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Diabetes 55:240-248, 2006
DOI: 10.2337/diabetes.55.01.06.db05-0648
© 2006 by the American Diabetes Association
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Pharmacology & Therapeutics

Muraglitazar, a Novel Dual ({alpha}/{gamma}) Peroxisome Proliferator–Activated Receptor Activator, Improves Diabetes and Other Metabolic Abnormalities and Preserves ß-Cell Function in db/db Mice

Thomas Harrity1, Dennis Farrelly1, Aaron Tieman1, Cuixia Chu1, Lori Kunselman1, Liqun Gu1, Randolph Ponticiello1, Michael Cap1, Fucheng Qu2, Chunning Shao2, Wei Wang2, Hao Zhang2, William Fenderson3, Sean Chen2, Pratik Devasthale2, Yoon Jeon2, Ramakrishna Seethala1, Wen-Pin Yang3, Jimmy Ren1, Min Zhou1, Denis Ryono2, Scott Biller2, Kasim A. Mookhtiar1, John Wetterau1, Richard Gregg1, Peter T. Cheng2, and Narayanan Hariharan1

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

Address correspondence and reprint requests to Narayanan Hariharan, PhD, Metabolic Diseases, HWP-21-2.02, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543. E-mail: narayanan.hariharan{at}bms.com

Abbreviations: ACO, acyl coenzyme-A oxidase; FFA, free fatty acid; HMW, high molecular weight; LMW, low molecular weight; MMW, medium molecular weight; PPAR, peroxisome proliferator–activated receptor; WAT, white adipose tissue


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muraglitazar, a novel dual ({alpha}/{gamma}) peroxisome proliferator–activated 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 (0.03–50 mg · kg–1 · day–1 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 · kg–1 · day–1 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 · kg–1 · day–1 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 high–molecular 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 ({alpha}/{gamma}) 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 proliferator–activated receptor (PPAR){gamma} and PPAR{alpha} are ligand-activated nuclear hormone receptors that regulate the transcription of genes involved in carbohydrate and lipid metabolism pathways (1–4). Activation of PPAR{gamma}, which is predominantly expressed in adipose tissue, results in insulin-sensitizing antidiabetic effects (5,6). Activation of PPAR{alpha}, 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{gamma} and PPAR{alpha} 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{gamma} and PPAR{alpha} 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 ({alpha}/{gamma}) PPAR activator, have been recently described (13). Muraglitazar binds with high affinity to both human PPAR{gamma} and PPAR{alpha} ligand binding domain protein (IC50 for binding = 0.19 and 0.25 µmol/l, respectively) and potently transactivates full-length human PPAR{gamma}- or PPAR{alpha}-mediated reporter gene activity (EC50 for transactivation = 0.11 and 0.32 µmol/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 (14–17). In these mice, the clinically used PPAR{gamma} selective activators (e.g., rosiglitazone and piogitazone) have been reported to show antidiabetic effects, and the PPAR{alpha} selective activators (e.g., gemfibrozil) have been reported to lower plasma triglyceride levels and also show some improvement in insulin sensitivity (14–17). 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
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 lepr–/–) 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 µg 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 0.16–0.20 mg · min–1 · 100 g body wt–1, 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 – the slope of the background rate)/mg protein] was measured according to a published method (19).

Plasma chemistry analysis.
About 50 µl tail vein blood from overnight-fasted or ad libitum–fed 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.

High–, medium–, and low–molecular weight adiponectin complexes.
The high–molecular weight (HMW), medium–molecular weight (MMW), and low–molecular 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 µl 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 4–15% 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 –20°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 µl 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
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Muraglitazar modulates PPAR target gene expression in mice.
As previously described, muraglitazar potently stimulates full-length human PPAR{gamma}- and PPAR{alpha}-mediated reporter gene expression (EC50 for PPAR{gamma} and PPAR{alpha} transactivation = 0.11 and 0.32 µmol/l, respectively; 13) The ability of muraglitazar to transactivate full-length mouse PPAR{gamma} or PPAR{alpha} receptor has not been determined. However, in a chimeric Gal4/mouse PPAR-mediated reporter gene assay, muraglitazar shows mouse PPAR{gamma} agonist activity at levels comparable with its human PPAR{gamma} activity (EC50 for mouse PPAR{gamma} = 0.09 µmol/l for muraglitazar and 0.08 µmol/l for the PPAR{gamma} selective activator rosiglitazone; the PPAR{alpha} selective activator fenofibric acid was inactive) and mouse PPAR{alpha} agonist activity that is weaker than its human PPAR{alpha} activity (observed EC50 for mouse PPAR{alpha} = 23.8 µmol/l for muraglitazar and 16.3 µmol/l for fenofibric acid; rosiglitazone was inactive). The disparity between the mouse and human PPAR{alpha} 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{alpha} 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 · kg–1 · day–1 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 · kg–1 · day–1 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 · kg–1 · day–1 for 3 days) stimulate aP2 and lipoprotein lipase mRNA levels and suppress 11ß-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 · kg–1 · day–1 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 · kg–1 · day–1) 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 · kg–1 · day–1), 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{alpha} 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.


Figure 1
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  		FIG. 1. Effect of muraglitazar on PPAR target gene expression and VLDL secretion in mice. A and B: WAT and liver: db/db mice. Mice (8-week-old males, three to four animals per group) were treated with vehicle alone or muraglitazar (10 mg · kg–1 · day–1 for 2 weeks). ACO activity was determined in liver homogenates. C and D: WAT and liver: normal mice. Mice (8-week-old males, three to four animals per group) were treated with vehicle alone, muraglitazar, or rosiglitazone (10 mg · kg–1 · day–1 for 3 days). E and F: Inhibition of triglycerides/VLDL secretion and liver weight change in normal mice. Mice (8-week-old females, five animals per group) were administered vehicle alone, muraglitazar, or rosiglitazone (3, 10, or 30 mg · kg–1 · day–1) or fenofibrate (30, 50, and 100 mg · kg–1 · day–1) for 7 days. At the end of the treatment period, mice were fasted overnight and injected with Triton-WR1339. The triglyceride/VLDL secretion rate was determined by the amount of triglycerides accumulated in the plasma 2.5 h after Triton injection. Liver weight is presented as percent body weight. (RNA data are presented as a percentage compared with vehicle-treated animals, which were defined as 100% [dotted line]). For WAT, tissue samples from three to four animals were pooled for RNA preparation. Data are presented as means ± SE. *Difference between vehicle- and muraglitazar-treated groups, P < 0.05. Feno, fenofibrate, Mura, muraglitazar, Rosi, rosiglitazone. Glut-4 mediates insulin-stimulated glucose uptake, aP2 binds to fatty acids, lipoprotein lipase promotes triglyceride lipolysis, and 11ß-HSD1 suppresses insulin sensitivity in adipocytes; ACO stimulates peroxisomal fatty acid oxidation in hepatocytes; and ApoCIII inhibits VLDL catabolism in plasma.

 

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  		TABLE 1 Effect of muraglitazar and rosiglitazone on WAT gene expression in db/db 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; 0.03–50 mg · kg–1 · day–1 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 · kg–1 · day–1 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 · kg–1 · day–1 for 2 weeks; rosiglitazone at 10 mg · kg–1 · day–1 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. 2A–E). 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{alpha} 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 · kg–1 · day–1, respectively, and in fed animals on day 15 are 0.5 and 1.3 mg · kg–1 · day–1, respectively. As observed with PPAR{gamma} activators (4), muraglitazar-treated mice (at 10 and 50 mg · kg–1 · day–1) experience a trend toward increased body weight gain in comparison with the vehicle-treated mice (Fig. 2F).


Figure 2
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  		FIG. 2. Effect of muraglitazar on plasma glycemic and lipid parameters and body weight in young hyperglycemic db/db mice. A–E: db/db mice (8-week-old males, five animals per group) were administered vehicle alone or muraglitazar (0.03–50 mg · kg–1 · day–1 for 2 weeks). Plasma samples were collected after 7 or 14 days (fasted overnight; 14-day data not shown), and 15 days (fed) and analyzed for glycemic and lipid parameters. Fasted cholesterol data are from 14-day treated mice (cholesterol levels were not determined for 0.03-mg · kg–1 · day–1 group). Animals were weighed pre- and posttreatment (F). Data are presented as means ± SE. *Difference between vehicle- and muraglitazar-treated groups, P < 0.05. Fasted values in C57BL/6J mice: glucose = 100–125 mg/dl, triglycerides = 75–100 mg/dl, FFAs = 0.5–1.0 meq/l, insulin = 0.5–1.0 ng/ml, and cholesterol = 75–100 mg/dl. Vh, vehicle.

 
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 ß-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. 3A–C). Muraglitazar-treated mice also show significant improvements in other metabolic parameters such as fasted FFA (–49%), fed glucose (–60%), fasted triglyceride (–31%), and fed triglyceride (–47%) levels (Fig. 3D–F).


Figure 3
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  		FIG. 3. Effect of long-term treatment of muraglitazar on time-dependent deterioration of glycemic control and insulin deficiency in old hyperglycemic db/db mice. db/db mice (12-week-old females, five animals per group) were administered vehicle alone or muraglitazar (30 mg · kg–1 · day–1 for 4 weeks). At the end of each week, the vehicle- and muraglitazar-treated animals were fasted overnight and bled, and the plasma glucose and insulin levels were determined. A: Pretreatment of fasted blood glucose. B and C: Fasted plasma glucose and insulin levels. D and F: Fasted plasma FFA and triglyceride levels (day 28). E and F: Fed plasma glucose and triglyceride levels (day 29). Data are presented as means ± SE. *Difference between vehicle- and drug-treated groups, P < 0.05.

 
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 (–51%), insulin (–55%), and FFA (–33%) levels as well as homeostasis model assessment index (–63%) (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 ß-cell function. Neither drug shows any significant impact on the weight of the pancreas in this study (Fig. 4D).


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  		TABLE 2 Effect of muraglitazar on glycemic parameters in severely hyperglycemic db/db mice

 

Figure 4
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  		FIG. 4. Effect of muraglitazar on glucose tolerance, plasma insulin levels, and pancreatic insulin content in severely hyperglycemic db/db mice. db/db mice (~10-week-old females, five animals per group) were administered vehicle alone, muraglitazar, or rosiglitazone (10 mg · kg–1 · day–1 for 2 weeks). At the end of treatment, animals were fasted overnight, and baseline (t = 0 min) plasma glucose and insulin levels were determined (A and B). Animals were then given a bolus of glucose, and blood samples were drawn after 15, 30, 60, and 90 min, and plasma glucose and insulin levels were determined (A and B). At the termination of the study, mice were fasted overnight, pancreata were harvested and weighed, and pancreatic insulin content (C) was determined. Weight of pancreas is presented as percent of body weight (D). Data are presented as means ± SE. *Difference between vehicle- and drug-treated groups, P < 0.05. Mura, muraglitazar; Rosi, rosiglitazone.

 
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 · kg–1 · day–1 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).


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  		TABLE 3 Effect of muraglitazar on diet-induced hyperglycemia and hyperlipidemia in DIO-mice

 
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 (20,25–30). 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 · kg–1 · day–1 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).


Figure 5
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  		FIG. 5. Effect of muraglitazar treatment on plasma adiponectin, HMW adiponectin complex, and corticosterone levels in db/db mice. A–D: Plasma adiponectin and corticosterone levels were determined by enzyme-linked immunosorbent assay. A and B: Treated with vehicle alone or muraglitazar (30 mg · kg–1 · day–1 for 4 weeks). C and D: Treated with vehicle alone, muraglitazar, or rosiglitazone (10 mg · kg–1 · day–1 for 2 weeks). E and F: Immunoblot analysis for adiponectin and adiponectin complex. Plasma samples (0.5 µl) from vehicle- and muraglitazar-treated db/db mice (10 mg · kg–1 · day–1 for 2 weeks, three animals per group; plasma samples were from an independent study) were analyzed by reducing/denaturing (for total adiponectin) and nonreducing/nondenaturing gel electrophoresis (for adiponectin complex) followed by immunoblotting. Data are presented as means ± SE. *Difference between vehicle- and drug-treated groups, P < 0.05. Plasma adiponectin and corticosterone concentration in normal mice is 25 ± 3 and 400 ± 50 ng/ml, respectively. Mura, muraglitazar; Rosi, rosiglitazone.

 
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 · kg–1 · day–1 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
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Muraglitazar is a novel dual ({alpha}/{gamma}) PPAR activator that selectively binds to and activates human PPAR{gamma} and human PPAR{alpha} (13,33–37). 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 (1–4,25,38–39). The HDL cholesterol lowering in muraglitazar-treated mice is most likely the result of a rodent-specific PPAR{alpha}-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{alpha} activators (e.g., fenofibrate, gemfibrozil), has demonstrated plasma HDL cholesterol–raising effects (7,8,40–42).

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 ß-cells and 2) prevent apoptosis of ß-cells, respectively. These effects may help to prevent deterioration of ß-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 (20,25–28). 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 11ß-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 ({alpha}/{gamma}) 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 (0.5–20 mg for 28 days) lowers glucose, insulin, triglyceride, FFA, and apolipoprotein CIII levels and increases HDL cholesterol levels in type 2 diabetic patients (40–42). 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{alpha} activity, relative to its human PPAR{alpha} 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 ({alpha}/{gamma}) 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.

Received for publication May 23, 2005 and accepted in revised form September 26, 2005


   REFERENCES
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
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