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Endocrinology Vol. 147, No. 9 4252-4262
Copyright © 2006 by The Endocrine Society

Peroxisome Proliferator-Activated Receptor (PPAR)-{alpha} Agonism Prevents the Onset of Type 2 Diabetes in Zucker Diabetic Fatty Rats: A Comparison with PPAR{gamma} Agonism

Raynald Bergeron, Jun Yao, John W. Woods, Emanuel I. Zycband, Cherrie Liu, Zhihua Li, Alan Adams, Joel P. Berger, Bei B. Zhang, David E. Moller and Thomas W. Doebber

Departments of Metabolic Disorders (R.B., J.Y., C.L., Z.L., J.B.P., B.B.Z., D.E.M., T.W.D.), Immunology (J.W.W., E.I.Z.), and Medicinal Chemistry (A.A.), Merck Research Laboratories, Rahway, New Jersey 07065

Address all correspondence and requests for reprints to: Raynald Bergeron, Ph.D., Professeur agrégé, Département de kinésiologie, Université de Montréal, C.P. 6128, Montréal, Québec, Canada H3C 3J7. E-mail: raynald.bergeron{at}umontreal.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptor (PPAR)-{gamma} agonists are insulin sensitizers, whereas PPAR{alpha} agonists are lipid-lowering agents in humans. Chronic treatment with PPAR{gamma} agonists has been shown to prevent the onset of diabetes in young Zucker diabetic fatty (ZDF) rats; however, the effects of PPAR{alpha} agonists have not been well characterized in this model. Here we investigated chronic efficacy of PPAR{alpha} and nonthiazolidinedione (nTZD) PPAR{gamma} agonists on the onset of diabetes in 6-wk-old male ZDF rats. Whereas treatment with the nTZD PPAR{gamma} agonist completely prevented development of hyperglycemia, PPAR{alpha} activation was associated with lowering of food intake and body weight and reductions in fed and fasting hyperglycemia, with prevention of the hyperinsulinemic peak preceding the development of hyperglycemia in ZDF rats. Both compounds improved glucose tolerance during an oral glucose tolerance test with concomitant increases in insulin response. Such improvements of insulin secretion were associated with increased islet to total pancreatic area ratio and pancreatic insulin contents. Hyperinsulinemic-euglycemic clamp studies demonstrated that nTZD PPAR{gamma} reduced basal endogenous glucose production and increased insulin-stimulated glucose disposal, consistent with an improved insulin action as a cause of the improved glucose homeostasis. In contrast, activation of PPAR{alpha} did not significantly improve glucose metabolism during the hyperinsulinemic-euglycemic clamp. In conclusion, chronic treatment of ZDF rats with a PPAR{gamma} agonist completely prevented the onset of diabetes by improving both insulin action and secretion, whereas PPAR{alpha} agonism was partially effective, primarily by improving the pancreatic islet insulin response. Unlike the PPAR{gamma} agonist, the PPAR{alpha} agonist demonstrated efficacy without inducing body weight gain and cardiomegaly. This study suggests a possible role for PPAR{alpha} agonists in the prevention of type 2 diabetes mellitus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME PROLIFERATOR-activated receptor (PPAR)-{gamma} agonists have the ability to augment insulin-stimulated adipose tissue uptake of free fatty acids, thereby diminishing the muscle and liver exposure to circulating nonesterified fatty acids (NEFAs), which in turn reduces steatosis, a likely cause of insulin resistance (1, 2). In addition, PPAR{gamma} ligands may modulate insulin sensitivity through their effects on cytokine and adipokine release such as TNF{alpha}, IL-6, resistin, and adiponectin (3, 4, 5). PPAR{gamma} agonists such as rosiglitazone and pioglitazone are effectively used in the clinic to treat type 2 diabetes mellitus (T2DM). In contrast, the scientific literature on the effects of PPAR{alpha} agonists on glycemic control in diabetics is inconclusive. On the one hand, the recent Fenofibrate Investigation and Event Lowering in Diabetes (FIELD) study, a cardiovascular disease outcome trial, did not support a beneficial role for fenofibrate on T2DM (6). On the other hand, there are clinical reports supporting beneficial effects of fibrates such as bezafibrate, clofibrate, and gemfibrozil (weak PPAR{alpha} agonists) on glucose control in type 2 diabetics, although antihyperglycemic effects of these drugs are rather mild and interpretation of the data may be misled by the lack of PPAR{alpha} selectivity or potency of some of these fibrates (7, 8). A number of previously published studies (7, 9, 10, 11) also point to an improvement in insulin sensitivity as a result of fibrate treatment in humans, although information on the mode of action of fibrates on glucose metabolism in humans is scarce. On the other hand, there is preclinical data from rodent studies showing that potent selective PPAR{alpha} agonists can ameliorate hyperglycemia through enhanced insulin action (12, 13). Improvement of insulin sensitivity by PPAR{alpha} agonists likely involves their ability to enhance hepatic expression of genes implicated in the transport and oxidation of fatty acids (13, 14, 15). Accordingly, PPAR{alpha} ligands have been shown to reduce liver as well as muscle steatosis in rodent models of insulin resistance (12, 16, 17).

Insulin sensitization by dual PPAR{alpha}/{gamma} agonists has been reported recently in obese nondiabetic subjects (18, 19). Activation of both PPAR{alpha} and PPAR{gamma} has been associated with the improvement of insulin action, including decreased liver and muscle lipid contents coupled with enhanced insulin-induced suppression of endogenous glucose production (EGP) and stimulation of muscle glucose disposal (20, 21).

Although the predominant antihyperglycemic effects of PPAR ligands may be a consequence of the amelioration of insulin resistance in adipocytes, liver, and muscle, there is growing evidence supporting a role for PPAR{gamma} agonists in the regulation of pancreatic function in T2DM (22, 23). Beneficial effects of PPAR{gamma} agonist treatment on pancreatic islet morphology in rodents have been demonstrated (24, 25). Recently it was shown that PPAR{alpha} activation plays a role in insulin secretion in isolated islets (26, 27, 28). However, the effects of PPAR{alpha} agonists on islet function are not clearly known to ameliorate T2DM. Moreover, the effects of PPAR{alpha} in the context of the transition from normal glycemia to overt T2DM are not known and have been proposed to have a preventative role in the progression of the disease (29).

With the current rise in the incidence of T2DM in industrialized countries, there is a growing need for early intervention to delay the progression of the disease. PPAR{gamma} agonists have been shown to slow or halt the onset of diabetes in rodent models (30, 31); there is also evidence suggesting that a PPAR{alpha}/{gamma} dual agonist may have similar beneficial effects (32). In contrast, little is known about the potential prophylactic effect of PPAR{alpha} agonists on the development of T2DM. The results of a 6.2-yr follow-up study suggested that PPAR{alpha} agonists may prevent T2DM. However, bezafibrate, the compound used in that study, has not only weak PPAR{alpha} activity but also significant activity on other PPAR isoforms as well (33). The present study aimed at determining the effects and mode of action of a novel PPAR{alpha} ligand on the onset of diabetes in ZDF rats in comparison with the effects of a novel nTZD PPAR{gamma} agonist.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and experimental protocol
Five-week-old male Zucker diabetic fatty (ZDF; Charles River Laboratories, Raleigh, NC) and their lean littermates were housed three per cage and fed Purina rat chow 5008 (protein: 26.8%; fat: 16.7%; carbohydrates: 56.5%, kilocalories per volume) and tap water ad libitum for 7 d after they were received in our laboratory. The 12-h light, 12-h dark cycle started at 0700 h, and the room was maintained at 20–23 C and 40–70% humidity. One week after their arrival, rats were treated orally once a day with a potent PPAR{alpha} selective agonist (introduced as Compound 10 in Ref. 34), a nonthiazolidinedione (nTZD) PPAR{gamma} selective agonist (introduced as COOH in Ref. 35) (Table 1Go), or vehicle (0.5% methylcellulose) for 9 wk (n = 12/group). The PPAR{alpha} agonist used in the present experiment was previously shown to achieve dose-dependent reversal of hyperglycemia [91% correction with 30 mg/kg (34)] over an 11-d once-daily treatment of db/db mice (model described in Ref. 36). Other known PPAR{alpha} agonists achieved little or no efficacy in this same db/db mice model [Wy-14,643: ~20% correction at 30 mg/kg, 20-fold less potent than the present PPAR{alpha} agonist in transactivation assay (human{alpha} TA inflection point): respectively, 300 vs. 15 nM; and Fenofibrate: 17% correction at 150 mg/kg]. Comparatively, our nTZD PPAR{gamma} agonist at a dose of 10 mg/kg q.d. administered orally corrected hyperglycemia by 80%, whereas 10 mg/kg rosiglitazone corrected it by approximately 60% (our unpublished data).


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  		TABLE 1.
 
Body weight and food intake were measured twice a week. Tail blood was sampled once a week in the morning in the postprandial state for determination of plasma glucose, insulin, NEFAs, and triglycerides. Plasma fructosamine concentrations were also determined on wk 3, 5, and 8 of treatment. Overnight fasted plasma glucose, insulin, and ß-hydroxybutyrate concentrations were determined after 8 wk of treatment. On that same day, an oral glucose tolerance test (OGTT) was performed. The last dose of agonist given before the OGTT was administered approximately 24 h before the glucose load. On the following day, seven of 12 animals from each group were subjected to surgery aimed at implanting chronic jugular vein and carotid artery catheters as previously described (37). Rats were allowed to recover for a period of 4–6 d during which body weight and overall appearance were monitored daily. Drug treatment was continued during the entire recovery from the surgery. The other five rats per group were killed after 9 wk of treatment using carbon dioxide. The pancreas was carefully excised. Approximately two thirds of the whole pancreas was carefully spread out into a thin sheet and immediately frozen in tissue freezing medium (EMS, Hatfield, PA) filled tissue molds for further microscopic immunohistochemical analysis. The last third of the pancreas was used to determine insulin content. Insulin content was then calculated as picomoles per milligram tissue. Liver, epididymal white adipose tissue (EWAT), and heart were dissected and weighed. Gastrocnemius muscle was also collected and frozen for further triglyceride content assessment as described earlier (38).

Hyperinsulinemic euglycemic clamp
On the day before the experiment, rats had their food withdrawn around 1800 h. A 120-min primed continuous infusion of [3-3H]glucose (NEN Life Science Products, Boston, MA) was initiated for assessment of glucose turnover rate. The infusion rate of the tracer was doubled during the 120-min hyperinsulinemic euglycemic clamp during which insulin was infused at a rate of 25 mU/kg·min. Arterial blood was sampled initially every 5 min during the first 30 min and every 10 min thereafter. Plasma glucose-specific activity used in the determination of whole-body glucose turnover was assessed from blood samples taken every 10 min over the last 30 min of both the basal and hyperinsulinemic periods. Simultaneously a 25% glucose solution was infused at a variable rate to maintain euglycemia. At the end of the hyperinsulinemic phase, a larger blood sample was taken for determination of plasma glucose and insulin concentrations. Immediately afterward, rats were anesthetized with an iv injection of ketamine/xylazine and killed by pneumothorax and exsanguination. Plasma samples were stored at –80 C until analyses were performed. The protocol of study was reviewed and approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.

Immunohistochemical pancreatic analysis
The sheet of pancreatic tissue was cut en face to yield the largest possible area and hence the best representation of the original tissue. Cryostat sections (7 µm) were cut on a Reichert-Jung Cryocut 1800 equipped with a cryostat sectioning aid (Instrumedics, Inc., Hackensack, NJ) and mounted on adhesive-coated slides (also from Instrumedics). Before immunolabeling, the sections were first fixed for 30 min in 2% paraformaldehyde in 0.01 M phosphate buffer (pH 7.3), washed, and treated sequentially with 3% hydrogen peroxide, citrate-based antigen retrieval solution (Biogenex, San Ramon, CA), 0.1% Triton X-100, and TSA blocking solution (PerkinElmer, Boston MA). Cryostat sections were then immunolabeled with a mixture of antiglucagon (Biogenex) antiinsulin (Dako, Carpinteria CA), and antisomatostatin (Dako). The primary antibodies were visualized using the TSA-CY3 immunodetection system following the vendor’s instructions (PerkinElmer). All antibody incubations and washes were facilitated using the Shandon cover plate system (Thermo Electron Corp., Pittsburgh PA). The penultimate wash also contained 0.005% 4',6'-diamino-2-phenylindole (DAPI) to facilitate visualization of cell nuclei in both the endocrine and exocrine pancreas. After a final wash, coverslips were mounted using PermaFluor aqueous mounting media (ThermoElectron). The resultant fluorescently labeled slides were examined and digital images captured using a Zeiss Axioplan II microscope equipped with a Slidebook Image analysis workstation (Intelligent Imaging Innovations, Denver, CO). The calculation of relative islet area was facilitated by using Slidebook to construct a montage of CY3 and DAPI digital images, which included the entire area of the section (~2 cm2). Using the Slidebook’s software threshold function, a mask was constructed that corresponded to the CY3-labeled islets. A separate mask, corresponding to the total section area, was drawn by an operator using the DAPI-labeled nuclei as a guide. The areas of the resultant masks were then calculated by the Slidebook software and exported to an Excel spreadsheet for statistical analysis. By first flattening the pancreatic tissue before freezing and then cutting the sections en face, very large sections from each pancreas were obtained, thereby eliminating the need to cut, label, and analyze multiple sections from each animal. In these experiments tissue was collected from five animals per treatment group and one section per animal was immunolabeled and analyzed.

Analytical methods
During the clamp studies, glucose concentrations were measured with an automatic glucose analyzer (model 2300; Yellow Springs Instruments, Yellow Springs, OH) using the glucose oxidase method. Weekly plasma glucose (Wako Chemicals USA, Richmond, VA), NEFAs and triglycerides (both kits from Roche Diagnostics, Mannheim, Germany), ß-hydroxybutyrate, and fructosamine (both kits from Sigma Diagnostics, St. Louis, MO) concentrations were measured with commercially available colorimetric assays. Assay protocols were adapted for use in a microtiter plate format. Insulin was determined by ELISA (ALPCO, Windham, NH) and glucagon by RIA (Linco Research, St. Charles, MO). Biochemical assay of pancreatic insulin content was performed by homogenizing approximately 100 mg of tissue in 1 ml of acid alcohol (0.1 N HCl in 70% ethanol alcohol) using a Polytron. After centrifugation, the supernatant was frozen for 48 h and subjected to homogenization, centrifugation, and freezing three more times. The four fractions of supernatant were pooled and used to determine insulin content by ELISA.

Plasma [3-3H]glucose concentration was determined by deproteinizing plasma by Ba(OH)2 and ZnSO4 precipitation. Samples were dried overnight in a vacuum oven (30 C) to eliminate [3H]H2O resulting from glycolysis and radioactivity determined using a scintillation counter. Glucose turnover rates were calculated based on radio dilution technique using the formula for non-steady-state conditions developed by Steele (39) and validated by Radziuk et al. (40). EGP was estimated by subtracting the glucose infusion rate (mean of the last 30 min of the hyperinsulinemic period) from the tracer calculated rate of glucose disposal.

Statistical analyses
All data are reported as mean ± SEM. Data from blood sampling and substrate turnover measurements were analyzed by a two-way ANOVA for repeated measures. These analyses were followed by Tukey test for post hoc comparisons. Data obtained from tissue samples were analyzed by Student’s t test. Differences were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Food intake and whole-body and tissue weights are differentially affected by PPAR{alpha} and PPAR{gamma} agonists
During the 8-wk chronic study, ZDF rats treated with the PPAR{alpha} agonist manifested significantly attenuated body weight gain (by 14%), compared with ZDF rats treated with the vehicle (Fig. 1AGo). In contrast, the nTZD PPAR{gamma} agonist caused a significant 70% increase in body weight in the same study. These changes in body weight were parallel to changes in food intake (Fig. 1BGo). The mean food intake was significantly increased in the nTZD PPAR{gamma} agonist-treated group by 41% over ZDF vehicle, whereas PPAR{alpha} agonist treatment caused an approximately 20% diminution of food intake (Fig. 1BGo). EWAT mass was insignificantly reduced by 22% by the PPAR{alpha} agonist (P = 0.098) treatment, compared with ZDF vehicle-treated rats but markedly increased by approximately 240% by the nTZD PPAR{gamma} agonist treatment (Table 2Go). The latter treatment was also associated with a 30% increase in cardiac mass, whereas PPAR{alpha} activation resulted in significantly smaller heart mass, compared with the ZDF vehicle group. Liver mass was significantly increased in the ZDF vehicle rats, compared with the lean animals. As expected, PPAR{alpha} agonist treatment caused a significant elevation of liver mass, a result of peroxisome proliferation induction that is consistently observed with PPAR{alpha} agonists in rodents. The nTZD PPAR{gamma} agonist treatment did not change liver mass, compared with vehicle-treated ZDF rats.


Figure 1
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  		FIG. 1. The PPAR{alpha} agonist reduces whereas nTZD PPAR{gamma} agonist treatment causes increases in food intake and body weight gain. Body weight (A) and food intake (B) were recorded in the morning postprandial state biweekly from d 4 to 56 of treatment. Data are reported as means ± SEM (n = 11–12/group). *, P < 0.01 vs. all three ZDF rats groups; §, P < 0.05, §§, P < 0.005 vs. ZDF vehicle group. Statistical differences determined by two-way ANOVA for repeated measures (time * treatment effect).

 

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  		TABLE 2. Anthropometric data over the course of the PPAR agonist treatments

 
PPAR{alpha} agonist blunts the onset of hyperglycemia
Postprandial plasma glucose concentrations in all ZDF rats (before treatment) at 6 wk old were no different from the glucose observed in lean rats of the same age (Fig. 2AGo). In contrast, postprandial plasma insulin levels ranged from 6.3 to 14.0 ng/ml in the ZDF groups before the start of the treatment at wk 0 and were all significantly higher than those in the lean rats (Fig 2BGo). Plasma glucose levels increased steadily in the ZDF vehicle group to become overtly diabetic (~400 mg/dl) by wk 3 of the study when the rats were 9 wk old (Fig. 2AGo). The rise in postprandial glycemia was significantly blunted in PPAR{alpha} agonist-treated rats in which glycemia reached a plateau at approximately 200–250 mg/dl, corresponding to a 67% correction of hyperglycemia. The nTZD PPAR{gamma} agonist treatment completely prevented the rise of glycemia throughout the study. Overnight fasting plasma glucose levels (Table 3Go) measured after 8 wk of treatment were in line with postprandial glycemia. Furthermore, fructosamine, which has been used clinically as an alternative to hemoglobin A1c levels as an index of long-term glucose control (41), was also measured. The PPAR{alpha} agonist treatment corrected plasma fructosamine by 39%, compared with the vehicle group (Table 2Go), whereas the nTZD PPAR{gamma} agonist treatment completely normalized plasma fructosamine to levels seen in the lean control group.


Figure 2
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  		FIG. 2. The PPAR agonists delay the onset of hyperglycemia in ZDF rats. Time course of postprandial plasma glucose (A) and insulin (B) concentrations determined weekly during either PPAR{alpha} or PPAR{gamma} agonist treatment. Values are reported as means ± SEM (n = 11–12/group). *, P < 0.01 vs. all three ZDF rats groups; £, P < 0.05 vs. lean vehicle group; §, P < 0.01 vs. ZDF vehicle group. Statistical differences determined by two-way ANOVA for repeated measures (time * treatment effect).

 

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  		TABLE 3. Fasting plasma parameters

 
Plasma insulin concentrations peaked approximately 2 wk after the initiation of the study in the ZDF vehicle group (Fig. 2BGo). As insulin resistance worsens, the pancreas fails to compensate resulting in diminished insulin levels (Fig 2BGo) and hyperglycemia ensues. In contrast, treatment with either PPAR agonist prevented the large increase in plasma insulin levels seen in the vehicle-treated group at approximately 2–3 wk of treatment and maintained plasma insulin concentrations close to those observed at the initiation of the study. Most notably, nTZD PPAR{gamma} agonist caused a decrease in postprandial plasma insulin within 1 wk of treatment. Plasma insulin concentration in both PPAR agonist-treated groups remained at higher levels than those in the lean-vehicle group. Elevated fasting insulin concentrations typically found in ZDF rats were not significantly different among the various groups when measured after 8 wk of treatment when rats were 14 wk of age (Table 3Go).

Plasma NEFA concentrations were approximately doubled in the ZDF vehicle rats, compared with those observed in the lean rats (P < 0.05) (Table 2Go). PPAR{alpha} agonist treatment did not consistently alter NEFA levels, whereas the nTZD PPAR{gamma} agonist significantly decreased plasma NEFAs to concentrations below those observed in the lean rats. Plasma triglyceride levels were elevated (Table 2Go) in all ZDF groups before the initiation of treatments, compared with levels found in the lean rats. Triglyceride concentrations rose progressively with time in the ZDF vehicle group, reaching concentrations that were approximately 5-fold higher than concentrations seen in the lean-vehicle group. PPAR{alpha} agonist treatment significantly decreased triglyceride by 30–45% after 5 wk of treatment, and the effect was sustained until the end of the study. The nTZD PPAR{gamma} agonist completely normalized hypertriglyceridemia after only 1 wk of treatment, an effect that was sustained for the duration of the treatment. Fasting concentrations of ß-hydroxybutyrate, which is a product of hepatic fatty acid oxidation, were increased 6- to 7-fold in the ZDF rats, compared with lean littermates (Table 3Go). The PPAR{alpha} agonist increased ß-hydroxybutyrate concentrations further by 144%. In contrast, the nTZD PPAR{gamma} agonist lowered the ketone body concentrations to a level that was 60% below that observed in the ZDF vehicle group.

OGTT and pancreatic morphology are improved by PPAR{alpha} agonism
Glucose tolerance was impaired in the ZDF rats, compared with their lean littermates (Fig. 3AGo). PPAR{alpha} agonist significantly improved glucose tolerance, leading to a 55% correction of the plasma glucose area under the curve (AUC) (Fig. 3BGo). The nTZD PPAR{gamma} corrected glucose excursion by 89%. In addition, the decreased insulin secretion during the OGTT seen in the ZDF vehicle group, as determined by the AUC above basal levels, was ameliorated by PPAR{alpha} agonist treatment as well as the nTZD PPAR{gamma} agonist (Fig. 3DGo). This improvement of the insulin response is supported by morphological analysis of the pancreatic islets performed by immunohistochemistry. (Fig. 4Go). Using a cocktail of antiinsulin, antiglucagon, and antisomatostatin antibodies, we were able to immunolabel the pancreatic islets to determine the percent of the pancreatic area occupied by islets. Both PPAR{alpha} and PPAR{gamma} agonist-treated groups showed greater islet/pancreas relative area, compared with both lean and ZDF vehicle groups (Fig. 5AGo). In addition, postprandial pancreatic insulin content measured biochemically from tissue sampled after 8 wk of treatment was significantly diminished in the ZDF vehicle, compared with the lean vehicle, group. The nTZD PPAR{gamma} agonist treatment significantly elevated pancreatic insulin content, compared with ZDF vehicle group, also suggesting the preservation of functional ß-cells by nTZD PPAR{gamma} (Fig. 5BGo). There was a trend for improvement of the pancreatic insulin content in PPAR{alpha} agonist-treated, compared with the ZDF vehicle, group (P = 0.090).


Figure 3
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  		FIG. 3. Glucose tolerance and insulin response during OGTT are improved by PPAR{alpha} and PPAR{gamma} agonist treatments of ZDF rats. Plasma glucose concentrations (A) and AUC (B) and insulin concentrations (C) and AUC above baseline (D) in lean and ZDF rats fasted overnight before a 2 g/kg OGTT. Values are reported as means ± SEM (n = 11–12 in each group). *, P < 0.05; **, P < 0.005 vs. lean vehicle group; §, P < 0.05; §§, P < 0.005 vs. ZDF vehicle group.

 

Figure 4
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  		FIG. 4. Immunohistochemical micrograph of ZDF rat pancreatic islets. Pancreas from ZDF rats treated for 9 wk with vehicle, PPAR{alpha}, or nTZD PPAR{gamma} agonists as well as pancreas from lean vehicle animals were immunolabeled with a mixture of antiinsulin, antiglucagon, and antisomatostatin. All three primary antibodies were visualized with a CY3 (red) fluor. The overall section area (delineated in blue) was highlighted by an operator using DAPI-labeled nuclei as a guide. See Materials and Methods for more details. Values are reported as means ± SEM. Magnification, x5; n = 5/group.

 

Figure 5
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  		FIG. 5. Pancreatic insulin content and islet area of ZDF rats. Relative density of islet expressed as percent of total pancreatic longitudinal section (A) and biochemically determined pancreatic insulin content (B). Islet density was determined by image analysis of immunohistochemical micrograph of pancreatic sections. Values are reported as means ± SEM n = 5 per group. *, P < 0.05 vs. lean vehicle; §, P < 0.05 vs. ZDF vehicle.

 
Insulin action during hyperinsulinemic euglycemic clamp is not improved by PPAR{alpha} contrarily to nTZD PPAR{gamma} treatment
To further evaluate the effect of the PPAR ligands on insulin sensitivity, we performed hyperinsulinemic euglycemic clamp studies. Mean plasma glucose over the last 60 min of the clamp period varied between 95 and 99 mg/dl across the groups (N.S.). Plasma insulin levels during a 25 mU/kg·min constant infusion were similar in all of the ZDF groups. Fasting EGP was elevated in the ZDF vehicle, compared with lean vehicle, group (Fig. 6AGo). The nTZD PPAR{gamma} treatment significantly reduced fasting EGP (P < 0.05), compared with ZDF vehicle. PPAR{alpha} agonist treatment, on the other hand, did not decrease fasting EGP. No significant difference in the insulin-suppressed EGP was observed among the various groups, although there was a tendency for the lean vehicle group to have a lower EGP rate during hyperinsulinemia (Fig. 6BGo). Insulin-stimulated glucose disposal was significantly higher in the lean vehicle, compared with the ZDF vehicle, group as expected. Among the two PPAR agonist treatments, only the nTZD PPAR{gamma} agonist significantly improved insulin-stimulated glucose disposal in the ZDF rats at this high rate of insulin infusion (Fig. 6CGo).


Figure 6
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  		FIG. 6. Glucose turnover during hyperinsulinemic-euglycemic clamp of ZDF rats. Hyperinsulinemic-euglycemic clamp studies were performed in overnight fasted rats after 9 wk of treatment. Basal EGP (A), insulin-suppressed EGP (B), and insulin-stimulated glucose disposal (C) were determined using [3-3H]glucose radio dilution technique. Insulin infusion rate was 25 mU/kg·min. All data are expressed in milligrams per kilogram per minute. Values are reported as means ± SEM n = 4–6/group. *, P < 0.05 vs. all three ZDF rats groups; §, P < 0.05 vs. ZDF vehicle.

 
Muscle triglyceride content
Muscle triglyceride contents were significantly increased in the ZDF vehicle group, compared with levels observed in the lean vehicle group (16.7 ± 0.8 vs. 8.5 ± 0.5 mg per 100 g tissue, respectively). Both PPAR{alpha} and nTZD PPAR{gamma} agonists significantly diminished muscle triglyceride contents, compared with levels observed in the ZDF vehicle group (12.7 ± 0.5. and 11.7 ± 1.0, respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PPAR{alpha} agonist treatments in rodents have already been reported to increase insulin sensitivity (12, 13, 17, 42). Here we show that chronic treatment with a novel PPAR{alpha} agonist substantially prevented the onset of diabetes in ZDF rats, in comparison with the complete prevention of the disease onset by a novel nTZD PPAR{gamma} agonist. Our results suggest different effects of the PPAR{alpha} and PPAR{gamma} agonist on body weight, food intake, and other metabolic parameters (see summary in Table 4Go). Both PPAR agonists affected pancreatic insulin responsiveness, whereas only nTZD PPAR{gamma} agonist treatment improved peripheral insulin action. Importantly, the improvement of glucose control with the nTZD PPAR{gamma} agonist was associated with increased body weight gain and cardiomegaly. In contrast, PPAR{alpha} agonist treatment decreased food intake, body weight, and cardiac mass. By extension, these data suggest that PPAR{alpha} activation may be beneficial in delaying or preventing the onset of T2DM in humans, although clinical studies will be necessary.


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  		TABLE 4. Summary of results

 
The magnitude of the diminution in hyperglycemia was smaller in the PPAR{alpha}-treated cohort than the PPAR{gamma} agonist-treated group. Nevertheless, the improvement of glucose control with PPAR{alpha} agonist treatment was substantial as demonstrated by the approximately 67% and approximately 47% correction of fed and fasting plasma glucose, respectively. In the only other diabetes prevention study previously reported (43), another PPAR{alpha} agonist had only a very modest lowering effect on postprandial glucose (glucose lowering of 21 vs. 230 mg/dl in the current study) in Otsuka Long-Evans Tokushima fatty rats, which are mildly diabetic. Therefore, the current findings demonstrate that early therapeutic intervention with a potent PPAR{alpha} agonist considerably attenuates the progression toward diabetes in a robust rodent model of T2DM.

Reductions in food intake and resulting body weight gain alone can result in lowering of hyperglycemia in diabetics. For this reason, a limitation of the present study is the absence of a pair-fed group matching the food intake of the PPAR{alpha} agonist-treated group. The inclusion of a pair-fed group would have allowed to more convincingly demonstrate the glucose lowering effect of the PPAR{alpha} agonist beyond the lowering that could be accounted for by the reductions of food intake and weight loss. Nonetheless, previous studies reporting 14–20% reductions of food intake in pair-fed groups, as in the present PPAR{alpha} agonist-treated rats, showed correction of hyperglycemia in the range of 21–33% (44, 45). Although the present 15% reduction of daily caloric intake in the PPAR{alpha} group accounted for a portion of the 67% correction of hyperglycemia, it is unlikely that it explains all of the antihyperglycemic effects caused by the PPAR{alpha} treatment. In addition, there is evidence supporting that PPAR{alpha}-related reductions of food intake could be mechanism based because hepatic fatty acid oxidation has been shown to have a role in the regulation of food intake via afferent signals mediated to the central nervous system through the hepatic vagus nerve (46). Furthermore, oleoylethanolamide, a high-affinity PPAR{alpha} ligand, suppresses food intake in wild-type but not PPAR{alpha}–/– mice, also supporting a role of PPAR{alpha} in the modulation of food intake (47). However, recent data suggest that this effect of oleoylethanolamide could be in part mediated by a novel G protein-coupled receptor GPR119 (48).

Histological analysis of pancreatic sections revealed that the improvement of glucose control as measured by lowering of fed and fasting plasma glucose and fed fructosamine as well as the improvement of glucose tolerance could be accounted for, at least in part, by the beneficial effects of PPAR{alpha} agonists on pancreatic islets. Indeed, the islets/total pancreatic surface area ratio was increased in PPAR{alpha} and the nTZD PPAR{gamma} agonist-treated animals, compared with the ZDF vehicle-treated group (Fig. 5AGo). These data are better understood in light of the evolution of islet dysfunction during the development of diabetes in the ZDF rats. In the early (prediabetic) stage, pancreatic islets undergo hypertrophy in response to whole-body insulin resistance. As insulin resistance further increases (usually occurs around 7–8 wk of age), the islets eventually become disorganized morphologically (infiltration of vascular cells and collagen into islets and disintegration of normal spherical shape) and severely degranulated (loss of insulin secretory granules) by the time hyperglycemia appears (49, 50). The present data suggest that the natural hypertrophy of islets occurred in the PPAR agonist-treated groups but that the late degeneration of the islets may have been prevented by both PPAR{alpha} and -{gamma} agonist treatments. However, the present data do not address whether proliferation and/or apoptosis of ß-cells were affected by the PPAR agonists treatments.

PPAR{gamma}-induced improvement of pancreatic parameters have been reported in preclinical models. Specifically, ZDF rats treated with rosiglitazone showed higher pancreatic insulin content and better conserved islet morphology (30). In addition, chronic treatment of db/db mice with pioglitazone led to enhanced ß-cell mass (51) and glucose-stimulated insulin secretion, which was also observed after treatment with a dual-PPAR{gamma}/{alpha} agonist. Improvement of pancreatic function in human T2DM subjects after PPAR{gamma} agonist treatment has also been described (22, 23). The mechanism(s) of action leading to the improvement of the pancreatic function in PPAR{gamma} agonist-treated patients is unknown, but one hypothesis put forward is that ß-cell mass is increased (52).

A beneficial role for PPAR{alpha} on insulin secretion and progression of diabetes has been suggested (27, 53). One hypothesis is that PPAR{alpha} activation causing increases in fatty acid oxidation capacity and lowering of islet lipid content, which is elevated in ZDF rats (54), would lead to improved pancreatic insulin response. Such increases in fatty acid oxidation and lowering of triglyceride content in pancreatic islets has been observed in Otsuka Long-Evans Tokushima fatty rats after chronic in vivo treatment with fenofibrate (43). Further supporting this concept, the present study demonstrates that chronic treatment of ZDF rats with a potent PPAR{alpha} agonist-induced improvements of islets/total pancreatic surface area ratio and pancreatic insulin content. These adaptations were associated with improved insulin responsiveness during an OGTT. However, establishment of a direct effect of PPAR{alpha} activation on ß-cells function was not achieved in the present study, nor is this demonstration clearly established in the published scientific literature. Elevation of fatty acid oxidation by PPAR{alpha} activation by adenoviral overexpression of PPAR{alpha} or clofibrate treatment in INS-1 cells has been shown to cause a diminution of glucose-stimulated insulin secretion in vitro (55), data which are in absolute opposition to the findings of Ravnskjaer et al. (26) obtained in INS-1E cells. Moreover, there was no up-regulation of bona fide PPAR{alpha} genes nor activation of fatty acid oxidation in isolated pancreatic islets of ZDF rats submitted to in vitro clofibrate treatment (28). Therefore, the possibility that systemic effect of PPAR{alpha} agonism in vivo may have contributed to the improvement of ß-cell function will need to be further examined. Lipid partitioning between skeletal muscle, adipose tissues, and the islets may be an important component of the antidiabetic effect caused by chronic treatment with a PPAR{alpha} agonist in diabetic models (56).

The nTZD PPAR{gamma} agonist treatment led to a reduction of EGP in overnight-fasted ZDF rats. The data are in line with previous findings using rosiglitazone (31). Lowered basal EGP in the nTZD PPAR{gamma} group was associated with lower plasma fatty acid concentrations, a parameter that is often invoked as a causative factor in the pathogenesis of T2DM. In the present study, the PPAR{alpha} agonist did not significantly affect basal EGP nor did it improve insulin-mediated suppression of EGP, which is in agreement with a previous report (12). In line with this finding, it has been proposed that PPAR{alpha}, through up-regulation of PGC-1{alpha}, may be associated with impaired insulin-mediated suppression of hepatic glucose production through up-regulation of Tribbles 3 (57). However, although not investigated here, the Tribbles 3 pathway is most likely not operative in the present study because regulation of hepatic glucose production by insulin was unchanged by the PPAR{alpha} treatment.

Insulin-stimulated glucose disposal was augmented by the nTZD PPAR{gamma} treatment in the current study as reported previously with TZD PPAR{gamma} agonists (12, 21, 58). In contrast, PPAR{alpha} agonist treatment did not significantly increase insulin-stimulated glucose disposal. This result contrasts with that from a previous study demonstrating that treatment of high-fat diet-fed Wistar rats with the PPAR{alpha} agonist Wy-14,643 for 2 wk improved glucose disposal in a study in which insulin was infused at a low rate, causing plasma insulin concentrations to increase by 2-fold (12), a condition that assesses insulin sensitivity. The present hyperinsulinemic clamp studies were performed at an insulin infusion rate of 25 mU/kg·min, which should provoke a maximal insulin response. We conclude that insulin responsiveness was not modified by PPAR{alpha} agonist treatment. The effects of lower insulin infusion rates would need to be studied to further determine whether insulin sensitivity might have been improved by our PPAR{alpha} activation regimen. Alternatively, muscle-specific overexpression of PPAR{alpha} in mice caused impaired insulin-stimulated muscle glucose uptake in vitro and reduced glucose disposal in vivo (59). However, this transgenic mice line is not a model of diabetes and may therefore not be predictive of what would happen in diabetic patients. Our data clearly demonstrate that the pharmacological administration of a selective and potent PPAR{alpha} synthetic ligand has the net effect of significantly reducing hyperglycemia.

Data from several studies performed in rodents suggest that increasing fatty acid oxidation in key insulin-sensitive tissues, such as liver and muscle, may be the predominant effect by which PPAR{alpha} agonists improve glucose metabolism. The ability of such ligands to reduce triglycerides and long chain acyl-CoA content in these tissue as well as elevate expression of genes involved in fatty acid oxidation support this hypothesis (12, 17). In the present study, muscle triglyceride content was decreased by both PPAR agonists, although there was no improvement of insulin responsiveness in the PPAR{alpha} agonist-treated group.

It has been proposed that PPAR{alpha} activation increases liver fatty acid oxidation, which is likely consequent to increased expression of a number of genes involved in fatty acid metabolism (13, 14, 15). Supporting this concept is the data from the present study showing elevation of plasma concentrations of the ketone body, ß-hydroxybutyrate. In addition to diminishing body weight, PPAR{alpha} agonist treatment also reduced epididymal fat (EWAT) mass in comparison with the vehicle group, thereby also suggesting an effect on lipid metabolism. Our data are inconclusive as to whether synthesis or oxidation of lipids was altered by PPAR{alpha} in EWAT. In contrast to the effect of our PPAR{alpha} agonist, activation of PPAR{gamma} led to significant increases in EWAT and whole-body weight. Consequently, PPAR{alpha} agonism has a distinct advantage over that of PPAR{gamma} with respect to these two parameters.

In conclusion, we have shown that chronic treatment with a novel and potent PPAR{alpha} agonist blunted the development of diabetes in ZDF rats mainly by improving the pancreatic insulin response without demonstrating the adverse effects on body weight gain and cardiomegaly typically seen with PPAR{gamma} agonists. This study suggests a potential therapeutical use of PPAR{alpha} agonists in the prevention of T2DM in human subjects suffering from metabolic syndrome.


   Acknowledgments
 
We gratefully acknowledge the efforts of the members of the Merck Research Laboratories Medicinal Chemistry and Process Chemistry Departments, who provided the ligands used in the studies presented in this manuscript. Authors are greatly indebted to Drs. Cai Li and Yun-Ping Zhou for their insightful review of the manuscript. Finally, we thank members of the Department of Metabolic Disease, who performed the in vitro assays that quantified the PPAR binding and transcriptional activation potencies of the ligands used here.


   Footnotes
 
Present address for D.E.M.: Lilly Research Laboratories, Lilly, Corporate Center, Indianapolis, Indiana 46285 (mollerda@lilly.com).

Author Disclosure Summary: All authors are employed by Merck & Co. with the exception of T.D. and D.M. who were previously employed by Merck & Co. D.M. is currently employed by Eli Lilly & Co.

First Published Online May 25, 2006

Abbreviations: AUC, Area under the curve; DAPI, 4',6'-diamino-2-phenylindole; EGP, endogenous glucose production; EWAT, epididymal white adipose tissue; NEFA, nonesterified fatty acid; nTZD, nonthiazolidinedione; OGTT, oral glucose tolerance test; PPAR, peroxisome proliferator-activated receptors; T2DM, type 2 diabetes mellitus; ZDF, Zucker diabetic fatty.

Received December 2, 2005.

Accepted for publication May 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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