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Keywords:

  • hyperlipidemia;
  • type 2 diabetes;
  • peroxisome proliferator-activated receptor alpha;
  • peroxisome proliferator-activated receptor gamma

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Objective: Preclinical evaluation of DRF 2655, a peroxisome proliferator-activated receptor alpha (PPARα) and PPARγ agonist, as a body-weight lowering, hypolipidemic and euglycemic agent.

Research Methods and Procedures: DRF 2655 was studied in different genetic, normal, and hyperlipidemic animal models. HEK 293 cells were used to conduct the reporter-based transactivation of PPARα and PPARγ. To understand the biochemical mechanism of lipid-, body-weight-, and glucose-lowering effects, activities of key β-oxidation and lipid catabolism enzymes and gluconeogenic enzymes were studied in db/db mice treated with DRF 2655. 3T3L1 cells were used for adipogenesis study, and HepG2 cells were used to study the effect of DRF 2655 on total cholesterol and triglyceride synthesis using [14C]acetate and [3H]glycerol.

Results: DRF 2655 showed concentration-dependent transactivation of PPARα and PPARγ. In the 3T3L1 cell-differentiation study, DRF 2655 and rosiglitazone showed 369% and 471% increases, respectively, in triglyceride accumulation. DRF 2655 showed body-weight lowering and euglycemic and hypolipidemic effects in various animal models. db/db mice treated with DRF 2655 showed 5- and 3.6-fold inhibition in phosphoenolpyruvate carboxykinase and glucose 6-phosphatase activity and 651% and 77% increases in the β-oxidation enzymes carnitine palmitoyltransferase and carnitine acetyltransferase, respectively. HepG2 cells treated with DRF 2655 showed significant reduction in lipid synthesis.

Discussion: DRF 2655 showed excellent euglycemic and hypolipidemic activities in different animal models. An exciting finding is its body-weight lowering effect in these models, which might be mediated by the induction of target enzymes involved in hepatic lipid catabolism through PPARα activation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The regulation of lipid and carbohydrate metabolism is central to energy homeostasis in man. Obesity is a complex disorder, which is attributable to imbalance of energy intake and expenditure leading to an excessive accumulation of body fat. A high-calorie diet coupled with limited physical activity contributes to the accumulation of excess calories in the form of fat. Obesity is now assuming epidemic proportions, not only in the developed nations, but also in the developing ones. Obesity is associated with several risk factors such as dyslipidemia, hypertension, cardiovascular complications, insulin resistance, and type 2 diabetes (1)(2). The primary goal of obesity treatment is weight loss. However, obesity is associated with metabolic disorders, which also need to be taken into consideration. Current research effort is aimed at discovering new antiobesity agents that not only achieve weight control but also improve metabolic and cardiovascular functions.

Peroxisome proliferator-activated receptor alpha (PPARα)1 and gamma (PPARγ) are members of the nuclear receptor superfamily that exert diverse effects on fat and carbohydrate metabolism and are targets for therapeutic agents in metabolic diseases (3)(4). The PPARγ agonists, thiazolidinediones (TZDs), promote adipocyte differentiation and improve insulin action in peripheral tissues by diverting the lipid supply from muscle and liver and increasing the uptake of fatty acids in adipose tissue (5)(6)(7). Consequently, although PPARγ ligands decrease circulating lipids, they increase accumulation of fat and, thereby, body weight. TZDs in the market also exhibit this undesirable increase of body weight, which is of particular concern in already obese type 2 diabetes patients (8). Fibrates are PPARα agonists that mediate the expression of genes regulating lipid metabolism. These drugs are used to treat hypertriglyceridemia and reduce cardiovascular risk (9). It is believed that PPARα ligands might have a positive effect on body weight through increased catabolism of fat. Fibrate treatment has been reported to reduce weight gain in rodents without affecting the food intake (10)(11)(12). Glaxo Wellcome (Research Triangle Park, NC) has disclosed ureidofibrate derivatives that are claimed to be PPARα selective and useful for treating obesity (13)(14).

Although some of the genetic factors responsible for body-weight regulation are known, most of the therapeutic agents have been reported to have undesirable side effects (15)(16). Only two antiobesity agents, Sibutramine, an appetite suppressant, and Orlistat, an inhibitor of fat absorption, have been introduced into the market recently. Although both the drugs are active, their efficacy is limited and their tolerability is less than desirable. Therefore, there is an unmet need to develop better body weight-reducing agents with alternative mechanisms. Based on comparative study of the mechanisms of actions of PPARα and PPARγ, it was hypothesized that a compound that is a coligand for both these PPAR isoforms could be a better choice for overall treatment of metabolic syndrome (17). Several compounds of this series are now in clinical development (18)(19)(20)(21)(22). Reports available so far indicate that these dual activators have clearly shown their efficacy in insulin resistance and dyslipidema, but not much is known about their effects on body weight. A few preliminary reports (23)(24)(25) indicate that some of these dual activators showed attenuation of high-fat diet-induced body weight increase. Considering the important role of PPARα in catabolism of fat, we have initiated a program to discover a dual PPAR agonist with greater specificity toward the alpha isoform not only for treating weight gain, but also for improving associated metabolic syndromes. Here we report a dual activator of PPARα and PPARγ, DRF 2655, an alkoxy propanoic acid analogue with hypolipidemic, insulin-sensitizing, and body weight-lowering effects in different animal models of obesity and dyslipidemia.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Compounds

DRF 2655 (Figure 1) was synthesized by the Discovery Chemistry group of DRF, Hyderabad, India. Rosiglitazone and WY14643 were synthesized following the published procedure and were found to be 99% pure. Fenofibrate was obtained from Sigma Chemical Co. (St. Louis, MO).

image

Figure 1. Structure of DRF 2655.

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Animals

All animal experimental protocols were approved by the Animal Ethics Committee of Dr. Reddy's Research Foundation (DRF). Male C57BL/KsJ-db/db and C57BL/6J-ob/ob mice (Jackson Laboratory, Bar Harbor, ME), Swiss albino mice (SAM), and monosodium glutamate-injected obese Swiss albino mice (MSG-SAM; DRF animal house) were used at 10 weeks of age. MSG-SAM was developed according to reported protocol (26). Zucker fa/fa rats (Institut Francais de la Fievre Aphtheuse—Centre de Recherche et d'Elevage Des Oncins, L'Arbresle Cedex, France) were used at 13 weeks of age. Sprague Dawley (SD) rats (150 to 180 g) were made hypercholesterolemic by feeding a high fat diet (2% cholesterol and 1% sodium cholate mixed with normal diet) for 6 days. Golden Syrian hamsters (National Institute of Nutrition, Hyderabad, India) weighing 80 to 100 g were fed 1% cholesterol mixed with normal diet for 15 days along with the start of compound treatment. All animals were maintained on normal laboratory chow (National Institute of Nutrition), ad libitum water, and a 12-hour light/dark cycle at 25 ± 1 °C.

Drug Administration and Blood Sampling

DRF 2655 was administered to db/db mice for 9 days at 0.03, 0.1, 1, and 3 mg/kg per oral doses and plasma parameters were measured. For studying the body weight changes, 30 mg/kg of DRF 2655 and rosiglitazone were administered for 30 days. For studying body weight changes, ob/ob mice were treated with DRF 2655 for 30 days at 10 mg/kg per oral dose. Zucker fa/fa rats were administered DRF 2655 and rosiglitazone at 1 and 3 mg/kg, respectively, for 6 days. DRF 2655 was administered at different doses to SAM for 6 days. MSG-SAM was treated with 3 mg/kg of DRF 2655 and 30 mg/kg of fenofibrate, respectively, for 30 days. High fat-fed SD rats were treated with DRF 2655 at 0.1, 0.3, 1, 3, and 10 mg/kg doses for 3 days, while they were on the same feed. After 6 days of treatment, animals were administered 250 mg/kg body weight of tylaxopol (Triton WR 1339) (5 mL/kg in saline; Sigma) through an intravenous route, and blood was collected at 0, 2, 4, 6, and 24 hours after administration. For the lipid tolerance test, animals were administered 20% intralipid (5 mL/kg intravenous; Amersham Biosciences, Piscataway, NJ), and blood samples were collected at 0, 1, 10, 30, 60, and 120 minutes for tissue triglyceride (TG) measurement. ApoCIII was measured 6 days after treatment. Male golden Syrian hamsters were treated with DRF 2655 at 10 mg/kg and fenofibrate at 100 mg/kg, respectively, for 15 days. All the control groups received vehicle alone (0.25% carboxymethyl cellulose, 10 mL/kg). Blood samples were collected from the retro-orbital sinus of the animals in the fed state, under mild ether anesthesia, 1 hour after drug administration. In all experiments, dosing of the compounds was done on a once-a-day schedule. (Preliminary experiments were performed to determine the optimum dosing schedule of each compound.) Rosiglitazone and fenofibrate have been used as reference drugs. Preliminary experiments were conducted to determine the optimum dose of these drugs in individual models.

Triglyceride Content in Tissue

TG content was measured according to reported protocol (27). Tissue homogenates (100 mg each) were extracted with chloroform:methanol (2:1). On centrifugation, the chloroform phase was separated and evaporated to dryness under nitrogen. The residue was reconstituted in 200 μL of chloroform. A 2.5-μL aliquot was used to determine TG content with the enzymatic method in a final reaction volume of 1 mL.

Plasmids, Transfection, and PPAR Transactivation Assay

The response element (UASGAL4 × 5) is present upstream of the pFR-Luc (Promega, Madison, WI) reporter that contains the Simian virus early promoter for luciferase assay. GAL4 fusions were made by fusing human PPARγ, PPARα, or PPARδ ligand-binding domain (amino acids 174 to 475) to the C-terminal end of the yeast GAL4 DNA-binding domain (amino acids 1 to 147) of the pM1 vector. pAdVantage vector (Promega) was used to enhance luciferase expression.

HEK293 cells were grown in Dullbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. Before transfection (1 day), cells were plated to 50% to 60% confluence in DMEM containing 10% delipidated FBS (DFBS). Cells were transfected with the expression vectors for the respective PPAR chimera along the reporter and enhancer constructs by Superfect (QIAGEN, Hilden, Germany) as per the supplier's instruction. After transfection (3 hours), the reagent was removed and cells were maintained in DMEM-DFBS. After transfection (42 hours), cells were placed in phenol red-free DMEM-DFBS and treated for 18 hours with the test compounds or 0.1% dimethyl sulfoxide (DMSO). The cells were lysed and assayed for luciferase activity. Luciferase activity was determined as fold activation relative to untreated cells by using the Luclite kit in a Top Count (Packard, Meriden, CT).

Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose 6-Phosphatase (G-6-Ptase), Carnitine Palmitoyltransferase (CPT1), and Carnitine Acetyltransferase (CAT) Activity in db/db Mice Liver

db/db mice were treated with DRF 2655, rosiglitazone, and fenofibrate at 3, 10, and 30 mg/kg, respectively, for 10 days. After the treatment, the mice were killed and liver samples were collected in liquid nitrogen and stored at −80 °C until further use. All procedures were performed at 0 to 4 °C unless otherwise specified. A 20% homogenate was prepared in 10 mM Tris-HCl buffer (pH 7.5) containing 0.35 M sucrose and centrifuged at 3300 rpm for 5 minutes. The supernatant was collected and subjected to another centrifugation at 10, 000 rpm for 15 minutes. The pellet containing mitochondria was used for the assay of CPT1 and CAT activity. The supernatant was further subjected to ultracentrifugation at 100, 000 g for 75 minutes. The microsomal pellet obtained was used for G-6-Ptase assay and the cytosol for PEPCK assay.

PEPCK, G-6-Ptase, CPT1, and CAT activity were measured by established procedures (28)(29)(30)(31)(32).

Lipoprotein Lipase (LPL) Activity in High Fat-Fed Rat Adipose Tissue

LPL activity was measured in adipose tissue of high fat-fed rats treated with DRF 2655 at 3 mg/kg per dose. LPL activity in tissue homogenate was measured as described (33).

Lipid Biosynthesis in Human Hepatoma (HepG2) Cells

HepG2 cells were cultured in DMEM containing 10% FBS. Cells were seeded at a density of 3.5 × 105 cells per six-well plate for total cholesterol (TC) synthesis. For triglyceride synthesis, HepG2 cells were seeded in a 24-well plate and maintained in DMEM + 10% FBS. After 24 hours, when the cells were 70% confluent, the medium was replaced with fresh medium containing 30 μM concentration of DRF 2655 in 100% DMSO. The final concentration of DMSO in the medium was 0.1%. An equal concentration of DMSO was added in the control wells. For the 24-hour treatment schedule, the cells were pre-incubated with the compound for 18 hours. Later the cells were changed into DMEM with 10% delipidated serum and 5 μCi of [14C] acetate (41.8 mCi/mmole; BRIT, Mumbai, India) to a final concentration of 0.5 mM along with the compound for TC synthesis. In the case of triglyceride synthesis, the media were removed and fresh medium containing DMEM, 10% delipidated serum, 2.5 μCi [3H] glycerol (1.10 Ci/mmol; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK), 1 mM glycerol, and 1 mM sodium oleate, along with the compound in DMSO, was added. After 6 hours of incubation at 37 °C, the medium was removed and the cells were washed with phosphate-buffered saline. The cells were lyzed in 0.1% Igepal (NP-40; Sigma) in 0.1 N NaOH, and the synthesized cholesterol was extracted according to known procedure (34). For triglyceride extraction, an aliquot (0.4 mL) of the lysate was used with chloroform:methanol (2:1, vol/vol) followed by 0.88% KCl. The organic phase was separated, dried, and reconstituted in a 100-μL volume of chloroform:methanol. The radioactivity incorporated into the lipids was quantified in a scintillation counter (Top Count, Packard) and the final results were expressed as counts per minute per milligram protein.

Analytical Methods

Plasma glucose (PG), TG, TC, and free fatty acids (FFAs) were measured spectrophotometrically using commercially available kits (Point Scientific, Lincoln Park, MI, and Roche Diagnostics, Mannheim, Germany). Insulin and leptin were measured using a radioimmunoassay kit from Linco Research Inc. (St. Charles, MO) ApoCIII was measured by immunoturbidimetric method with antihuman ApoCIII antibody, which has an 85% cross-reactivity with rodent ApoCIII (Daiichi Pure Chemical Co., Tokyo, Japan).

Data Analysis and Statistics

All data are presented as mean ± SE. The percent reduction was calculated according to the formula: [1 − [(Test Day treated/Day 0 treated)/(Test Day control/Day 0 control)] × 100].

The statistical analyses were performed using ANOVA or Student's t test. p < 0.05 was considered significant to control.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Effect on PPAR Transactivation and Differentiation of 3T3L1 Preadipocytes to Adipocytes

In a transactivation assay, DRF 2655 was 20-fold more potent in activating PPARα than WY14643 [50% effective dose (EC50) = 1.08 vs. 22.1 μM for WY14643), and the transactivation potential of this compound was somewhat higher than that of WY14643 (Figure 2A). However, the potency of transactivation at PPARγ of DRF 2655 (EC50 = 2.10 vs. 0.201 μM for rosiglitazone) was 10-fold less than that of rosiglitazone (Figure 2B). The transactivation potential of DRF 2655, PPARα to PPARγ was found to be 2:1. The compound did not have any effect on PPARδ activation. When murine PPARα and PPARγ constructs were used, DRF 2655 showed similar dual activation potential (20-fold activation as compared with 15-fold by rosiglitazone for PPARγ and 5-fold as compared with 2-fold by WY14643 for PPARα). All compounds were used at 10 μM concentration.

image

Figure 2. (A) Activation of PPARα by DRF 2655 and WY14643. HEK 293 cells were transfected with Gal4-PPARα-LBD, pGL2(Gal4 × 5)-SV40-Luc reporter construct and PAdVantage. Luciferase activity was plotted as fold activation relative to untreated cells. Values are an average of three experiments conducted in triplicate. (B) Activation of PPARγ by DRF 2655 and rosiglitazone. HEK 293 cells were transfected with Gal4-PPARγ1-LBD, pGL2(Gal4 × 5)-SV40-Luc reporter construct and PAdVantage. Luciferase activity was plotted as fold activation relative to untreated cells. Values are an average of three experiments conducted in triplicate.

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Induction of adipogenesis is a well-documented response of PPARγ agonists both in vitro and in vivo. 3T3L1 preadipocytes were differentiated to adipocytes in the presence of DRF 2655 following standard protocol (35)(36). The compound showed 369% increase (at 1 μM) in TG accumulation as compared with untreated cells (58.95 ± 0.85 vs. 12.58 ± 0.92 μg TG/mg protein), whereas in rosiglitazone-treated cells the TG level increased to 471% as compared with untreated cells (71.86 ± 4.08 vs. 12.58 ± 0.92 μg TG/mg protein) at a maximum concentration of 1 μM.

Effects in Zucker fa/fa Rats and db/db and ob/ob Mice

Zucker fa/fa rats treated with DRF 2655 at a 1 mg/kg dose showed significant decrease in TG (49.8 ± 1.6 vs. 365.27 ± 33.10 mg/dL of vehicle-treated control group), TC (147.56 ± 6.67 vs. 203.12 ± 6.45 mg/dL of control), FFA (0.142 ± 0.04 vs. 0.369 ± 0.06 mM/L of control), leptin (14.13 ± 2.6 μg/mL vs. 37 ± 3.8 μg/mL of control), and insulin (99.2 ± 3.8 μg/mL vs. 1739.8 ± 88.7 μg/mL of control), and no change in PG levels (120.3 ± 4.1 mg/dL vs. 121.8 ± 8.19 mg/dL of control) after 6 days of treatment (Table 1). Rosiglitazone was used at a 3 mg/kg dose for comparison, where the compound showed relatively less effect on these plasma parameters.

Table 1.  Effect of DRF 2655 and rosiglitazone on plasma parameters in Zucker fa/fa rats
Plasma parametersDRF 2655 (1 mg/kg): percent reductionRosiglitazone (3 mg/kg): percent reduction
  • Values are expressed as mean ± SE of percentage reduction (n = 5). Percentage reduction is measured as per the formula in the text.

  • *

    Treatment related effects in plasma parameters are statistically significant as compared with the untreated control (p < 0.05; one-way ANOVA followed by Dunnett's test).

Glucose1 ± 0.11 ± 0.1
Triglyceride85.4 ± 2.44*45.51 ± 11.45*
TC27.25 ± 3.90*No effect
FFA61.40 ± 11.33*57.57 ± 7.84*
Insulin96.40 ± 0.68*79.70 ± 8.24*

Administration of DRF 2655 for 10 days in db/db mice at 0.01, 0.03, 0.1, 1, and 3 mg/kg per dose showed dose-dependent decreases in PG, TG, FFA, and insulin. The ED50 values for PG, TG, FFA, and insulin were 0.25, 0.52, 1.63, and 0.1 mg/kg, respectively, with the maximum effect observed at 1 mg/kg per dose. DRF 2655 showed a significant decrease in the TG content in heart and liver (Table 2), and the levels were comparable with lean controls. Rosiglitazone at 10 mg/kg per dose showed a similar effect in heart, but failed to show any significant effect in liver. In db/db mice, 30 days of treatment with DRF 2655 at 30 mg/kg showed 19% reduction in body weight, whereas rosiglitazone at a similar dose showed a 17% increase in body weight by the 15th day of treatment (Table 3).

Table 2.  Effect of DRF 2655 on tissue TG in db/db mice
GroupHeart (mg TG/g tissue)Liver (mg TG/g tissue)
  • Values are expressed as mean ± SE (n = 5).

  • *

    p < 0.05 as compared with thedb/db control (one-way ANOVA followed by Dunnett's test).

Lean control2.96 ± 0.86*7.40 ± 1.53*
db/db control6.7 ± 0.7115.89 ± 1.43
DRF 2655 (3 mg/kg)3.27 ± 0.69*6.30 ± 1.68*
Rosiglitazone (10 mg/kg)3.27 ± 0.62*13.95 ± 2.29
Table 3.  Effect of DRF 2655 on body weight in db/db mice
Group0 Day30th Day
  • Body weight was measured at the end of the treatment (30 days for DRF 2655 and 15 days for rosiglitazone) and expressed as mean ± SE (n = 5).

  • *

    p < 0.001 as compared with the 0 day value (Student's t test).

Control35.88 ± 0.9738.00 ± 1.88
DRF 2655 (30 mg/kg)36.50 ± 1.6629.75 ± 0.48*
Rosiglitazone (30 mg/kg)40.4 ± 0.9847.2 ± 1.07

In ob/ob mice, administration of DRF 2655 at 10 mg/kg for 30 days showed 12% decrease in body weight (47.8 ± 1.1 g on Day 0 vs. 41.9 ± 1.6 g on the Day 30), whereas rosiglitazone showed an 11% increase in body weight (50 ± 1.3 g on Day 0 vs. 55.6 ± 1.0 on Day 14) after 14 days of treatment. There was also a significant reduction (44%) in epididymal fat weight (3.60 ± 0.10 g for control vs. 2.01 ± 0.11 g for the treated group) in the DRF 2655-treated group.

No significant difference in food intake was observed between the compound- and vehicle-treated groups in any of these animal models.

Effect in Cholesterol-Fed SD Rats and Hamsters

To further substantiate the lipid-lowering effect of DRF 2655 in genetic models, the high fat-fed rat model was used. These animals have increased levels of plasma TG and TC as compared with normal rats. Treatment with DRF 2655 at different concentrations for 3 days significantly reduced plasma TG and TC (Figure 3 A and B). The effect of DRF 2655 was much better than that of fenofibrate (Table 4). Rosiglitazone did not have any effect in this model. Animals treated with DRF 2655 at 3 mg/kg for 6 days when injected with Triton WR 1339 showed almost total inhibition in TG secretion rate (Figure 4A) as compared with control animals (11.0 ± 3.1 vs. 283.9 ± 22.4 mg/dL per hour). After 8 days of treatment when challenged with exogenous lipid, these animals showed 63% improvement in lipid tolerance (Figure 4B) over control animals (area under the curve units: 13, 729 ± 1389 vs. 36, 882 ± 5562). DRF 2655 treatment showed 42% reduction in plasma apoCIII levels (6.1 ± 0.4 vs. 3.5 ± 0.4 mg/dL in treated animals; p < 0.05). DRF 2655-treated rats at 3 mg/kg per dose for 10 days showed a 93% increase in adipose tissue LPL activity (1, 527, 472 ± 235, 501 vs. 790, 508 ± 115, 604 counts per minute/g tissue; p < 0.05) as compared with vehicle-treated animals. No treatment-related change in food consumption was observed.

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Figure 3. Effect of DRF 2655 on plasma triglyceride (A) and TC (B) in high fat-fed rats. Animals were kept on high-fat diet and compound treatment was done at 0.1, 0.3, 1, 3, and 10 mg/kg for 3 days. Values are expressed as mean ± SE (n = 5). *, p < 0.05 as compared with the control group (one-way ANOVA followed by Dunnett's test).

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Table 4.  Comparative effect of DRF 2655 and fenofibrate in high fat-fed rats
 DRF 2655Fenofibrate
Plasma parametersED50 (mg/kg)EmaxED50 (mg/kg)Emax
  1. Measurements were done after 3 days of treatment. ED50 values were calculated according to the regression analysis of the dose-response curve.

Triglyceride0.11 ± 0.0192% at 3 mg12 ± 148% at 60 mg
Total cholesterol0.08 ± 0.00469% at 0.1 mg45 ± 265% at 60 mg
image

Figure 4. Effect of DRF 2655 on hepatic triglyceride secretion (A) and plasma triglyceride (B) clearance in high fat-fed rats. Animals were treated with DRF 2655 at 3 mg/kg for 6 days and injected with Triton WR1339 or 20% intralipid as described in the methods. Values are expressed as mean ± SE (n = 5). *, p < 0.05 as compared with control (Student's t test).

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Golden Syrian hamsters on high-fat diets are an excellent model to assess the body weight-lowering potential of PPARα agonists (13). These animals, when fed high-fat diets, show significant increases in TC, TG, and body weight. Hamsters were treated with DRF 2655 at 10 mg/kg and fenofibrate at 100 mg/kg, respectively, for 15 days. DRF 2655 treatment showed a significant (p < 0.05) decrease in body weight (129.1 ± 3.8 vs. 106.8 ± 4.4 and 127.6 ± 6.02 g for control, DRF 2655, and fenofibrate, respectively), plasma TG, and TC, whereas fenofibrate-treated animals showed only negligible effect (Figure 5). A dose-response study was also performed with DRF 2655 in this model. The compound showed 5%, 12%, 16%, and 21% reduction in the body weight at 0.3, 1, 3, and 10 mg/kg per dose, respectively. No change in food consumption was observed in the treated group as compared with control. Rosiglitazone had no effect in this model.

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Figure 5. Effect of DRF 2655 and fenofibrate on body weight, plasma triglyceride, and TC lowering in high fat-fed hamsters. Hamsters were treated with 10 mg/kg DRF 2655 and 100 mg/kg fenofibrate for 15 days. Animals were maintained on a high-fat diet throughout the treatment period. Values are expressed as mean ± SE (n = 5).

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Effect in SAM and MSG-SAM

The mildly hyperlipidemic SAM were used to ascertain the lipid-lowering effect observed in the genetic models. Treatment (6 days) of SAM with DRF 2655 at 0.3, 1, 3, and 10 mg/kg showed dose-dependent decreases in plasma TG (51%, 72%, 72%, and 86%, respectively) and TC (13%, 19%, 30%, and 46%). A mild decrease in body weight was also observed, although it was not significant. Fenofibrate showed 74% reduction in TG at 300 mg/kg and no effect in TC and body weight. The PPARγ agonist rosiglitazone did not have any effect in this animal model.

Obesity was induced in SAM by chemical lesions to the hypothalamus by a single neonatal administration of monosodium glutamate. These animals showed significant increases in plasma TG, TC, and body weight compared with normal SAM. MSG-SAM when treated with DRF 2655 at 3 mg/kg per dose for 30 days showed significant (p < 0.05) decrease in plasma TG (144.5 ± 11.7 vs. 23.3 ± 7.2 mg/dL), TC (157.5 ± 24.7 vs. 58.80 ± 11.7 mg/dL), and body weight (40 ± 2.70 vs. 26.5 ± 1.19 g). Fenofibrate at 60 mg/kg showed relatively less effect on all these parameters (Figure 6). Interestingly, we did not observe any significant difference in the activity with fenofibrate at 30 and 60 mg/kg (reduction in TG: 56% vs. 57%, TC: 14% vs. 12%, and body weight: 9.25% vs. 9% at 30 and 60 mg/kg per dose, respectively).

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Figure 6. Effect of DRF 2655 and fenofibrate on body weight, plasma triglyceride, and TC lowering in MSG-SAM. Animals were treated with 3 mg/kg DRF 2655 and 60 mg/kg fenofibrate for 30 days. Values are expressed as mean ± SE (n = 5).

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Effect on Liver Enzyme Activities in db/db Mice

db/db mice treated with DRF 2655 at 3 mg/kg for 10 days showed significant inhibition in liver PEPCK (5-fold) and G-6-Ptase (3.6-fold) activity (Table 5). The effect of rosiglitazone at 10 mg/kg on these enzymes was much less (1.77- and 2.2-fold, respectively) as compared with DRF 2655. Fenofibrate at 30 mg/kg per dose showed an inhibitory effect on both these enzymes (2.5- and 1.76-fold, respectively).

Table 5.  Effect of DRF 2655, rosiglitazone, and fenofibrate on PEPCK, G-6-Ptase, CPT1, and CAT activity in db/db mice liver
GroupPEPCK (U/mg protein)G-6-Ptase (U/mg protein)CPT1 (U/mg protein)CAT (U/mg protein)
  • Compound treatment was done for 10 days. Values are expressed as mean ± SE (n = 5).

  • *

    p < 0.05 as compared with the respective control groups (one-way ANOVA followed by Dunnett's test).

Control64.24 ± 3.170.945 ± 0.07817.50 ± 0.6214.76 ± 1.55
DRF 2655 (3 mg/kg)13.01 ± 1.19*0.264 ± 0.018*30.97 ± 0.84*110.66 ± 5.39*
Rosiglitazone (10 mg/kg)36.21 ± 4.26*0.433 ± 0.042*16.43 ± 1.4618.34 ± 1.11
Fenofibrate (30 mg/kg)25.46 ± 2.18*0.537 ± 0.025*33.01 ± 1.11*61.02 ± 4.39*

db/db mice treated with DRF 2655 at 3 mg/kg for 10 days showed a tremendous increase in the CAT and CPT1 enzyme activity (651% and 77%, respectively) as compared with control animals (Table 5). Animals treated with 10 times higher dose of fenofibrate (30 mg/kg) also showed increase in activity (313% and 89%, respectively), whereas rosiglitazone had no significant effect on these enzymes.

Effect on TC and Triglyceride Synthesis in HepG2 Cells

HepG2 cells incubated with [14C] acetate and/or [3H] glycerol in presence of DRF 2655 at 30 μM concentration for 24 hours showed significant inhibition in TC and TG synthesis (Table 6).

Table 6.  Effect of DRF 2655 on cholesterol and triglyceride synthesis in HepG2 cells
GroupCholesterol synthesis (CPM/mg protein)Triglyceride synthesis (CPM/mg protein)
  • Compound treatment was done for 24 hours. Values are expressed as mean ± SE (n = 5).

  • *

    p < 0.05 as compared with the control (Student's t test).

Control64, 724 ± 1, 5648695 ± 351
DRF 2655 (30 μM)51, 025 ± 4, 646*5836 ± 218*

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Obesity continues to be a growing problem worldwide with no suitable treatment options without side effects. The PPAR family of nuclear receptors has been the subject of intense investigation during the last decade because of its fundamental role in regulating energy balance. We initiated a project to discover small organic molecules that can activate both PPARα and PPARγ receptors and modulate lipid and glucose metabolism, resulting in reduction of body weight. DRF 2655, a novel alkoxy propanoic acid analogue, is a coligand of both PPARα and PPARγ. This study reports the body weight-lowering, hypolipidemic, and euglycemic potential of DRF 2655 in different insulin-resistant animal models of obesity and noninsulin-resistant models of dyslipidemia.

In a cell-based transactivation assay, DRF 2655 significantly activated both PPARα and PPARγ. Although the fibrates in clinical use are agonists of PPARα, they are weak ligands of the receptor. Therefore, in our study, for comparison, a more potent PPARα ligand, WY14643, was used (10). Rosiglitazone, the most potent PPARγ ligand in the market (37)(38), was used as standard for the PPARγ transactivation assay. The results indicate that DRF 2655 was severalfold more potent than WY14643 in activating PPARα, whereas rosiglitazone was ∼10-fold more potent than DRF 2655 in activating PPARγ. When EC50 values were compared, DRF 2655 was found to be more active toward PPARα. It is well established that the PPARγ activators like TZDs and prostaglandins enhance adipocyte differentiation (39)(40). Although treatment of 3T3L1 cells with DRF 2655 resulted in differentiation of preadipocytes to adipocytes, it was less than that observed with rosiglitazone dosed at the same concentration (1μM), which correlates very well with the PPARγ transactivation results.

Obese Zucker fa/fa rats exhibit pronounced hyperinsulinemia, hyperlipidemia, and normoglycemia (41). In these animals, DRF 2655 showed significant (p < 0.05) reductions in plasma insulin, TG, TC, FFA, and leptin, and the data suggest that the compound is 3-fold more potent than rosiglitazone. Although Zucker fa/fa rat is a good model for studying body weight changes (42), no change in body weight was observed in both the treated groups because the animals were kept on normal diets and the period of treatment was very small. To understand the body weight-lowering effect, db/db and ob/ob mice were treated with DRF 2655 for a longer period of time. The db/db mice exhibited an initial phase of hyperinsulinemia, hyperphagia, and obesity. They progressively developed insulinopenia with age, a feature commonly observed in the late stage of type 2 diabetes (43). When administered orally to db/db mice, DRF 2655 showed a significant decrease in body weight. In contrast, rosiglitazone, a potent PPARγ agonist, at a similar dose, showed an increase in body weight. The compound also alleviated insulin resistance in this model. ob/ob mice are characterized by overt hyperinsulinemia, obesity, hyperphagia, severe insulin resistance, and impaired glucose tolerance that are evident before the development of hyperglycemia. The pathological status of ob/ob mice resembles the early stage of type 2 diabetes (44). As in the db/db mice, long-term treatment with DRF 2655 resulted in decreases in body weight and epididymal fat pad size in the ob/ob model. Rosiglitazone showed an increase in body weight in this model. These results in different animal models demonstrate the plasma insulin-, glucose-, lipid-, and body weight-lowering action of the dual PPARα and PPARγ agonist DRF 2655. However, the most interesting finding in these models is the body weight reduction by DRF 2655 that is in direct contrast to the weight gain observed with TZD treatment. This unique body weight-reducing property of DRF 2655 could be due to its potent PPARα activation. It is known that PPARα ligands not only affect apolipoprotein synthesis and secretion, but also increase the catabolism of lipids. These combined effects would lead to the depletion of body fat that will ultimately contribute to body-weight reduction.

DRF 2655 was 12-fold more potent than rosiglitazone (ED50 value = 0.25 vs. 3.2 with rosiglitazone) in its ability to lower PG in db/db mice. However, these results do not correlate well with the PPARγ activation. This anomaly between the in vitro and in vivo potency suggests the possibility of its antidiabetic action being mediated through mechanisms other than PPARγ activation alone (29). Our ex vivo results of DRF 2655 in db/db mice clearly demonstrate severalfold higher inhibition of liver PEPCK and G-6-Ptase activity than rosiglitazone. Although PEPCK is known to have a peroxisome proliferator response element, differential effects of DRF 2655 and rosiglitazone on these two enzymes indicate that it might be independent of PPARγ. The antihyperglycemic effect of DRF 2655 might be through the suppression of hepatic glucose output by inhibition of these two key enzymes in gluconeogenesis. Also, there are several recent observations suggesting an insulin-sensitizing effect of PPARα agonists (11)(45)(46). In our studies, fenofibrate showed significant inhibition in PEPCK and G-6-Ptase activity besides its lipid-lowering activity. The better insulin-sensitizing effect of DRF 2655 compared to rosiglitazone could be due to its combined effect of PPARγ and PPARα.

To substantiate the lipid- and body weight-lowering effect of DRF 2655 in the genetic insulin-resistant models, further studies were conducted in noninsulin-resistant hyperlipidemic models. The lipoprotein profile of the high fat-fed rat model resembles that of hyperlipidemic humans, and this model has been used previously to study the efficacy of fibrates (47). DRF 2655 treatment resulted in significant reduction in plasma TG and TC. The lipid-lowering effect of DRF 2655 was several times better than that of fenofibrate. Rosiglitazone did not have any effect in this model. DRF 2655 was tested for its ability to inhibit lipoprotein secretion and TG clearance in high fat-fed rats. This assay is based on the ability of intravenously injected Triton WR 1339 to prevent the catabolism of TG-rich lipoproteins (48). Thus, after administration of Triton, there is a linear increase in plasma TG levels, which reflects the TG secretion rates from the liver. DRF 2655 almost totally prevented the TG secretion in these animals. When DRF 2655-treated animals were challenged with exogenous lipid, they were able to rapidly clear the plasma TG. PPARα and PPARγ agonists are known to lower serum TG by the inhibition of apoCIII and induction of LPL activity (49)(50) LPL plays an important role in the removal of plasma TG by hydrolyzing the triglycerides of very-low-density lipoprotein and chylomicron particles (51). DRF 2655 treatment resulted in inhibition of plasma apoCIII levels and an increase in the LPL activity in the adipose tissue. Low levels of apoCIII and increased LPL activity might be responsible for the rapid clearance of circulating TG. Hamsters on a high-fat diet show an increase in body weight, TG, and TC, and its lipoprotein profile is considered to be comparable with that of humans (52). Treatment with DRF 2655 brought about significant reduction in body weight and these plasma parameters. In a moderately hyperlipidemic, normoglycemic substrain of SAM kept on normal diets, DRF 2655 showed significant reductions in TG and TC and a slight decrease in body weight. The nonsignificant decrease in body weight in SAM could be due to their small size and because they were on normal diets.

DRF 2655 exhibited PPARα transactivation potential, and this correlates well with its lipid lowering effect in genetic, high fat-fed, and normal animals. PPARα agonists increase the expression of peroxisomal and mitochondrial β-oxidation enzymes, in particular, CAT, CPT1, acyl-coenzyme A (CoA) oxidase, and several other related enzymes in hepatocellular compartments in rodents (53)(54). In humans, these agonists do not induce peroxisomal β-oxidation, but affect the fatty acid uptake, conversion, and catabolism through the mitochondrial β-oxidation pathway. Long-chain acyl-CoA/FFAs will not penetrate mitochondria and become oxidized unless they form acylcarnitines. CPT1 present in the mitochondrial membrane converts long-chain acyl-CoA to acylcarnitine, which is able to penetrate mitochondria and gain access to β-oxidation. Carnitine acts as an interorganellar shuttle for acetyl Co-A. Because CPT1 and CAT are the two major enzymes involved in mitochondrial β-oxidation, we studied the effect of DRF 2655 on them. The increased activity of CPT1 and CAT in the presence of the dual PPARα and γ agonist DRF 2655 in the liver is suggestive of its role in fatty acid uptake and catabolism in mitochondria.

DRF 2655 showed significant body weight-lowering effect in insulin-resistant and noninsulin-resistant animal models. Although MSG-SAM is an induced model of inappropriate hypothalamic function and abnormal feeding behavior, the treatment did not result in any change in feeding behavior in this model or any other models. In a preliminary experiment in fasted rats, DRF 2655 administered through an intracerebroventricular route failed to show any significant change in feeding behavior as compared with controls (data not shown). Because no change in feeding behavior was observed between control and treated groups in all the experiments, likelihood of the involvement of the neuronal mediation in body-weight lowering is less, although the possibility may not be completely ruled out. The increased hepatic oxidation of fatty acids by PPARα agonists would result in increased fatty acid flux from peripheral tissues to the liver, which would further result in decreased fatty acid synthesis and lowered delivery of TG to the peripheral tissues (11). DRF 2655-treated db/db mice showed significant reduction of TG content in liver and heart. The reduction in liver TG could be mediated through PPARα, mainly by the induction of target enzymes involved in hepatic peroxisomal and mitochondrial β-oxidation of fatty acids, thereby leading to an increase in resting metabolic rate and body weight reduction.

To understand the mechanism of lipid lowering further, studies were conducted in isolated cell lines. HepG2 cells treated with DRF 2655 showed significant decreases in TC and TG synthesis from its precursor acetate and glycerol, giving an indication that the lipid-lowering effect could be due to decreased synthesis of these lipids in the liver. The lipid- and body weight-lowering effect of DRF 2655 could be a combined effect of increased activity of β-oxidation enzymes, decreased synthesis, assembly and secretion of triglyceride from the liver and also increased clearance of the secreted triglyceride particles from the systemic circulation.

Overall, our study clearly indicates that DRF 2655, a potent coactivator of PPARα and PPARγ, shows excellent hypolipidemic and euglycemic activity in different animal models and shows great potential as a single agent capable of better overall management of metabolic syndrome. The additional exciting finding is the body weight-lowering potential of DRF 2655, which might be mediated by the induction of target enzymes involved in the hepatic lipid catabolism through PPARα activation. Because obesity is a common problem associated with insulin resistance and type 2 diabetes, we believe that the unique profile of DRF 2655 will make it a drug of choice for the treatment of metabolic syndrome.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The authors are thankful to the management of Dr. Reddy's Group for encouragement and funding this research. We are thankful to Novo Nordisk (Bagsvaerd, Denmark) for providing the PPAR constructs. This is DRF publication number 222.

Footnotes
  • 1

    Nonstandard abbreviations: PPARα, peroxisome proliferator-activated receptor α PPARγ, peroxisome proliferator-activated receptor gamma; TZD, thiazolidinedione; SAM, Swiss albino mice; MSG-SAM, monosodium glutamate-injected obese Swiss albino mice; SD, Sprague Dawley; TG, tissue triglyceride; DMEM, Dullbecco's Modified Eagle's medium; FBS, fetal bovine serum; DFBS, delipidated fetal bovine serum; DMSO, dimethyl sulfoxide; PEPCK, phosphoenolpyruvate carboxykinase; G-6-Ptase, glucose 6-Phosphatase; CPT, carnitine palmitoyltransferase; CAT, carnitine acetyltransferase; LPL, lipoprotein lipase; TC, total cholesterol; PG, plasma glucose; FFA, free fatty acid; CoA, coenzyme A; ED50, 50% effective dose.

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  6. Discussion
  7. Acknowledgment
  8. References
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