 mg · kgeC1 · dayeC1, respectively. As observed with PPAR activators (4), muraglitazar-treated mice (at 10 and 50 mg · kgeC1 · dayeC1) experience a trend toward increased body weight gain in comparison with the vehicle-treated mice (Fig. 2F).
In study 2, the vehicle- and muraglitazar-treated db/db mice were monitored weekly for changes in fasting glucose and insulin levels. As shown in Fig. 3A and B, the vehicle-treated control db/db mice show poor glycemic control throughout the duration of the study. The vehicle-treated db/db mice also show signs of further deterioration of glycemic control (higher fasting plasma glucose levels) and some -cell exhaustion (significant drop in plasma insulin levels) by the end of the 4-week treatment period (Fig. 3B and C). By contrast, the muraglitazar-treated mice show time-dependent improvement in glycemic control (fasting glucose levels are reduced to the levels observed in lean normal mice) and maintain reduced, but stable, plasma insulin levels during the entire 4-week period (Fig. 3AeCC). Muraglitazar-treated mice also show significant improvements in other metabolic parameters such as fasted FFA (eC49%), fed glucose (eC60%), fasted triglyceride (eC31%), and fed triglyceride (eC47%) levels (Fig. 3DeCF).
In study 3, at the end of the 2-week treatment period, the db/db mice were fasted overnight and, after collecting baseline (t = 0 min) plasma samples, were challenged with an oral bolus of glucose. Muraglitazar treatment results in significant reduction of baseline fasting plasma glucose (eC51%), insulin (eC55%), and FFA (eC33%) levels as well as homeostasis model assessment index (eC63%) (Table 2). When challenged with an oral bolus of glucose, muraglitazar-treated animals exhibit a reduced glucose excursion (indicating increased tolerance to glucose) and lower plasma insulin levels compared with vehicle-treated animals (Fig. 4A and B). The increased glucose tolerance, along with the concomitantly lowered insulin levels and reduced homeostasis model assessment index, indicate that insulin sensitivity has been improved in muraglitazar-treated mice. Rosiglitazone treatment also results in improved glycemic control; however, the effects are less pronounced than with muraglitazar at the same dose (Table 2, Figs. 4A and B). In addition to improvements in insulin sensitivity and glycemic control, pancreatic insulin content is increased by about fourfold by both muraglitazar and rosiglitazone treatment (Fig. 4C), which is suggestive of some preservation of -cell function. Neither drug shows any significant impact on the weight of the pancreas in this study (Fig. 4D).
Muraglitazar treatment lowers hyperglycemia and hyperlipidemia in DIO-mice.
C57BL/6J mice, when maintained on a diet high in fat and sucrose (DIO-mice), develop mild hyperglycemia and high plasma triglyceride and cholesterol levels (18). Consistent with its antidiabetic and lipid-lowering effects in db/db mice, muraglitazar treatment (10 mg · kgeC1 · dayeC1 for 2 weeks) lowers fasting glucose, triglyceride, and cholesterol levels of DIO-mice to the levels observed in mice on normal standard diet (Table 3).
Muraglitazar treatment increases low plasma adiponectin levels, increases HMW adiponectin complex levels, and lowers elevated corticosterone levels in db/db mice.
As in some type 2 diabetic patients, db/db mice exhibit abnormally low plasma adiponectin levels and high plasma corticosterone (the murine counterpart to cortisol in humans) levels compared with age-matched normal C57BL/6J mice. Diminished adiponectin levels and elevated corticosterone levels serve as biomarkers of tissue insulin resistance and increased hepatic glucose production (20,25eC30). Adiponectin exists as HMW, MMW, and LMW complexes in plasma (20,25,28). In patients and in animal models, increased levels of HMW adiponectin complex have been associated with improved insulin sensitivity (20,25,28).
In older hyperglycemic db/db mice, muraglitazar treatment (study 2) elevates plasma adiponectin levels and lowers plasma corticosterone levels to the levels observed in normal mice (Fig. 5A and B). In severely hyperglycemic db/db mice, muraglitazar treatment (study 3) elevates their plasma adiponectin levels above the levels observed in lean normal mice and significantly lowers plasma corticosterone levels (Fig. 5C and D). By comparison, rosiglitazone treatment elevates adiponectin levels to the levels observed in normal mice and lowers corticosterone to the levels comparable with muraglitazar-treated levels in this study (Fig. 5C and D). Furthermore, immunoblot analysis shows that in muraglitazar-treated db/db mice (10 mg · kgeC1 · dayeC1 for 2 weeks), their plasma total adiponectin levels as well as HMW adiponectin complex levels are substantially increased compared with the vehicle-treated mice (Fig. 5E and F).
Muraglitazar treatment lowers liver lipid content in db/db mice.
Obese patients with insulin resistance and type 2 diabetes frequently suffer from nonalcoholic fatty liver condition (31,32). Elevated lipid content in the liver has been implicated in hepatic insulin resistance, glucose overproduction, and increased VLDL synthesis and secretion (30,31). The db/db mice on a normal diet accumulate lipids (primarily triglycerides) in the liver and develop hepatic steatosis. In these mice, muraglitazar treatment (50 mg · kgeC1 · dayeC1 for 2 weeks) results in significant reductions of liver triglycerides content (76 ± 3 mg/g liver tissue in muraglitazar-treated vs. 100 ± 10 mg/g liver tissue in vehicle-treated mice).
DISCUSSION
Muraglitazar is a novel dual (/) PPAR activator that selectively binds to and activates human PPAR and human PPAR (13,33eC37). The in vivo pharmacological data in lean normal mice and in db/db mice demonstrate that muraglitazar modulates the expression of PPAR target genes implicated in the regulation of glucose and lipid metabolic pathways in WAT and in liver. The in vivo data also demonstrate that muraglitazar is a potent and efficacious antidiabetic and lipid-lowering agent in db/db mice. In young hyperglycemic db/db mice, muraglitazar lowers both fasted and fed glucose and triglyceride levels to the levels commonly observed in lean normal mice. In addition, muraglitazar treatment reduces fasted and fed insulin, FFA, and cholesterol levels. In older db/db mice, longer-term muraglitazar treatment prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment markedly reduces fasted plasma glucose and insulin levels as well as glucose excursion. In these animals, muraglitazar also increases the insulin content in the pancreas. Muraglitazar treatment elevates the low plasma adiponectin levels, increases the HMW adiponectin complex levels, and reduces the elevated plasma corticosterone levels of db/db mice. Muraglitazar treatment also significantly lowers triglyceride content in db/db mouse liver. Finally, in DIO-mice, muraglitazar treatment normalizes diet-induced mild hyperglycemia and hyperlipidemia, which corroborates the glucose and lipid-lowering effects in db/db mice. Muraglitazar treatment did not cause hypoglycemia in mice under the experimental conditions used.
The antidiabetic and lipid-lowering effects induced by muraglitazar treatment may result from one or more of the following PPAR-mediated mechanisms: 1) improved insulin action and enhanced glucose uptake in adipose tissue and skeletal muscle, 2) increased fatty acid uptake and storage in adipose tissue, 3) reduced plasma FFA levels, 4) increased plasma total adiponectin and HMW adiponectin complex levels, 5) suppression of glucose overproduction by liver, 6) enhanced VLDL catabolism in the plasma, and 7) reduced triglycerides/VLDL synthesis/secretion in the liver (1eC4,25,38eC39). The HDL cholesterol lowering in muraglitazar-treated mice is most likely the result of a rodent-specific PPAR-mediated mechanism that suppresses the production of apolipoprotein A1 (a major protein component of HDL particles) in the liver (24). In humans, muraglitazar, like other human PPAR activators (e.g., fenofibrate, gemfibrozil), has demonstrated plasma HDL cholesteroleCraising effects (7,8,40eC42).
The trend toward increased weight gain in muraglitazar-treated db/db mice is probably due to a combination of effects including 1) enhanced adipogenesis, 2) retention of calories that would otherwise be lost due to glucosuria, and 3) water retention due to the alleviation of the glucose-driven osmotic diuresis and/or increased plasma or extracellular fluid volume (43,44). The liver triglyceride-lowering effect of muraglitazar is possibly due to reduced plasma FFA and lipid levels, which would limit fatty acid substrate availability for lipid biosynthesis in the liver. Reduced lipid content in the liver will lower hepatic insulin resistance, glucose overproduction, and increased VLDL synthesis (31,32).
In muraglitazar-treated db/db mice, the improvement in insulin sensitivity and the concomitant reduction in plasma glucose and FFA levels are anticipated to 1) lower insulin secretory demand on -cells and 2) prevent apoptosis of -cells, respectively. These effects may help to prevent deterioration of -cell function, loss of pancreatic insulin content, development of insulin deficiency, and deterioration of glycemic control in muraglitazar-treated db/db mice.
The increase in both total adiponectin levels and HMW adiponectin complex levels are expected to stimulate fatty acid oxidation in liver and skeletal muscle as well as enhance insulin sensitivity and glucose uptake in skeletal muscle (20,25eC28). Interestingly, an inverse correlation has been recently described between plasma adiponectin levels and the rate of incidence of myocardial infarction in men irrespective of their glycemic status (47). The reduction of corticosterone levels by muraglitazar is likely due to reduced metabolic stress and/or PPAR-mediated suppression of the 11-hydroxy steroid desaturase 1 gene expression in WAT and liver. The reduced corticosterone levels may suppress hepatic glucose overproduction and enhance glucose uptake in peripheral tissues (29,30).
In conclusion, the novel dual (/) PPAR activator muraglitazar 1) exerts potent and efficacious insulin-sensitizing antidiabetic effects, 2) prevents time-dependent deterioration of glycemic control and development of insulin deficiency, 3) increases pancreatic insulin content and, 4) improves other metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and high corticosterone levels in db/db mice. In the clinical setting, muraglitazar treatment (0.5eC20 mg for 28 days) lowers glucose, insulin, triglyceride, FFA, and apolipoprotein CIII levels and increases HDL cholesterol levels in type 2 diabetic patients (40eC42). These clinical data emphasize the utility of db/db mice as a useful model for evaluating antidiabetic properties and antidyslipidemic properties of novel PPAR activators. However, the weak mouse PPAR activity, relative to its human PPAR activity, for muraglitazar may suggest that its antidyslipidemic effects are probably underrepresented in db/db mice. Finally, since type 2 diabetes patients often suffer from dyslipidemia and other metabolic abnormalities, thus putting them at high risk for cardiovascular disease, muraglitazar, with its dual (/) PPAR activity, has the potential to be useful for the treatment of these patients.
ACKNOWLEDGMENTS
We thank Drs. S. Taylor, R. Parker, J. Whaley, R. Mukherjee, J. Graham, and R. Belder for critically reading the manuscript.
FOOTNOTES
S.B. is currently affiliated with Novartis Institute Bio-Med Research, Cambridge, Massachusetts. K.A.M. is currently affiliated with Advinus Therapeutics, Pune, India. J.W. is currently affiliated with the Department of Cardiovascular Pharmaceuticals, Pfizer, Ann Arbor, Michigan.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Kersten S, Desvergne B, Wahli W: Roles of PPARs in health and disease. Nature 405:421eC424, 2000
Kliewer S, Xu E, Lambert M, Willson T: Peroxisome proliferator-activated receptors: from genes to physiology. Recent Prog Horm Res 56:239eC263, 2001
Berger J, Moller D: The mechanism of action of PPARs. Annu Rev Med 53:409eC435, 2002
Bays H, Mandarino L, DeFronzo R: Mechanisms of endocrine disease: role of adipocytes, free fatty acids, and ectopic fat in pathogenesis of type 2 diabetes mellitus: PPAR agonists provide a rational therapeutic approach. J Clin Endocrinol Metab 89:463eC478, 2004
Olefsky J: Treatment of insulin resistance with PPAR agonists. J Clin Inves 106:467eC472, 2000
Lebovitz H: Differentiating members of the thiazolidinedione class: a focus on safety. Diabetes Metab Res Rev 18 (Suppl. 2):S23eCS29, 2002
Robins S, Collins D, Wittes J, Papademetriou V, Deedwania P, Schaefer E, McNamara J, Kashyap M, Hershman J, Wexler L, Rubins H: Relation of gemfibrozil treatment and lipid levels with major coronary events. VA-HIT: a randomized controlled trial. J Am Med Assoc 285:1585eC1591, 2001
Diabetes Atherosclerosis Intervention Study Investigators: Effect of fenofibrate on progression of coronary artery disease in type 2 diabetes: the Diabetes Atherosclerosis Intervention Study, a randomised study. Lancet 357:905eC910, 2001
Buchan K, Hassall D: PPAR agonists as direct modulators of the vessel wall in cardiovascular disease. Med Res Rev 20:350eC366, 2000
Duval C, Chinetti G, Trottein F, Fruchart J, Staels B: The role of PPARs in atherosclerosis. Trends Mol Med 8:422eC430, 2002
Beckman J, Creager M, Libby P: Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. J Am Med Assoc 287:2570eC2581, 2002
Nesto R, Drexler A: Evaluating the cardiovascular effects of the thiazolidinediones and their place in the management of type 2 diabetes mellitus: proceedings of a symposium. November 6eC8, 2002, New York, New York, USA. Am J Med 115 (Suppl. 1):1SeC120S, 2003
Devasthale P, Chen S, Jeon Y, Qu F, Shao C, Wang W, Zhang H, Cap M, Farrelly D, Golla R, Grover G, Harrity H, Ma Z, Moore L, Ren J, Seethala R, Cheng L, Sleph P, Sun W, Tieman A, Wetterau J, Biller S, Ryono D, Selan F, Hariharan N, Cheng PTW: Design and synthesis of N-[(4-methoxyphenoxy)carbonyl]-N-[[4-[2-(5-methyl-2-phenyl-4-oxazolyl) ethoxy] phenyl]methyl]glycine [muraglitazar/BMS-298585), a novel PPAR/ dual agonist with efficacious glucose and lipid-lowering activities. J Med Chem 48:2248eC2250, 2005
Connor S, Hughes M, Moore G, Lister C, Smith S: Antidiabetic efficacy of BRL-49653, a potent orally active insulin sensitizing agent, assessed in the C57BL/KsJ db/db diabetic mouse my non-invasive 1H NMR studies of urine. J Pharm Pharmacol 49:336eC344, 1997
Diani A, Sawada G, Wyse B, Murray F, Khan M: Pioglitazone preserves pancreatic islet structure and insulin secretory function in three murine models of type 2 diabetes. Am J Physiol Endo Metab 286:E116eCE122, 2004
Ishida H, Takizawa M, Ozawa S, Nakamichi Y, Yamaguchi S, Katsuta F, Tanaka T, Maruyama M, Katahira H, Yoshimoto K, Itagaki E, Nagamatsu S: Pioglitazone improves insulin secretory capacity and prevents the loss of -cell mass in obese diabetic db/db mice: possible protection of -cells from oxidative stress. Metabolism 53:488eC494, 2004
Mukherjee R, Strasser J, Jow L, Hoener P, Paterniti J, Heyman R: RXR agonists activates PPAR-inducible genes, lower triglycerides, and raise HDL levels in vivo. Arterioscler Thromb Vasc Biol 18:272eC276, 1998
Rossmeisl M, Rim J, Koza R, Kozak L: Variation in type 2 diabetes-related traits in mouse strains susceptible to diet induced obesity. Diabetes 52:1958eC1966, 2003
Small G, Burdett K, Connock M: A sensitive spectrophotometric assay for peroxisomal acyl co-A oxidase. Biochem J 227:205eC210, 1985
Waki H, Yamauchi T, Kamon J, Ito Y, Uchida S, Kita S, Hara K, Hada Y, Vasseur F, Froguel P, Kimura S, Nagai R, Kadowaki T: Impaired multimerization of human adiponectin mutants associated with diabetes: molecular structure and multimer formation of adiponectin. J Biol Chem 278:40352eC40363, 2003
Yajima K, Hirose H, Fujita H, Seto Y, Ukeda K, Miyashita K, Kawai T, Yamamoto Y, Ogawa T, Yamada T, Saruta T: Combination therapy with PPAR and PPAR agonists increases glucose-stimulated insulin secretion in db/db mice. Am J Physiol Endo Met 284:E966eCE971, 2003
Boettcher B, Fanelli B, Stephen Z, Caplan S, Sabio M: Comparison of ligand binding affinities in the mouse and human PPAR and ligand binding domains. Keystone Symposium-PPARs, 2003 (Abstract no. 106)
Petit D, Bonnefis M, Rey C, Infante R: Effects of ciprofibrate and fenofibrate on liver lipids and lipoprotein synthesis in normo- and hyperlipidemic rats. Atherosclerosis 74:215eC225, 1988
Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart J: Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 10:2088eC2093, 1998
Kadowaki T, Yamauchi T: Adiponectin and adiponectin receptors. Endocrin Rev 26:439eC451, 2005
Combs T, Wagner J, Berger J, Doebber T, Wang W, Zhang B, Tanen M, Berg A, O’Rahilly S, Savage D, Chatterjee K, Weiss S, Larson P, Gottesdiener K, Gertz B, Charron M, Scherer P, Moller D: Induction of adipocyte complement related protein of 30 Kd by PPAR agonists: a potential mechanism of insulin sensitization. Endocrinology 143:998eC1007, 2002
Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno N, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T: Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762eC769, 2003
Pajvani U, Hawkins M, Combs T, Rajala M, Doebber T, Berger J, Wagner J, Wu M, Knopps A, Xiang A, Utzschneider K, Kahn S, Olefsky J, Buchanan T, Scherer P: Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity. J Biol Chem. 279:12152eC12162, 2004
Alberts P, Nilsson C, Selen G, Engblom L, Edling N, Norling S, Klingstrom G, Larsson C, Forsgren M, Ashkzari M, Nilsson C, Fiedler M, Bergqvist E, Ohman B, Bjorkstrand E, Abrahmsen L: Selective inhibition of 11 beta-hydroxysteroid dehydrogenase type 1 improves hepatic insulin sensitivity in hyperglycemic mice strains. Endocrinology 144:4755eC4762, 2003
Nielsen M, Caumo A, Chandramouli V, Schumann W, Cobelli C, Landau B, Vilstrup H, Rizza R, Schmitz O: Impaired basal glucose effectiveness but unaltered fasting glucose release and gluconeogenesis during short-term hypercortisolemia in healthy subjects. Am J Physio Endocrin Met 286:E102eCE110, 2004
Lind P: Interdependence of hepatic lipid and glucose metabolism: novel pharmaceutical targets for diabetes. Curr Opin Investig Drugs 5:395eC401, 2004
Bajaj M, Suraamornkul S, Hardies L, Pratipanawatr T, DeFronzo R: Plasma resistin concentration, hepatic fat content, and hepatic and peripheral insulin resistance in pioglitazone-treated type II diabetic patients. Int J Obes Relat Metab Disord 28:783eC789, 2004
Murakami K, Tobe K, Ide T, Mochizuki T, Ohashi M, Akanuma Y, Yazaki Y, Kadawaki T: A novel insulin sensitizer acts as coligand for PPAR and PPAR: effect of PPAR activation on abnormal lipid metabolism in liver of Zucker fatty rats. Diabetes 47:1841eC1847, 1998
Etriglyceridesen G, Oldham B, Johnson W, Broderick C, Montrose C, Brozinick J, Misener E, Bean J, Bensch W, Brooks D, Shuker A, Rito C, McCarthy J, Ardecky R, Tyhonas J, Dana S, Bilakovics J, Paterniti J, Ogilvie K, Liu, Kauffman R: A tailored therapy for the metabolic syndrome: the dual PPAR/ agonist LY465608 ameliorates insulin resistance and diabetic hyperglycemia while improving cardiovascular risk factors in pre-clinical models. Diabetes 51:1083eC1087, 2002
Chakrabarti R, Vikramadithyan R, Misra P, Hiriyan J, Raichur S, Damarla R, Gershome C, Suresh J, Rajagopalan R: Ragaglitazar: a novel PPAR and PPAR agonist with potent lipid-lowering and insulin sensitizing efficacy in animal models. Br J Pharmacol 140:527eC537, 2003
Ljung B, Bamberg K, Dahllof B, Kjellstedt A, Oakes N, Ostling J, Camejo G: AZ-242, a novel PPAR/ dual agonist with beneficial effect on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats. J Lipid Res 43:1855eC1863, 2002
Pickavance L, Brand C, Wassermann K, Wilding J: The dual PPAR/ agonist, ragaglitazar, improves insulin sensitivity and metabolic profile equally with pioglitazone in diabetic and dietary obese ZDF rats. Br J Pharmacol 144:308eC316, 2005
Chaput E, Saladin R, Silvestre M, Edgar A: Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight. Biochem Bioplys Res Commun 27:445eC450, 2000
Guerre-Millo M, Gervois P, Rape E, Madsen L, Poulain P, Derudas B, Herbert J-M, Winegar D, Wilson T, Fruchart J-C, Berge R, Staels B: PPAR activators improve insulin sensitivity and reduce adiposity. J Biol Chem 275:16638eC16642, 2000
Mosqueda-Garcia R, Frost C, Swaminathan A, Raymond R, Nepal S, Reeves R, Gregg R: Glucose lowering effects of multiple dose administration of muraglitazar (BMS-298585), a novel PPAR/ dual agonist, in type 2 diabetic patients (Abstract). Diabetes 53 (Suppl. 2):A32, 2004
Gregg R, Swaminathan A, Frost C, Nepal S, Raymond R, Mosqueda-Garcia R: Muraglitazar, a novel PPAR/ dual agonist, lowers fasting plasma glucose, triglycerides, NEFA and apoCIII after once a day administration in type 2 diabetic patients (Abstract no. 717). Abstract presented at the 40th Annual Meeting of the European Association for the Study of Diabetes, 5eC9 September 2004, Munich, Germany
Frost C, Swaminathan A, Raymond R, Nepal S, Gregg R, Reeves R, Mosqueda-Garcia R: Lipid lowering effects of multiple dose administration of muraglitazar (BMS-298585), a novel PPAR/ dual agonist, in type 2 diabetic patients (Abstract). Diabetes 53 (Suppl. 2):A475, 2004
Lebovitz H: Differentiating members of the thiazolidinedione class: a focus on safety. Diabetes Metab Res Rev 18 (Suppl. 2):S23eCS29, 2002
Larsen T, Toubro S, Astrup A: PPAR agonists in the treatment of type II diabetes: is increased fatness commensurate with long-term efficacy Int J Obes Relat Metab Disord 27:147eC161, 2003
Bergman R, Ader M: Free fatty acids and pathogenesis of type 2 diabetes mellitus. TEM 11:331eC356, 2000
Buchanan T, Xiang A, Peters R, Kjos S, Marroquin A, Goico J, Ochoa C, Tan S, Berkowitz K, Hodis H, Azen S: Preservation of pancreatic -cell function and prevention of type 2 diabetes by pharmacological treatment of insulin resistance in high-risk Hispanic women. Diabetes 51:2796eC2803, 2002
Pischon T, Girman C, Hotamisligil G, Rifai N, Hu F, Rimm E: Plasma adiponectin levels and risk of myocardial infarction in men. J Am Med Assoc 291:1730eC1737, 2004(Thomas Harrity, Dennis Fa)
2 Department of Metabolic Diseases Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
3 Department of Applied Genomics, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
ACO, acyl coenzyme-A oxidase; FFA, free fatty acid; HMW, high molecular weight; LMW, low molecular weight; MMW, medium molecular weight; PPAR, peroxisome proliferatoreCactivated receptor; WAT, white adipose tissue
ABSTRACT
Muraglitazar, a novel dual (/) peroxisome proliferatoreCactivated receptor (PPAR) activator, was investigated for its antidiabetic properties and its effects on metabolic abnormalities in genetically obese diabetic db/db mice. In db/db mice and normal mice, muraglitazar treatment modulates the expression of PPAR target genes in white adipose tissue and liver. In young hyperglycemic db/db mice, muraglitazar treatment (0.03eC50 mg · kgeC1 · dayeC1 for 2 weeks) results in dose-dependent reductions of glucose, insulin, triglycerides, free fatty acids, and cholesterol. In older hyperglycemic db/db mice, longer-term muraglitazar treatment (30 mg · kgeC1 · dayeC1 for 4 weeks) prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment (10 mg · kgeC1 · dayeC1 for 2 weeks) improves oral glucose tolerance and reduces plasma glucose and insulin levels. In addition, treatment increases insulin content in the pancreas. Finally, muraglitazar treatment increases abnormally low plasma adiponectin levels, increases higheCmolecular weight adiponectin complex levels, reduces elevated plasma corticosterone levels, and lowers elevated liver lipid content in db/db mice. The overall conclusions are that in db/db mice, the novel dual (/) PPAR activator muraglitazar 1) exerts potent and efficacious antidiabetic effects, 2) preserves pancreatic insulin content, and 3) improves metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and elevated corticosterone levels.
Peroxisome proliferatoreCactivated receptor (PPAR) and PPAR are ligand-activated nuclear hormone receptors that regulate the transcription of genes involved in carbohydrate and lipid metabolism pathways (1eC4). Activation of PPAR, which is predominantly expressed in adipose tissue, results in insulin-sensitizing antidiabetic effects (5,6). Activation of PPAR, which is highly expressed in the liver, results in the lowering of triglycerides and the elevation of plasma HDL cholesterol levels (7,8). In addition, both PPAR and PPAR selective activators have been demonstrated to suppress vessel wall inflammatory activity and reduce atherosclerosis in experimental animal models through complementary mechanisms (9,10). Since type 2 diabetic patients often develop dyslipidemia and other metabolic abnormalities, eventually resulting in atherosclerotic coronary heart disease, an agent that simultaneously activates both PPAR and PPAR has the potential to be useful for the treatment of these patients (11,12).
The discovery and preliminary biological and pharmacokinetic properties of muraglitazar (BMS-298585), a novel oxybenzylglycine dual (/) PPAR activator, have been recently described (13). Muraglitazar binds with high affinity to both human PPAR and PPAR ligand binding domain protein (IC50 for binding = 0.19 and 0.25 e蘭ol/l, respectively) and potently transactivates full-length human PPAR- or PPAR-mediated reporter gene activity (EC50 for transactivation = 0.11 and 0.32 e蘭ol/l, respectively). We assessed the effects of muraglitazar treatment on diabetes and other metabolic abnormalities in genetically obese, diabetic, and hyperlipidemic db/db mice. Untreated db/db mice exhibit progressive deterioration of glycemic control and develop insulin deficiency and loss of pancreatic insulin content (14eC17). In these mice, the clinically used PPAR selective activators (e.g., rosiglitazone and piogitazone) have been reported to show antidiabetic effects, and the PPAR selective activators (e.g., gemfibrozil) have been reported to lower plasma triglyceride levels and also show some improvement in insulin sensitivity (14eC17). Furthermore, we assessed the effects of muraglitazar treatment on diet-induced hyperglycemia and hyperlipidemia in C57BL/6J mice (diet-induced obese [DIO]) (18)
RESEARCH DESIGN AND METHODS
Compounds.
Muraglitazar and rosiglitazone were synthesized by BMS Medicinal Chemistry. Fenofibric acid was purchased from Sigma (St. Louis, MO).
Mice.
db/db mice (C57BL/6ks lepreC/eC) and age-matched lean normal C57BL/6J or Swiss Webster mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under controlled temperature (23°C) and lighting (12 h of light between 6 A.M. and 6 P.M.) with free access to water and standard mouse diet (18% protein rodent diet no. 2018; Harlan). The db/db mice were prebled, and those within a narrow range of fasted glucose levels were selected for studies to minimize variability between control and drug-treated groups. C57BL/6J mice on experimental diet were fed research diet no. 12327, which contains 40% sucrose/40% fat by calorie (Research Diets, New Brunswick, NJ) for 12 weeks before the start of the experiment and were maintained on this diet for the duration of the experiment. Mice were dosed daily by oral gavage in a vehicle composed of 20% polyethylene glycol (vol/vol), 5% N-methyl pyrrolidone, and 75% 10 mmol/l phosphate buffer, pH 7.4. Bristol-Myers Squibb study guidelines were strictly followed in the investigations.
Gene expression profiling.
Lean normal mice or db/db mice that were treated with vehicle or compounds, respectively, were killed, and their inguinal white adipose tissue (WAT) and liver were harvested. Total RNA was isolated from WAT or liver samples using RNeasy (Qiagen, Valencia, CA). For Northern blot analysis, 15 e蘥 RNA was subjected to MOPS-formaldehyde gel electrophoresis. The gels were blotted to nylon membranes and hybridized to 32P-cDNA probes according to standard procedures. The radioactivity in the hybridized bands was counted on an Instant Imager (Packard Instruments, Meridian, CT). Alternatively, SYBR-Green PCR analysis was carried out (Applied Biosystems, Foster City, CA). Oligonucleotide primers were designed using Primer Express, and RT-PCRs were carried out (primer sequences and protocol available upon request). The mRNA levels of target genes were normalized to control glyceraldehyde-3-phosphate dehydrogenase mRNA levels. WAT RNA samples from vehicle- and compound-treated mice were also analyzed by Affymetrix microarray for changes in gene expression pattern (protocol available upon request).
Triglyceride/VLDL secretion assay.
C57BL/6J mice that were treated with vehicle or compounds for 7 days were fasted overnight and intravenously injected with Triton-WR1339 (250 mg/kg) 1 h after the final dosing. The injection of Triton prevents the degradation of triglyceride-rich VLDL (triglyceride/VLDL) particles in plasma, resulting in an accumulation of triglycerides. The secretion rate (typically 0.16eC0.20 mg · mineC1 · 100 g body wteC1, linear for 5 h after Triton administration) was determined by calculating the amount of triglycerides accumulated 2.5 h after the Triton injection/100 g body wt. Triglyceride levels were determined using a Roche Cobas blood chemistry analyzer.
Acyl coenzyme-A oxidase activity.
The db/db mice that were treated with vehicle or compounds for 14 days were killed, and their liver was harvested. Liver acyl coenzyme-A oxidase activity (ACO) activity [(slope of the rate of A502 increase after addition of substrate eC the slope of the background rate)/mg protein] was measured according to a published method (19).
Plasma chemistry analysis.
About 50 e蘬 tail vein blood from overnight-fasted or ad libitumeCfed mice was collected in EDTA-coated tubes. Plasma glucose, triglyceride, free fatty acid (FFA), cholesterol, and HDL cholesterol levels were determined using a Roche Cobas blood chemistry analyzer; insulin, adiponectin, and corticosterone levels were determined by mouse enzyme-linked immunosorbent assay kits (Linco Research, St. Charles, MO). Corticosterone levels were assessed at 10 A.M. during regular 12-h diurnal light cycle. ED50 to normalization is calculated as the midpoint of the dose-response activity curve using a four-parameter-fit equation.
HigheC, mediumeC, and loweCmolecular weight adiponectin complexes.
The higheCmolecular weight (HMW), mediumeCmolecular weight (MMW), and loweCmolecular weight (LMW) adiponectin complexes in db/db mouse plasma were detected according to the method described by Waki et al. (20). A total of 0.5 e蘬 db/db mouse plasma samples were diluted (1:12) and incubated for 1 h at room temperature in reducing sample buffer (3% SDS, 50 mmol/l Tris-HCl, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, and 10 mmol/l dithiothreitol) or nonreducing sample buffer (3% SDS, 50 mmol/l Tris-HCl, pH 6.8, and 10% glycerol) and subjected to SDS-PAGE under reducing/heat-denaturing conditions (samples were heated at 95°C for 10 min) or nonreducing/nonheat-denaturing conditions, according to the standard Laemmli’s method with Criterion precast Tris-HCl 4eC15% gel (Bio-Rad, Hercules, CA). For immunoblotting, proteins separated by SDS-PAGE were transferred to nitrocellulose membranes, blocked with StartingBlock (Tris-buffered saline) Blocking Buffer (Pierce, Rockford, IL), and then incubated with 1:5,000 diluted anti-mouse adiponectin globular domain monoclonal antibody (Chemicon, Temecula, CA) in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature. After washing, the membranes were incubated with goat anti-mouse IRDye 800 (1:10,000) (Rockland, Gilbersville, PA) for 1 h at room temperature and then washed thoroughly. The membrane was scanned with the Odyssey Imaging System (Li-Cor, Lincoln, NE).
Oral glucose tolerance test.
The db/db mice, which were on a 2-week dosing regimen, were fasted overnight on day 13. On day 14, an oral dose of vehicle alone or compound was given in the morning, and blood samples were collected from the tail vein for determination of baseline values (t = 0 min). The mice were then gavaged with an oral bolus of glucose (2 g/kg), and additional blood samples were collected at regular intervals (t = 15, 30, 60, and 90 min) for glucose and insulin measurement. Homeostasis model assessment index values were calculated using the following equation: (the product of the fasting insulin levels [mU/l] x fasting glucose levels [mmol/l]/22.5).
Pancreatic insulin content.
Pancreata were harvested from overnight-fasted vehicle- and drug-treated mice, placed in liquid N2, then stored at eC20°C. Pancreata were homogenized in acid-ethanol (75% ethanol, 23.5% water, and 1.5% c-HCl in 1.8 ml volume) with a polytron homogenizer. The homogenates were stored at 4°C for 28 h and then centrifuged at 1500g for 30 min at 4°C. The supernatants were diluted (1:20,000), and insulin levels were determined by enzyme-linked immunosorbent assay (21).
Liver lipid analysis.
Liver triglyceride levels were determined using a Wako Kit (no. 997-69801). Frozen liver pieces were homogenized in saline and brought to a concentration of 0.05 mg/1 ml. Twenty microliters of the sample were solubilized with 20 e蘬 deoxycholate (1.6% wt/vol in water), and 1 ml of the Wako reagent was added. The mixture was incubated at 37°C for 15 min, and the absorbance was read at 505 nm.
Statistical analysis.
Unpaired, two-tailed Student’s t tests were performed for comparisons between compound-treated and vehicle control groups. Differences were considered significant at P < 0. 05.
RESULTS
Muraglitazar modulates PPAR target gene expression in mice.
As previously described, muraglitazar potently stimulates full-length human PPAR- and PPAR-mediated reporter gene expression (EC50 for PPAR and PPAR transactivation = 0.11 and 0.32 e蘭ol/l, respectively; 13) The ability of muraglitazar to transactivate full-length mouse PPAR or PPAR receptor has not been determined. However, in a chimeric Gal4/mouse PPAR-mediated reporter gene assay, muraglitazar shows mouse PPAR agonist activity at levels comparable with its human PPAR activity (EC50 for mouse PPAR = 0.09 e蘭ol/l for muraglitazar and 0.08 e蘭ol/l for the PPAR selective activator rosiglitazone; the PPAR selective activator fenofibric acid was inactive) and mouse PPAR agonist activity that is weaker than its human PPAR activity (observed EC50 for mouse PPAR = 23.8 e蘭ol/l for muraglitazar and 16.3 e蘭ol/l for fenofibric acid; rosiglitazone was inactive). The disparity between the mouse and human PPAR activity is likely due to mouse/rodent-specific differences in the interactions of muraglitazar with several amino acid residues that are altered between mouse and human PPAR ligand binding domains (22).
The effects of muraglitazar treatment on the expression of PPAR target genes in WAT and liver were determined in db/db and normal mice. In db/db mice, muraglitazar treatment (10 mg · kgeC1 · dayeC1 for 2 weeks) increases mRNA levels of fatty acid binding protein aP2, GLUT4 glucose transporter, and lipoprotein lipase in WAT and stimulates both mRNA and activity levels of ACO and suppresses apolipoprotein CIII mRNA levels in liver (Fig. 1A and B). Microarray analysis of WAT RNA from muraglitazar- or rosiglitazone (10 mg · kgeC1 · dayeC1 for 7 days)-treated db/db mice shows that expression levels of genes that are implicated in 1) adipocyte differentiation, 2) insulin signaling and glucose metabolism, 3) fatty acid transport, 4) fatty acid oxidation, 5) triglyceride synthesis, and 6) energy expenditure are modulated by both muraglitazar and rosiglitazone treatment (Table 1). Both muraglitazar and rosiglitazone treatment (10 mg · kgeC1 · dayeC1 for 3 days) stimulate aP2 and lipoprotein lipase mRNA levels and suppress 11-hydroxy steroid desaturase 1 mRNA levels in normal mouse WAT (Fig. 1C). Muraglitazar, but not rosiglitazone, stimulates ACO mRNA levels in normal mouse liver (Fig. 1D). Finally, in normal mice, muraglitazar treatment (3, 10, and 30 mg · kgeC1 · dayeC1 for 7 days) dose dependently inhibits triglyceride/VLDL secretion from the liver without promoting liver weight increase (Fig. 1E and F). Fenofibrate treatment (30, 50, and 100 mg · kgeC1 · dayeC1) also inhibits triglyceride/VLDL secretion (Fig. 1E and F). However, this effect is accompanied by dose-dependent increases in liver weight, which is a known fibrate-induced phenomenon in rodents (23). Rosiglitazone treatment (3, 10, and 30 mg · kgeC1 · dayeC1), by contrast, does not inhibit triglyceride/VLDL secretion (Fig. 1E and F). The gene expression data thus demonstrate that muraglitazar treatment results in modulation of PPAR target gene expression in WAT and liver. The PPAR agonist activity of muraglitazar may have contributed to the differences in the expression levels of various PPAR target genes in WAT and liver as well as inhibition of VLDL secretion in muraglitazar-treated compared with rosiglitazone-treated mice.
Muraglitazar treatment ameliorates diabetes and hyperlipidemia and increases pancreatic insulin content in db/db mice.
Muraglitazar was investigated in three separate studies for 1) dose-dependent lowering of fasted and fed glucose, insulin, FFA, triglyceride, and cholesterol levels in young hyperglycemic db/db mice (8-week-old males; 0.03eC50 mg · kgeC1 · dayeC1 orally for 2 weeks), 2) effect on time-dependent deterioration of glycemic control and plasma insulin levels in older db/db mice (12-week-old females; 30 mg · kgeC1 · dayeC1 for 4 weeks), and 3) improvements in hyperglycemia and glucose tolerance and effect on pancreatic insulin content in severely hyperglycemic db/db mice (10-week-old females with fasting plasma glucose >500 mg/dl; 10 mg · kgeC1 · dayeC1 for 2 weeks; rosiglitazone at 10 mg · kgeC1 · dayeC1 was used as a positive control in the study).
In study 1, muraglitazar treatment results in dose-dependent lowering of both fasted (day 7 data shown, similar data were also obtained after 14 days) and fed (on day 15) plasma glucose, FFA, insulin, triglyceride, and cholesterol levels (Fig. 2AeCE). Amelioration of hyperglycemia in the presence of reduced plasma insulin levels suggests that insulin sensitivity has been improved in muraglitazar-treated young db/db mice. As previously observed with PPAR activators in rodents, the cholesterol-lowering effect of muraglitazar is restricted to a reduction of the HDL cholesterol fraction (data not shown) (24). The ED50 to normalization of glucose and triglyceride levels in fasted animals on day 14 are 0.1 and 0.2 mg · kgeC1 · dayeC1, respectively, and in fed animals on day 15 are 0.5 and 1.3 mg · kgeC1 · dayeC1, respectively. As observed with PPAR activators (4), muraglitazar-treated mice (at 10 and 50 mg · kgeC1 · dayeC1) experience a trend toward increased body weight gain in comparison with the vehicle-treated mice (Fig. 2F).
In study 2, the vehicle- and muraglitazar-treated db/db mice were monitored weekly for changes in fasting glucose and insulin levels. As shown in Fig. 3A and B, the vehicle-treated control db/db mice show poor glycemic control throughout the duration of the study. The vehicle-treated db/db mice also show signs of further deterioration of glycemic control (higher fasting plasma glucose levels) and some -cell exhaustion (significant drop in plasma insulin levels) by the end of the 4-week treatment period (Fig. 3B and C). By contrast, the muraglitazar-treated mice show time-dependent improvement in glycemic control (fasting glucose levels are reduced to the levels observed in lean normal mice) and maintain reduced, but stable, plasma insulin levels during the entire 4-week period (Fig. 3AeCC). Muraglitazar-treated mice also show significant improvements in other metabolic parameters such as fasted FFA (eC49%), fed glucose (eC60%), fasted triglyceride (eC31%), and fed triglyceride (eC47%) levels (Fig. 3DeCF).
In study 3, at the end of the 2-week treatment period, the db/db mice were fasted overnight and, after collecting baseline (t = 0 min) plasma samples, were challenged with an oral bolus of glucose. Muraglitazar treatment results in significant reduction of baseline fasting plasma glucose (eC51%), insulin (eC55%), and FFA (eC33%) levels as well as homeostasis model assessment index (eC63%) (Table 2). When challenged with an oral bolus of glucose, muraglitazar-treated animals exhibit a reduced glucose excursion (indicating increased tolerance to glucose) and lower plasma insulin levels compared with vehicle-treated animals (Fig. 4A and B). The increased glucose tolerance, along with the concomitantly lowered insulin levels and reduced homeostasis model assessment index, indicate that insulin sensitivity has been improved in muraglitazar-treated mice. Rosiglitazone treatment also results in improved glycemic control; however, the effects are less pronounced than with muraglitazar at the same dose (Table 2, Figs. 4A and B). In addition to improvements in insulin sensitivity and glycemic control, pancreatic insulin content is increased by about fourfold by both muraglitazar and rosiglitazone treatment (Fig. 4C), which is suggestive of some preservation of -cell function. Neither drug shows any significant impact on the weight of the pancreas in this study (Fig. 4D).
Muraglitazar treatment lowers hyperglycemia and hyperlipidemia in DIO-mice.
C57BL/6J mice, when maintained on a diet high in fat and sucrose (DIO-mice), develop mild hyperglycemia and high plasma triglyceride and cholesterol levels (18). Consistent with its antidiabetic and lipid-lowering effects in db/db mice, muraglitazar treatment (10 mg · kgeC1 · dayeC1 for 2 weeks) lowers fasting glucose, triglyceride, and cholesterol levels of DIO-mice to the levels observed in mice on normal standard diet (Table 3).
Muraglitazar treatment increases low plasma adiponectin levels, increases HMW adiponectin complex levels, and lowers elevated corticosterone levels in db/db mice.
As in some type 2 diabetic patients, db/db mice exhibit abnormally low plasma adiponectin levels and high plasma corticosterone (the murine counterpart to cortisol in humans) levels compared with age-matched normal C57BL/6J mice. Diminished adiponectin levels and elevated corticosterone levels serve as biomarkers of tissue insulin resistance and increased hepatic glucose production (20,25eC30). Adiponectin exists as HMW, MMW, and LMW complexes in plasma (20,25,28). In patients and in animal models, increased levels of HMW adiponectin complex have been associated with improved insulin sensitivity (20,25,28).
In older hyperglycemic db/db mice, muraglitazar treatment (study 2) elevates plasma adiponectin levels and lowers plasma corticosterone levels to the levels observed in normal mice (Fig. 5A and B). In severely hyperglycemic db/db mice, muraglitazar treatment (study 3) elevates their plasma adiponectin levels above the levels observed in lean normal mice and significantly lowers plasma corticosterone levels (Fig. 5C and D). By comparison, rosiglitazone treatment elevates adiponectin levels to the levels observed in normal mice and lowers corticosterone to the levels comparable with muraglitazar-treated levels in this study (Fig. 5C and D). Furthermore, immunoblot analysis shows that in muraglitazar-treated db/db mice (10 mg · kgeC1 · dayeC1 for 2 weeks), their plasma total adiponectin levels as well as HMW adiponectin complex levels are substantially increased compared with the vehicle-treated mice (Fig. 5E and F).
Muraglitazar treatment lowers liver lipid content in db/db mice.
Obese patients with insulin resistance and type 2 diabetes frequently suffer from nonalcoholic fatty liver condition (31,32). Elevated lipid content in the liver has been implicated in hepatic insulin resistance, glucose overproduction, and increased VLDL synthesis and secretion (30,31). The db/db mice on a normal diet accumulate lipids (primarily triglycerides) in the liver and develop hepatic steatosis. In these mice, muraglitazar treatment (50 mg · kgeC1 · dayeC1 for 2 weeks) results in significant reductions of liver triglycerides content (76 ± 3 mg/g liver tissue in muraglitazar-treated vs. 100 ± 10 mg/g liver tissue in vehicle-treated mice).
DISCUSSION
Muraglitazar is a novel dual (/) PPAR activator that selectively binds to and activates human PPAR and human PPAR (13,33eC37). The in vivo pharmacological data in lean normal mice and in db/db mice demonstrate that muraglitazar modulates the expression of PPAR target genes implicated in the regulation of glucose and lipid metabolic pathways in WAT and in liver. The in vivo data also demonstrate that muraglitazar is a potent and efficacious antidiabetic and lipid-lowering agent in db/db mice. In young hyperglycemic db/db mice, muraglitazar lowers both fasted and fed glucose and triglyceride levels to the levels commonly observed in lean normal mice. In addition, muraglitazar treatment reduces fasted and fed insulin, FFA, and cholesterol levels. In older db/db mice, longer-term muraglitazar treatment prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment markedly reduces fasted plasma glucose and insulin levels as well as glucose excursion. In these animals, muraglitazar also increases the insulin content in the pancreas. Muraglitazar treatment elevates the low plasma adiponectin levels, increases the HMW adiponectin complex levels, and reduces the elevated plasma corticosterone levels of db/db mice. Muraglitazar treatment also significantly lowers triglyceride content in db/db mouse liver. Finally, in DIO-mice, muraglitazar treatment normalizes diet-induced mild hyperglycemia and hyperlipidemia, which corroborates the glucose and lipid-lowering effects in db/db mice. Muraglitazar treatment did not cause hypoglycemia in mice under the experimental conditions used.
The antidiabetic and lipid-lowering effects induced by muraglitazar treatment may result from one or more of the following PPAR-mediated mechanisms: 1) improved insulin action and enhanced glucose uptake in adipose tissue and skeletal muscle, 2) increased fatty acid uptake and storage in adipose tissue, 3) reduced plasma FFA levels, 4) increased plasma total adiponectin and HMW adiponectin complex levels, 5) suppression of glucose overproduction by liver, 6) enhanced VLDL catabolism in the plasma, and 7) reduced triglycerides/VLDL synthesis/secretion in the liver (1eC4,25,38eC39). The HDL cholesterol lowering in muraglitazar-treated mice is most likely the result of a rodent-specific PPAR-mediated mechanism that suppresses the production of apolipoprotein A1 (a major protein component of HDL particles) in the liver (24). In humans, muraglitazar, like other human PPAR activators (e.g., fenofibrate, gemfibrozil), has demonstrated plasma HDL cholesteroleCraising effects (7,8,40eC42).
The trend toward increased weight gain in muraglitazar-treated db/db mice is probably due to a combination of effects including 1) enhanced adipogenesis, 2) retention of calories that would otherwise be lost due to glucosuria, and 3) water retention due to the alleviation of the glucose-driven osmotic diuresis and/or increased plasma or extracellular fluid volume (43,44). The liver triglyceride-lowering effect of muraglitazar is possibly due to reduced plasma FFA and lipid levels, which would limit fatty acid substrate availability for lipid biosynthesis in the liver. Reduced lipid content in the liver will lower hepatic insulin resistance, glucose overproduction, and increased VLDL synthesis (31,32).
In muraglitazar-treated db/db mice, the improvement in insulin sensitivity and the concomitant reduction in plasma glucose and FFA levels are anticipated to 1) lower insulin secretory demand on -cells and 2) prevent apoptosis of -cells, respectively. These effects may help to prevent deterioration of -cell function, loss of pancreatic insulin content, development of insulin deficiency, and deterioration of glycemic control in muraglitazar-treated db/db mice.
The increase in both total adiponectin levels and HMW adiponectin complex levels are expected to stimulate fatty acid oxidation in liver and skeletal muscle as well as enhance insulin sensitivity and glucose uptake in skeletal muscle (20,25eC28). Interestingly, an inverse correlation has been recently described between plasma adiponectin levels and the rate of incidence of myocardial infarction in men irrespective of their glycemic status (47). The reduction of corticosterone levels by muraglitazar is likely due to reduced metabolic stress and/or PPAR-mediated suppression of the 11-hydroxy steroid desaturase 1 gene expression in WAT and liver. The reduced corticosterone levels may suppress hepatic glucose overproduction and enhance glucose uptake in peripheral tissues (29,30).
In conclusion, the novel dual (/) PPAR activator muraglitazar 1) exerts potent and efficacious insulin-sensitizing antidiabetic effects, 2) prevents time-dependent deterioration of glycemic control and development of insulin deficiency, 3) increases pancreatic insulin content and, 4) improves other metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and high corticosterone levels in db/db mice. In the clinical setting, muraglitazar treatment (0.5eC20 mg for 28 days) lowers glucose, insulin, triglyceride, FFA, and apolipoprotein CIII levels and increases HDL cholesterol levels in type 2 diabetic patients (40eC42). These clinical data emphasize the utility of db/db mice as a useful model for evaluating antidiabetic properties and antidyslipidemic properties of novel PPAR activators. However, the weak mouse PPAR activity, relative to its human PPAR activity, for muraglitazar may suggest that its antidyslipidemic effects are probably underrepresented in db/db mice. Finally, since type 2 diabetes patients often suffer from dyslipidemia and other metabolic abnormalities, thus putting them at high risk for cardiovascular disease, muraglitazar, with its dual (/) PPAR activity, has the potential to be useful for the treatment of these patients.
ACKNOWLEDGMENTS
We thank Drs. S. Taylor, R. Parker, J. Whaley, R. Mukherjee, J. Graham, and R. Belder for critically reading the manuscript.
FOOTNOTES
S.B. is currently affiliated with Novartis Institute Bio-Med Research, Cambridge, Massachusetts. K.A.M. is currently affiliated with Advinus Therapeutics, Pune, India. J.W. is currently affiliated with the Department of Cardiovascular Pharmaceuticals, Pfizer, Ann Arbor, Michigan.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Kersten S, Desvergne B, Wahli W: Roles of PPARs in health and disease. Nature 405:421eC424, 2000
Kliewer S, Xu E, Lambert M, Willson T: Peroxisome proliferator-activated receptors: from genes to physiology. Recent Prog Horm Res 56:239eC263, 2001
Berger J, Moller D: The mechanism of action of PPARs. Annu Rev Med 53:409eC435, 2002
Bays H, Mandarino L, DeFronzo R: Mechanisms of endocrine disease: role of adipocytes, free fatty acids, and ectopic fat in pathogenesis of type 2 diabetes mellitus: PPAR agonists provide a rational therapeutic approach. J Clin Endocrinol Metab 89:463eC478, 2004
Olefsky J: Treatment of insulin resistance with PPAR agonists. J Clin Inves 106:467eC472, 2000
Lebovitz H: Differentiating members of the thiazolidinedione class: a focus on safety. Diabetes Metab Res Rev 18 (Suppl. 2):S23eCS29, 2002
Robins S, Collins D, Wittes J, Papademetriou V, Deedwania P, Schaefer E, McNamara J, Kashyap M, Hershman J, Wexler L, Rubins H: Relation of gemfibrozil treatment and lipid levels with major coronary events. VA-HIT: a randomized controlled trial. J Am Med Assoc 285:1585eC1591, 2001
Diabetes Atherosclerosis Intervention Study Investigators: Effect of fenofibrate on progression of coronary artery disease in type 2 diabetes: the Diabetes Atherosclerosis Intervention Study, a randomised study. Lancet 357:905eC910, 2001
Buchan K, Hassall D: PPAR agonists as direct modulators of the vessel wall in cardiovascular disease. Med Res Rev 20:350eC366, 2000
Duval C, Chinetti G, Trottein F, Fruchart J, Staels B: The role of PPARs in atherosclerosis. Trends Mol Med 8:422eC430, 2002
Beckman J, Creager M, Libby P: Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. J Am Med Assoc 287:2570eC2581, 2002
Nesto R, Drexler A: Evaluating the cardiovascular effects of the thiazolidinediones and their place in the management of type 2 diabetes mellitus: proceedings of a symposium. November 6eC8, 2002, New York, New York, USA. Am J Med 115 (Suppl. 1):1SeC120S, 2003
Devasthale P, Chen S, Jeon Y, Qu F, Shao C, Wang W, Zhang H, Cap M, Farrelly D, Golla R, Grover G, Harrity H, Ma Z, Moore L, Ren J, Seethala R, Cheng L, Sleph P, Sun W, Tieman A, Wetterau J, Biller S, Ryono D, Selan F, Hariharan N, Cheng PTW: Design and synthesis of N-[(4-methoxyphenoxy)carbonyl]-N-[[4-[2-(5-methyl-2-phenyl-4-oxazolyl) ethoxy] phenyl]methyl]glycine [muraglitazar/BMS-298585), a novel PPAR/ dual agonist with efficacious glucose and lipid-lowering activities. J Med Chem 48:2248eC2250, 2005
Connor S, Hughes M, Moore G, Lister C, Smith S: Antidiabetic efficacy of BRL-49653, a potent orally active insulin sensitizing agent, assessed in the C57BL/KsJ db/db diabetic mouse my non-invasive 1H NMR studies of urine. J Pharm Pharmacol 49:336eC344, 1997
Diani A, Sawada G, Wyse B, Murray F, Khan M: Pioglitazone preserves pancreatic islet structure and insulin secretory function in three murine models of type 2 diabetes. Am J Physiol Endo Metab 286:E116eCE122, 2004
Ishida H, Takizawa M, Ozawa S, Nakamichi Y, Yamaguchi S, Katsuta F, Tanaka T, Maruyama M, Katahira H, Yoshimoto K, Itagaki E, Nagamatsu S: Pioglitazone improves insulin secretory capacity and prevents the loss of -cell mass in obese diabetic db/db mice: possible protection of -cells from oxidative stress. Metabolism 53:488eC494, 2004
Mukherjee R, Strasser J, Jow L, Hoener P, Paterniti J, Heyman R: RXR agonists activates PPAR-inducible genes, lower triglycerides, and raise HDL levels in vivo. Arterioscler Thromb Vasc Biol 18:272eC276, 1998
Rossmeisl M, Rim J, Koza R, Kozak L: Variation in type 2 diabetes-related traits in mouse strains susceptible to diet induced obesity. Diabetes 52:1958eC1966, 2003
Small G, Burdett K, Connock M: A sensitive spectrophotometric assay for peroxisomal acyl co-A oxidase. Biochem J 227:205eC210, 1985
Waki H, Yamauchi T, Kamon J, Ito Y, Uchida S, Kita S, Hara K, Hada Y, Vasseur F, Froguel P, Kimura S, Nagai R, Kadowaki T: Impaired multimerization of human adiponectin mutants associated with diabetes: molecular structure and multimer formation of adiponectin. J Biol Chem 278:40352eC40363, 2003
Yajima K, Hirose H, Fujita H, Seto Y, Ukeda K, Miyashita K, Kawai T, Yamamoto Y, Ogawa T, Yamada T, Saruta T: Combination therapy with PPAR and PPAR agonists increases glucose-stimulated insulin secretion in db/db mice. Am J Physiol Endo Met 284:E966eCE971, 2003
Boettcher B, Fanelli B, Stephen Z, Caplan S, Sabio M: Comparison of ligand binding affinities in the mouse and human PPAR and ligand binding domains. Keystone Symposium-PPARs, 2003 (Abstract no. 106)
Petit D, Bonnefis M, Rey C, Infante R: Effects of ciprofibrate and fenofibrate on liver lipids and lipoprotein synthesis in normo- and hyperlipidemic rats. Atherosclerosis 74:215eC225, 1988
Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart J: Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 10:2088eC2093, 1998
Kadowaki T, Yamauchi T: Adiponectin and adiponectin receptors. Endocrin Rev 26:439eC451, 2005
Combs T, Wagner J, Berger J, Doebber T, Wang W, Zhang B, Tanen M, Berg A, O’Rahilly S, Savage D, Chatterjee K, Weiss S, Larson P, Gottesdiener K, Gertz B, Charron M, Scherer P, Moller D: Induction of adipocyte complement related protein of 30 Kd by PPAR agonists: a potential mechanism of insulin sensitization. Endocrinology 143:998eC1007, 2002
Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno N, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T: Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762eC769, 2003
Pajvani U, Hawkins M, Combs T, Rajala M, Doebber T, Berger J, Wagner J, Wu M, Knopps A, Xiang A, Utzschneider K, Kahn S, Olefsky J, Buchanan T, Scherer P: Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity. J Biol Chem. 279:12152eC12162, 2004
Alberts P, Nilsson C, Selen G, Engblom L, Edling N, Norling S, Klingstrom G, Larsson C, Forsgren M, Ashkzari M, Nilsson C, Fiedler M, Bergqvist E, Ohman B, Bjorkstrand E, Abrahmsen L: Selective inhibition of 11 beta-hydroxysteroid dehydrogenase type 1 improves hepatic insulin sensitivity in hyperglycemic mice strains. Endocrinology 144:4755eC4762, 2003
Nielsen M, Caumo A, Chandramouli V, Schumann W, Cobelli C, Landau B, Vilstrup H, Rizza R, Schmitz O: Impaired basal glucose effectiveness but unaltered fasting glucose release and gluconeogenesis during short-term hypercortisolemia in healthy subjects. Am J Physio Endocrin Met 286:E102eCE110, 2004
Lind P: Interdependence of hepatic lipid and glucose metabolism: novel pharmaceutical targets for diabetes. Curr Opin Investig Drugs 5:395eC401, 2004
Bajaj M, Suraamornkul S, Hardies L, Pratipanawatr T, DeFronzo R: Plasma resistin concentration, hepatic fat content, and hepatic and peripheral insulin resistance in pioglitazone-treated type II diabetic patients. Int J Obes Relat Metab Disord 28:783eC789, 2004
Murakami K, Tobe K, Ide T, Mochizuki T, Ohashi M, Akanuma Y, Yazaki Y, Kadawaki T: A novel insulin sensitizer acts as coligand for PPAR and PPAR: effect of PPAR activation on abnormal lipid metabolism in liver of Zucker fatty rats. Diabetes 47:1841eC1847, 1998
Etriglyceridesen G, Oldham B, Johnson W, Broderick C, Montrose C, Brozinick J, Misener E, Bean J, Bensch W, Brooks D, Shuker A, Rito C, McCarthy J, Ardecky R, Tyhonas J, Dana S, Bilakovics J, Paterniti J, Ogilvie K, Liu, Kauffman R: A tailored therapy for the metabolic syndrome: the dual PPAR/ agonist LY465608 ameliorates insulin resistance and diabetic hyperglycemia while improving cardiovascular risk factors in pre-clinical models. Diabetes 51:1083eC1087, 2002
Chakrabarti R, Vikramadithyan R, Misra P, Hiriyan J, Raichur S, Damarla R, Gershome C, Suresh J, Rajagopalan R: Ragaglitazar: a novel PPAR and PPAR agonist with potent lipid-lowering and insulin sensitizing efficacy in animal models. Br J Pharmacol 140:527eC537, 2003
Ljung B, Bamberg K, Dahllof B, Kjellstedt A, Oakes N, Ostling J, Camejo G: AZ-242, a novel PPAR/ dual agonist with beneficial effect on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats. J Lipid Res 43:1855eC1863, 2002
Pickavance L, Brand C, Wassermann K, Wilding J: The dual PPAR/ agonist, ragaglitazar, improves insulin sensitivity and metabolic profile equally with pioglitazone in diabetic and dietary obese ZDF rats. Br J Pharmacol 144:308eC316, 2005
Chaput E, Saladin R, Silvestre M, Edgar A: Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight. Biochem Bioplys Res Commun 27:445eC450, 2000
Guerre-Millo M, Gervois P, Rape E, Madsen L, Poulain P, Derudas B, Herbert J-M, Winegar D, Wilson T, Fruchart J-C, Berge R, Staels B: PPAR activators improve insulin sensitivity and reduce adiposity. J Biol Chem 275:16638eC16642, 2000
Mosqueda-Garcia R, Frost C, Swaminathan A, Raymond R, Nepal S, Reeves R, Gregg R: Glucose lowering effects of multiple dose administration of muraglitazar (BMS-298585), a novel PPAR/ dual agonist, in type 2 diabetic patients (Abstract). Diabetes 53 (Suppl. 2):A32, 2004
Gregg R, Swaminathan A, Frost C, Nepal S, Raymond R, Mosqueda-Garcia R: Muraglitazar, a novel PPAR/ dual agonist, lowers fasting plasma glucose, triglycerides, NEFA and apoCIII after once a day administration in type 2 diabetic patients (Abstract no. 717). Abstract presented at the 40th Annual Meeting of the European Association for the Study of Diabetes, 5eC9 September 2004, Munich, Germany
Frost C, Swaminathan A, Raymond R, Nepal S, Gregg R, Reeves R, Mosqueda-Garcia R: Lipid lowering effects of multiple dose administration of muraglitazar (BMS-298585), a novel PPAR/ dual agonist, in type 2 diabetic patients (Abstract). Diabetes 53 (Suppl. 2):A475, 2004
Lebovitz H: Differentiating members of the thiazolidinedione class: a focus on safety. Diabetes Metab Res Rev 18 (Suppl. 2):S23eCS29, 2002
Larsen T, Toubro S, Astrup A: PPAR agonists in the treatment of type II diabetes: is increased fatness commensurate with long-term efficacy Int J Obes Relat Metab Disord 27:147eC161, 2003
Bergman R, Ader M: Free fatty acids and pathogenesis of type 2 diabetes mellitus. TEM 11:331eC356, 2000
Buchanan T, Xiang A, Peters R, Kjos S, Marroquin A, Goico J, Ochoa C, Tan S, Berkowitz K, Hodis H, Azen S: Preservation of pancreatic -cell function and prevention of type 2 diabetes by pharmacological treatment of insulin resistance in high-risk Hispanic women. Diabetes 51:2796eC2803, 2002
Pischon T, Girman C, Hotamisligil G, Rifai N, Hu F, Rimm E: Plasma adiponectin levels and risk of myocardial infarction in men. J Am Med Assoc 291:1730eC1737, 2004(Thomas Harrity, Dennis Fa)