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ENDOCRINE AND DIABETES

The Dual Peroxisome Proliferator-Activated Receptor / Activator Muraglitazar Prevents the Natural Progression of Diabetes in db/db Mice

Metabolic and Cardiovascular Diseases Discovery Biology (E.T., R.P., J.S., D.F., R.Z., G.W., D.E., L.K., A.P., L.G., A.S., T.H., J.W., S.T., N.H.), Discovery Toxicology (M.F., E.J.), Metabolic Diseases Discovery Chemistry (S.C., P.D., P.T.C.), Global Clinical Research (R.B.), Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey

Received October 11, 2006; accepted January 24, 2007.

	Abstract

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There are two major defects in type 2 diabetes: 1) insulin resistance and 2) insulin deficiency due to loss of -cell function. Here we demonstrated that treatment with muraglitazar (a dual peroxisome proliferator-activated receptor / activator), when initiated before or after the onset of diabetes in mice, is effective against both defects. In study 1, prediabetic db/db mice were treated for 12 weeks. The control mice developed diabetes, as evidenced by hyperglycemia, hyperinsulinemia, reduced insulin levels in the pancreas, blunted insulin response to glucose, and impaired glucose tolerance. The muraglitazar-treated mice had normal plasma glucose, and insulin levels, equivalent or higher pancreatic insulin content than normal mice, showed a robust insulin response to glucose and exhibited greater glucose tolerance. In study 2, diabetic db/db mice were treated for 4 weeks. The control mice displayed increased glucose levels, severe loss of islets, and their isolated islets secreted reduced amounts of insulin in response to glucose and exendin-4 compared with baseline. In muraglitazar-treated mice, glucose levels were reduced to normal. These mice showed reduced loss of islets, and their isolated islets secreted insulin at levels comparable to baseline. Thus, muraglitazar treatment decreased both insulin resistance and preserved -cell function. As a result, muraglitazar treatment, when initiated before the onset of diabetes, prevented development of diabetes and, when initiated after the onset of diabetes, prevented worsening of diabetes in db/db mice.

Muraglitazar (BMS-298585), a dual PPAR/ activator, has been demonstrated to lower insulin resistance and hyperglycemia and improve dyslipidemia in animal models of type 2 diabetes (e.g., db/db mice) and type 2 diabetic patients (Buse et al., 2005; Devasthale et al., 2005; Harrity et al., 2006; Hosagrahara et al., 2006; Kendall et al., 2006). In previously published work, muraglitazar was shown to directly stimulate both PPAR and PPAR activity in vitro (Devasthale et al., 2005). Furthermore, in db/db mice, muraglitazar treatment has been shown to modulate the expression of target genes of both PPAR (e.g., apoCIII and acyl-CoA oxidase) and PPAR (e.g., aP2, GLUT4, lipoprotein lipase, and 11-HSD1) in liver and fat, respectively (Harrity et al., 2006). The db/db mouse, with a loss of function mutation in the leptin receptor gene, is a useful model for human type 2 diabetes because, upon aging, it progressively develops insulin resistance, hyperinsulinemia, hyperglycemia, and loss of -cell function (Leiter, 1989; Kobayashi et al., 2000; Kjorholt et al., 2005). In these mice, muraglitazar was assessed for its effects on the development and worsening of diabetes when drug treatment was initiated before (i.e., in young prediabetic mice) or after (i.e., in mature diabetic mice) the onset of diabetes, respectively.

	Materials and Methods

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Animals. Female db/db mice (C57BLKS/J-m-Lepr^–/–) and corresponding age-matched female normal C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed under a controlled climate (23°C, 12-h light:12-h dark cycle) with free access to water and food (standard mouse diet number 2018; Harlan, Indianapolis, IN). Female mice were used in the studies, because male mice occasionally developed pyelonephritis in our hands. However, it should be noted that, based on prior experience with male and female db/db mice, no gender differences were anticipated either in terms of severity of diabetes or the response to muraglitazar treatment (Harrity et al., 2006). All experimental procedures were conducted according to Bristol-Myers Squibb Co. animal care guidelines.

Study 1. The prediabetic db/db mice (approximately 4 weeks old, fasting plasma glucose levels of 83–84 mg/dl) were randomized into four groups (45 mice per group) and gavaged orally once a day with vehicle (5% v/v 1-methyl-2-pyrrolidinone, 20% polyethylene glycol 400, and 75% 20 mM dibasic sodium phosphate, pH 7.2) alone or with muraglitazar at 1, 3, or 10 mg/kg/day for 12 weeks. After 4, 8, and 12 weeks, mice were fasted overnight for 16 h, and plasma glucose, insulin, and C-peptide levels were measured from tail-vein blood samples. At these time points, six mice per group were sacrificed, and their pancreatic insulin content was measured. At the 8-week time point, six mice each from the vehicle and muraglitazar-treated (10 mg/kg/day) groups were sacrificed for immunohistochemical analysis of islets. At the end of the 12-week treatment period, hemoglobin A1c (HbA1c) levels were measured for the remaining mice. In addition, nine mice per group underwent an oral glucose tolerance test (oGTT). In an independent pilot study, normal female C57BL/6J mice (4-week period between 10 and 14 weeks of age; 10 mice per group) and diabetic female db/db mice (10-week period between 8 and 18 weeks of age; six mice per group) were also analyzed for pancreatic insulin content.

Study 2. Diabetic db/db mice (approximately 10 weeks old, fasting plasma glucose levels of approximately 150 mg/dl) were randomized into three groups: 12 mice for baseline, 6 mice for the 2-week time point, and 12 mice for the 4 week time point. The mice were gavaged orally once a day with vehicle alone or muraglitazar (10 mg/kg/day) for 4 weeks. At baseline and after 2 and 4 weeks of treatment, fed plasma glucose, HbA1c, insulin, free fatty acids (FFA), triglycerides, and adiponectin levels were measured. Islets were isolated at baseline and at the 2- and 4-week time points to determine islet number and size as well as in vitro insulin secretion in response to glucose or exendin-4 (Wang and Brubaker, 2002).

Plasma Chemistry Analysis. Glucose, HbA1c, triglyceride, and FFA levels were measured by a Cobas automatic analyzer (Roche Diagnostics Corp., Indianapolis, IN). Plasma insulin was determined using a mouse ELISA (ALPCO Diagnostics, Salem, NH). Area under the glucose and insulin curves (AUCs) were determined from time 0 (baseline) values using SigmaPlot software. C-peptide levels were measured using a rat radioimmunoassay, which is reported to cross-react 100% with mouse C-peptide (Linco Research Inc., St Louis, MO). Adiponectin levels were determined using a mouse ELISA (Linco Research Inc., St Louis, MO).

oGTT. After an overnight fast, blood samples were collected from the tail vein for determination of 12-week plasma chemistry. The mice were gavaged with a dose of glucose (2 g/kg), and tail-vein blood samples were collected at 0 (immediately after glucose ingestion), 15, 30, 60, and 120 min for glucose and insulin measurements.

Insulin Content in Isolated Pancreata. Using methodology adapted from Yajima et al. (2003), pancreata were harvested from overnight fasted db/db mice or age-matched normal C57BL/6J mice immediately after sacrifice, frozen in liquid N₂, and stored at –20°C. Pancreata were homogenized in acidic/ethanol solution (75% ethanol, 23.5% distilled water, and 1.5% concentrated HCl; 1.8 ml/pancreas) with a Polytron homogenizer. The homogenates were stored at 4°C for 28 h and centrifuged at 1500g for 30 min at 4°C. The supernatants were diluted 1:20,000, and insulin levels were determined by ELISA (ALPCO Diagnostics).

Islet Histology. Immunohistochemistry was conducted on 5-µm thick sections of formalin-fixed, paraffin-embedded pancreata (six mice per group). Representative sections in which pancreas was fully exposed were used for immunohistochemical analysis. In these analyses, the avidin-biotin peroxidase method was used with guinea pig anti-insulin antibody (DAKO, Carpinteria, CA) as the primary antibody, biotinylated goat anti-guinea pig antibody (Vector Laboratories, Burlingame, CA) as the secondary antibody, 3,3-diaminobenzidine (DAKO) as the chromogen substrate, and Gill's hematoxylin (Vector Laboratories) as the counterstain. Image analysis was performed using the Ariol SL-50 (Applied Imaging, San Jose, CA). Five islets, comprising a minimum of 10 cells each, were selected from each pancreas for image analysis. The islet cells were classified semiquantitatively into either high-intensity or low-intensity insulin-staining cells. Islet images were captured using three automated scan passes: the first scan at 1.25x objective for tissue identification; the second scan at 5x objective for islet selection; and the third scan at 20x objective for image capture and analysis.

Islet Isolation. Islets were isolated from fed db/db mice by the Liberase RI method (Roche Diagnostics Corp.) and purified using Histopaque-1077. Mice were sacrificed, and the pancreas was inflated by injecting 3 ml of 0.17 mg/ml Liberase RI in Hanks' balanced salt solution into the common bile duct. The pancreata were placed in 15-ml tubes and digested at 37°C for 25 min in a water bath. Islets were purified on a Histopaque-1077 (Sigma, St. Louis, MO) gradient and hand-picked under a Leica WILD M3Z stereomicroscope (Leica, Wetzlar, Germany). Islets were sized using an eyepiece graticule calibrated against a stage micrometer and grouped as small (diameter = 100–150 µm), medium (diameter = 150–350 µm), and large (diameter = 350–650 µm).

Insulin Secretion Assay. The medium-size islets were incubated overnight in Dulbecco's minimal Eagle's medium supplemented with 5% fetal bovine serum (Gibco/Invitrogen Corp., Carlsbad, CA). The following day, 10 size-matched islets per well were placed into 96-well Multiscreen nylon mesh plates (60 µM pore size; Millipore Corp., Burlington, MA) for static insulin secretion assays. The incubation medium was Krebs-Ringer bicarbonate buffer (KRB; 118.5 mM NaCl, 2.54 mM CaCl₂·2H₂0, 1.19 mM KH₂PO₄, 4.74 mM KCl, 25 mM NaHCO₃, 1.19 mM MgSO₄·7H₂O, and 10 mM HEPES). Islets were preincubated in KRB containing 0.2% w/v bovine serum albumin and 2.8 mM glucose (Sigma) for 1 h followed by one more hour in fresh medium. Insulin content was measured in the media to establish baseline static insulin secretion for each well, using the Mercodia High-Range rat insulin ELISA, which is reported to cross-react 100% with mouse insulin (ALPCO Diagnostics). Fresh KRB plus 0.2% bovine serum albumin, supplemented with 16.8 mM glucose or 16.8 mM glucose + 20 nM exendin-4, was added for the final 1 h, after which insulin content was measured in the media. Insulin secretion was calculated as the change () in insulin secretion (nanograms/10 islets/h), which is the quantity of insulin secreted after 1 h of incubation with high glucose or exendin-4 minus the quantity of insulin secreted with low glucose during a 1-h preincubation period.

Statistical Analysis. Statistical analysis was performed using analysis of variance followed by Dunnett's test (12-week study) or unpaired two-tailed Student's t test (4-week study).

Compound. Muraglitazar was synthesized by Metabolic Diseases Discovery Chemistry, Bristol-Myers Squibb (Devasthale et al., 2005).

	Results

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Muraglitazar Treatment Prevents Development of Diabetes in db/db Mice. In study 1, the impact of muraglitazar treatment on the development of insulin resistance, hyperglycemia, and -cell failure was determined. In this study, approximately 4-week-old prediabetic db/db mice were treated with vehicle alone or muraglitazar at doses of 1, 3, or 10 mg/kg/day for 12 weeks.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Effect of muraglitazar treatment in prediabetic db/db mice on the development of diabetes. Approximately 4-week-old prediabetic db/db mice were treated with vehicle or muraglitazar for 12 weeks. Plasma samples were collected from overnight fasted animals at the 0-, 4-, 8-, and 12-week time points. A, glucose. B, HbA1c (12-week time point only). C, body weight. D and E, insulin and C-peptide (4-, 8-, and 12-week time points only). Numbers of mice per group were as follows: 45 at week 0, 45 at week 4, 33 at week 8, and 21 at week 12 (reduced number of animals during the course of the study was the result of animals sacrificed for harvesting of pancreas). Fasting plasma glucose levels before treatment began were 83 to 84 mg/dl. Data are presented as mean ± S.E.M. *, difference between vehicle- and muraglitazar-treated groups, p < 0.05; t, difference between 4 weeks versus 8 or 12 weeks, p < 0.05. Normal C57BL/6J mice (8–10-week-old, fasted): glucose, 100–125 mg/dl; HbA1c, 4%; insulin, 0.5–2.0 ng/ml; and C-peptide, 4 ng/ml.

The vehicle-treated db/db mice maintained approximately 2- to 3-fold higher fasting insulin levels at the 4-, 8-, and 12-week time points (6.3 ± 0.6, 4.0 ± 0.8, and 4.7 ± 0.8 ng/ml, respectively) than age-matched normal C57BL/6J mice (0.5 to 2.0 ng/ml; unpublished data) (Fig. 1D). In comparison, the muraglitazar-treated mice displayed reduced fasting plasma insulin levels at the 4-, 8-, and 12-week time points (Fig. 1D). At the conclusion of the 12-week study, the fasting plasma insulin levels were at levels close to the levels observed in normal C57BL/6J mice (at the 10-mg/kg/day dose: 4.4 ± 0.3, 2.3 ± 0.3, and 2.2 ± 0.2 ng/ml at the 4-, 8-, and 12-week time points, respectively). Consistent with higher plasma insulin levels, the vehicle-treated mice showed relatively high plasma C-peptide levels that remained stable over time (10.7 ± 1.6, 11.6 ± 1.7, and 9.9 ± 2.1 ng/ml at 4, 8, and 12 weeks of treatment, respectively). In accordance with the decreased fasting insulin levels, the muraglitazar-treated mice showed reduced C-peptide levels relative to the vehicle-treated mice and showed a nonsignificant trend for increase over time (at the 10-mg/kg/day dose: 5.4 ± 0.4, 5.7 ± 0.4, and 7.0 ± 1.0 ng/ml at the 4-, 8-, and 12-week time points, respectively) (Fig 1E). Hyperglycemia, in the presence of elevated insulin levels (2–3-fold above the levels in normal C57BL/6J mice), in vehicle-treated db/db mice indicates their severely insulin-resistant state. The data also suggest that, in these mice, their -cells could not adequately compensate for severe insulin resistance. On the other hand, the normoglycemia and reduced or normalized plasma insulin levels in muraglitazar-treated mice indicate improvement of insulin sensitivity and/or delay of worsening of insulin resistance.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Effect of muraglitazar treatment in prediabetic db/db mice on pancreatic insulin content. Approximately 4-week-old prediabetic db/db mice were treated with vehicle or muraglitazar for 12 weeks. At the 4-, 8-, and 12-week time points, pancreata were harvested from mice fasted overnight, and insulin content was determined. A, pancreatic insulin levels in normal C57BL/6J mice (age 10–14 weeks; 10 mice per group) and db/db mice (age 8 through 18 weeks; six mice per group) in a pilot study. B, pancreatic insulin levels in vehicle- and muraglitazar-treated db/db mice at the 4-, 8-, and 12-week time points (six mice per group). C to F, insulin immunohistochemistry of islets after 8 weeks of treatment. Magnification, five times (C–D) and 20 times (E–F). G, percentage of high-intensity insulin-staining cells in islets from vehicle- or muraglitazar-treated mice (five islets per pancreas, six animals per group). Data presented as mean ± S.E.M. **, difference between vehicle- and muraglitazar-treated groups, p = 0.06; *, difference between vehicle- and muraglitazar-treated groups, p < 0.05.

At the end of study 1, the vehicle- and muraglitazar-treated db/db mice were fasted overnight and, after collecting tail-vein blood for determination of week 12 fasting plasma chemistry, were challenged with an oral bolus of glucose in an oGTT. Plasma glucose and insulin levels were determined immediately after the glucose challenge (defined as time 0) and at regular intervals for up to 120 min after ingestion of glucose. As presented in Fig. 3, A, B, and C, the muraglitazar-treated mice showed markedly reduced glucose excursions (the glucose AUC was reduced by 28 and 43% at the 3- and 10-mg/kg/day doses, respectively) and a trend for reduced insulin AUC levels compared with the vehicle-treated mice. The reduced glucose excursion demonstrates that, in the muraglitazar-treated db/db mice, their glucose tolerance has been improved. During oGTT, the vehicle-treated db/db mice were hyperinsulinemic at time 0 (Fig. 3D). However, their plasma insulin levels, both as absolute levels and as a percentage of time 0 values, remained relatively unchanged for up to 120-min postglucose ingestion (Fig. 3, D and E). The glucose-stimulated first-phase insulin secretion was completely absent in the vehicle-treated mice (Fig. 3, D and E). In contrast, the muraglitazar-treated db/db mice, with nearly normal baseline plasma insulin levels, showed robust glucose-stimulated first-phase insulin secretion, as well as significantly elevated plasma insulin levels throughout the oGTT (Fig. 3, D and E). Therefore, these data indicate that, in the muraglitazar-treated db/db mice, a robust -cell insulin secretory response to glucose was maintained, whereas in the vehicle-treated db/db mice, the normal -cell insulin secretory response to glucose was lost. It is possible that the reduced glucose excursion in muraglitazar-treated mice is due, at least in part, to the preservation of normal insulin secretory response by -cells to the glucose challenge.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Effect of muraglitazar treatment in prediabetic db/db mice on glucose excursion and insulin secretory response after a glucose challenge. Approximately 4-week-old prediabetic db/db mice were treated with vehicle or muraglitazar for 12 weeks. At the end of the 12-week study, animals were fasted overnight and, after collecting tail-vein blood for week 12 fasting plasma chemistry, were given an oral bolus of glucose, and plasma glucose and insulin levels were determined immediately (time 0) and after 15, 30, 60, and 120 min after the gavage. A, glucose. B and C, glucose AUC and insulin AUC based on respective time 0 values. D, insulin levels postglucose challenge. E, insulin as a percentage of respective time 0 values postglucose challenge. Number of mice per group: 9. Data presented as mean ± S.E.M. *, difference between vehicle- and muraglitazar-treated groups, p < 0.05; **, difference between time 0 and postglucose challenge, p < 0.05.

Muraglitazar Treatment Prevents Deterioration of Diabetes in db/db Mice. In study 2, the impact of muraglitazar treatment, when initiated after the onset of diabetes, on hyperglycemia and deterioration of -cell function was assessed. In this study, a cohort of approximately 10-week-old diabetic db/db mice was treated with muraglitazar (10 mg/kg/day) or vehicle alone for 4 weeks. The analyses were done with ad libitum fed mice to increase the survival rate of the islets and to preserve glucose stimulated insulin secretory function in isolated islets in vitro (Bouman et al., 1979; Trus et al., 1980).

At the start of the 4-week study, the average baseline fed plasma glucose levels in the untreated db/db mice were 567 ± 13 versus 225 mg/dl in age-matched normal C57BL/6J mice. Over the course of the study, the vehicle-treated db/db mice showed signs of worsening of hyperglycemia (fed plasma glucose = 651 ± 32 mg/dl at the 2-week time point), whereas in muraglitazar-treated db/db mice, the fed plasma glucose levels were lowered (fed glucose = 256 ± 39 mg/dl at the 2-week time point) close to the levels observed in normal C57BL/6J mice (Fig. 4A). Consistent with the glucose levels, HbA1c values were higher in control mice (HbA1c, 7.4 ± 0.3 versus 6.1 ± 0.03% at baseline) and were lower in drug-treated mice (HbA1c, 5.5 ± 0.3%) after 2 weeks of treatment (Fig. 4B). The fed plasma insulin levels tended to be lower in muraglitazar-treated db/db mice in comparison with the vehicle-treated db/db mice; however, the differences between the vehicle- and drug-treated groups failed to reach statistical significance (Fig. 4C). The normalized glucose level in the presence of either equal or reduced insulin levels is indicative of improved insulin sensitivity in muraglitazar-treated mice. The muraglitazar-treated db/db mice also showed improvements in other metabolic parameters. For example, after 2 weeks of treatment, the plasma FFA levels were decreased to 0.4 ± 0.03 mEq/l, triglyceride levels were decreased to 78.0 ± 2.0 mg/dl, and the levels of adiponectin, an adipokine that plays an important role in insulin sensitivity, were increased to 80.7 ± 8.2 µg/ml from baseline values of 1.2 ± 0.14 mEq/l, 181.0 ± 19.0 mg/dl and 17.0 ± 0.8 µg/ml, respectively (Fig. 4, D–F). In the vehicle-treated db/db mice, after 2 weeks on treatment, the plasma FFA levels were unchanged (1.0 ± 0.1 mEq/l), the triglyceride levels were increased (368.0 ± 31.0 mg/dl), and adiponectin levels were decreased (8.6 ± 0.3 µg/ml) from the corresponding baseline levels (Fig. 4, D–F). Consistent with the 2-week data, the muraglitazar-treated mice maintained improved glucose levels and other metabolic parameters at the 4-week time point (Fig. 4, A–F). Thus, these data show that muraglitazar treatment, when initiated after the onset of diabetes, improved insulin sensitivity and corrected hyperglycemia, whereas the control mice exhibited worsening of hyperglycemia.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Effect of muraglitazar treatment in diabetic db/db mice on hyperglycemia and metabolic abnormalities. Approximately 10-week-old diabetic db/db mice were treated with vehicle or muraglitazar for 4 weeks. Plasma samples were collected from untreated mice (food was removed 1 h before samples were collected) at baseline and after 2 and 4 weeks of treatment. Shown are plasma glucose (A), HbA1c (B), insulin (C), FFA (D), triglycerides (E), and adiponectin (F) levels. Number of mice per group: 12 mice for baseline, 6 mice for the 2-week time point, and 12 mice for the 4-week time point. Data presented as mean ± S.E.M. *, difference between vehicle- and muraglitazar-treated groups, p < 0.05. t, difference between baseline and 2- or 4-week time points, p < 0.05.

Islets were harvested from the pancreata of untreated db/db mice at baseline and from vehicle- or muraglitazar-treated db/db mice at the 2- and 4-week time points. The size of the islets in the pooled samples were categorized as small (diameter = 100–150 µM), medium (diameter = 150–350 µM), and large (diameter = 350–650 µm) and counted. The medium-size islets, which make up the largest volume in the pancreas, are typically used for insulin secretion experiments (Diani et al., 2004; Ishida et al., 2004). As shown in Fig. 5, A and B, in the vehicle-treated db/db mice, both the total number of islets per mouse (–52% after 2 weeks) and the number of medium-sized islets per mouse (–40% after 2 weeks) were decreased after 4 weeks compared with baseline values. In the muraglitazar-treated db/db mice, the corresponding reduction in the total number of islets per mouse was blunted (–18% after 2 weeks) (Fig. 5A), and the number of medium-sized islets per mouse was increased (+26% after 2 weeks) (Fig. 5B). These data suggest that muraglitazar treatment reduced the loss of islets and stabilized the number of medium-sized islets in the pancreata of diabetic db/db mice.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effect of muraglitazar treatment in diabetic db/db mice on islet number in the pancreas and on glucose and exendin-4-induced insulin secretion from islets in vitro. Approximately 10-week-old diabetic db/db mice were treated with vehicle or muraglitazar for 4 weeks. Islets were isolated from the pancreata of untreated mice at baseline and from vehicle- or muraglitazar-treated mice at the 2- and 4-week time points. A, total number of islets. B, number of medium-size islets. Number of mice per group: 12 for baseline, 6 for the 2-week time point, and 11 for the 4-week time point. Since islets were pooled for isolation, the islet data are reported as the average number of islets per mouse without further statistical analysis. C to D, in vitro insulin secretion response by medium-size islets, isolated at baseline or after 2 and 4 weeks of treatment. C, insulin levels (ng/ml) in the incubation buffer containing low glucose (2.8 mM [2.8 mM (1) and 2.8 mM (2) represent islets used in subsequent high glucose and high glucose + exendin-4 studies, respectively]). D, the difference () between low glucose (2.8 mM) and high glucose (16.8 mM) or high glucose (16.8 mM) + exendin-4 (20 mM). Number of islets per well: 10 islets; six wells per group. Data presented as mean ± S.D. *, difference between low glucose and 16.8 mM glucose; **, difference between 16.8 mM glucose and 16.8 mM glucose + 20 nM exendin-4; t, difference between vehicle- and muraglitazar-treated mice, p < 0.05.

The microscopically similar medium-size islets (as judged by appearance and size), which were isolated at baseline and after treatment with vehicle alone or muraglitazar for 2 and 4 weeks, were analyzed next in vitro for their insulin secretory response to high glucose (16.8 mM) or high glucose (16.8 mM) + exendin-4 (20 nM). Exendin-4, a 39-amino acid peptide originally isolated from Gila monster salivary gland with 53% homology to the glucagon like peptide-1, is a ligand of the glucagon like peptide-1 receptor and exerts incretin action in animals and in humans and is a direct insulin secretagogue (Gallwitz, 2006). In these analyses, the insulin secretory response was determined as the change () in the insulin level in the incubation media after 1 h of incubation in high glucose or high glucose + exendin-4 minus insulin level during a 1-h preincubation under low glucose conditions (2.8 mM). As shown in Fig. 5C, during a 1-h preincubation, islets isolated at baseline or after 2 and 4 weeks of drug treatment secreted insulin in response to a low concentration of glucose (2.8 mM). As shown in Fig. 5D, the islets isolated at baseline responded to high-glucose or high-glucose + exendin-4 and secreted increased amounts of insulin (=+2.9 and +7.4 ng/10 islets/h, respectively, versus low glucose). At the 2-week time point, the islets from the vehicle-treated mice showed reduced insulin secretory response to high glucose or high glucose + exendin-4 (=+0.1 and +1.6 ng/10 islets/h, respectively, versus low glucose) (Fig. 5D) compared with baseline levels. In contrast, the islets from muraglitazar-treated mice secreted insulin in response to high glucose or high glucose + exendin-4 at levels higher than the vehicle-treated mice and comparable to baseline levels ( +2.3 and +8.7 ng/10 islets/h, respectively, versus low glucose) (Fig. 5D). The increased insulin secretory response to high glucose was maintained at the 4-week time point in muraglitazar-treated mice (Fig. 5D). Thus, in vehicle-treated diabetic db/db mice, the glucose-stimulated islet -cell insulin secretory function had deteriorated compared with baseline during the course of the treatment period. Over the same treatment period, muraglitazar treatment prevented the deterioration of islet -cell insulin secretory function.

	Discussion

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We have confirmed the previous observation that muraglitazar treatment corrects insulin resistance and hyperglycemia in db/db mice (Harrity et al., 2006). We now have shown that muraglitazar prevents loss of islet mass and -cell function in db/db mice. Thus, muraglitazar favorably affects the two major defects in type 2 diabetes, namely, insulin resistance and loss of -cell function. As a result, it prevents development of diabetes in prediabetic db/db mice and the worsening of diabetes in diabetic db/db mice.

Muraglitazar treatment, when initiated before the onset of diabetes, prevents the subsequent characteristic development of diabetes in db/db mice. This was evidenced by 1) improved insulin sensitivity (i.e., maintenance of normal insulin and glucose levels), 2) preservation of insulin levels in the pancreas, 3) preservation of islet structural integrity and insulin content, 4) robust insulin secretory response after an oral glucose challenge, and 5) improved glucose tolerance. Muraglitazar treatment, when initiated after the onset of diabetes, prevents or possibly delays the further deterioration of the diabetic state in db/db mice. This was demonstrated by 1) normalization of glucose levels, 2) the reduced loss of islets in the pancreas, and 3) preservation of the isolated islet insulin secretory response to glucose. Consistent with previous observations, muraglitazar improves other metabolic abnormalities, such as the elevated plasma FFA and triglyceride levels, as well as reduced plasma adiponectin levels seen in vehicle-treated control db/db mice (Harrity et al., 2006).

Previously, some -cell protective effects have been reported for the clinically used PPAR activators (e.g., pioglitazone) in animal models of type 2 diabetes (Yajima et al., 2003; Diani et al., 2004; Ishida et al., 2004). These protective effects in animal models and clinical trials against diabetes (Troglitazone in Prevention of Diabetes, Troglitazone in Diabetes Prevention Trial, and Pioglitazone in Prevention of Diabetes) have been attributed to improvement of insulin sensitivity and glycemic control by these agents, which reduced insulin secretory demand and preserved -cell function (Buchanan et al., 2002; The Diabetes Prevention Program Research Group, 2005; Xiang et al., 2006). The data reported here are consistent with these published data and demonstrate that muraglitazar improves both insulin sensitivity and preserved -cell function. Furthermore, the data indicate that muraglitazar has the potential to prevent both the development and worsening of diabetes.

It is possible that multiple mechanisms, both indirect and direct, play a role in muraglitazar-mediated prevention of diabetes development and/or worsening of diabetes in db/db mice. First, muraglitazar, by correcting insulin resistance, either prevents development of hyperglycemia or reduces hyperglycemia. Second, muraglitazar-mediated improvement in insulin sensitivity may result in a reduced demand for insulin secretion from -cells to maintain glycemic control and thus lengthen their productive lifespan (Buchanan et al., 2002; Diani et al., 2004; Ishida et al., 2004; The Diabetes Prevention Program Research Group, 2005; Xiang et al., 2006). Third, by lowering both hyperglycemia and hyperlipidemia, muraglitazar may protect -cells from glucotoxicity- and lipotoxicity-induced premature apoptosis (Bergman and Ader, 2000; Cnop et al., 2005; Kjorkolt et al., 2005). Fourth, the muraglitazar-mediated stimulation of plasma adiponectin levels, through enhancement of insulin sensitivity, may help to preserve -cell function (Kadowaki and Yamauchi, 2005; Furuta et al., 2006).

The muraglitazar-treated mice with normal fasting insulin levels, maintained normal fasting glucose levels and showed improved glucose tolerance (i.e., improved insulin sensitivity). The vehicle-treated mice, in contrast to the muraglitazar-treated mice, with 2- to 3-fold above the normal fasting insulin levels, showed fasting hyperglycemia and glucose intolerance (i.e., insulin resistance). The reduced fasting plasma insulin levels and C-peptide levels in muraglitazar-treated mice compared with the vehicle-treated mice is indicative of reduced insulin secretory demand from the pancreas. Interestingly, in muraglitazar-treated mice, the plasma C-peptide levels showed a nonsignificant trend for increase over time in the presence of reduced or normal plasma insulin levels. Although no data are available to support, it is possible that this is, at least in part, due to the changes in the relative rates of clearance of insulin (faster) versus clearance of C-peptide (slower) in muraglitazar-treated mice as a result of improved insulin sensitivity and glucose control (Katz and Rubenstein, 1973; Kim et al., 2005).

Finally, pancreatic islets have been shown to express both PPAR and PPAR (Yoshikawa et al., 2001; Rosen et al., 2003; Lin et al., 2005; Park et al., 2006; Richardson et al., 2006). It is possible that muraglitazar may protect -cells directly by favorably altering the expression of PPAR-or PPAR-target genes, which are implicated in important functions, such as glucose sensing, fatty acid transport, control of cell differentiation, apoptosis, and insulin secretion in -cells (Yoshikawa et al., 2001; Rosen et al., 2003; Lin et al., 2005; Park et al., 2006; Richardson et al., 2006). The preclinical and clinical data with PPAR activators indicate that the predominantly PPAR-mediated mechanisms, such as stimulation of adiponectin levels, correction of insulin resistance, and lowering of hyperglycemia, may play a dominant role and protect the -cells by reducing excessive insulin secretory demand (Buchanan et al., 2002; Rosen et al., 2003; Diani et al., 2004; Ishida et al., 2004; Lin et al., 2005; The Diabetes Prevention Program Research Group, 2005; Richardson et al., 2006; Xiang et al., 2006). It is also possible that the beneficial PPAR-mediated effects, such as improvements in hyperlipidemia, lipotoxicity, and oxidative stress, also play a role in the protection of the -cells (Yoshikawa et al., 2001; Park et al., 2006). It is tempting to speculate that the above normal insulin content in the pancreas of muraglitazar-treated db/db mice may be due to the combined PPAR-and PPAR-mediated effects of this drug in the -cells. Clearly, further work would be necessary to understand the relative importance of the role of each one of the above mechanisms in the muraglitazar-mediated prevention of diabetes development in prediabetic db/db mice and the worsening of diabetes in diabetic db/db mice.

In conclusion, the results of these studies demonstrate that muraglitazar treatment prevents the natural progression of diabetes in db/db mice by correcting insulin resistance, lowering hyperglycemia, and preserving islet mass and -cell function. These preclinical data suggest that muraglitazar treatment, 1) if initiated before the onset of diabetes, may prevent the development of diabetes in high risk humans and, 2) if initiated after the onset of diabetes, may attenuate loss of islet mass and -cell function, in addition to improving insulin resistance and hyperglycemia in patients with type 2 diabetes.

	Acknowledgements

 
We are grateful to members of the Metabolic and Cardiovascular Diseases Department for reviewing the manuscript.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.115337.

ABBREVIATIONS: BMS-298585, muraglitazar; PPAR, peroxisome proliferator-activated receptor; AUC, area under the curve; FFA, free fatty acids; HbA1c, hemoglobin A1C; KRB, Krebs-Ringer bicarbonate buffer; ELISA, enzyme-linked immunosorbent assay; Mura, muraglitazar; oGTT, oral glucose tolerance test; Veh, vehicle.

¹ Current affiliation: Hoffmann-La-Roche Pharmaceuticals, Basel, Switzerland.

² Current affiliation: Pharmacopeia, Princeton, New Jersey.

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

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