1 Rheoscience, Rødovre, Denmark
2 Center for Clinical and Basic Research, Ballerup, Denmark
3 Discovery Pharmacology, Novo Nordisk, Måløv, Denmark
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ABSTRACT |
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The nuclear receptors, peroxisome proliferatoractivated receptors (PPARs), constitute a family of three genes, PPAR, -
, and -ß(
), all of which are involved in control of energy homeostasis (1,2). Unequivocal evidence of endogenous ligands for PPAR
and -
is lacking, but a number of synthetic PPAR activating ligands exist, of which hypolipidemic fibric acids are typical examples of PPAR
activators while hypoglycemic thiazolidinediones are typical examples of PPAR
activators.
The antidiabetic effects of PPAR agonists are partly mediated via increased insulin sensitivity of adipose tissue and skeletal muscle. From clinical experience, PPAR
agonists are associated with weight gain, whereas PPAR
agonists appear body weight neutral (3). Part of the body weight increase may be caused by their oedema-inducing class effect, but activators of PPAR
also induce adipogenesis (4,5). They act preferentially on subcutaneous adipocytes, which in comparison to intrabdominal adipocytes express higher levels of PPAR
(6). The long-term metabolic consequences of the increased fat accumulation accompanying treatment with PPAR
agonists are not fully elucidated. Intrabdominal body fat accumulation is one of several hallmarks typifying the metabolic syndrome and, as such, an independent risk factor of type 2 diabetes (7,8,9). Many clinical trials of oral antidiabetic agents (including PPAR
agonists) are conspicuous by their absence of obese test subjects. Thus, it may be that the apparent lack of effect by PPAR
activation on intra-abdominal adipocytes is specific to normal-weight subjects, leaving an enhanced intra-abdominal fatty acid accumulation in PPAR
agonisttreated obese type 2 diabetic patients an ill-fated possibility.
Also, PPAR activation is associated with decreased plasma levels of leptin despite increased body adiposity (10,11). The reason for this utter paradox is uncertain, but lowered leptin levels may increase food consumption. However, the reported effects of PPAR
agonists on feeding are ambiguous. In lean rodents, short-term (1-week) PPAR
activation increases feeding with no overt effect on body weight (11), whereas leptin signalingdeficient mice and rats markedly increase food intake and body adiposity when treated with PPAR
agonists (12,13). In contrast, PPAR
agonists decrease body weight in both leptin receptordeficient fa/fa rats and high-fatfed rodents (14,15). In a recent study, we have observed that the dual PPAR
and -
activator, ragaglitazar (16), appears much less adipogenic compared with the known PPAR
activators rosiglitazone and pioglitazone (17). To further study the metabolic consequences of combined PPAR
and -
activation, food intake, body composition, and metabolic surrogate markers were analyzed in low-fatand high-fatfed obesity-prone rats treated for 32 days with either the dual PPAR
/
activator ragaglitazar or the reference PPAR
and -
activators pioglitazone and fenofibrate, respectively. We have chosen this animal model because it clearly differentiates itself from monogenic or inbred rodent models of obesity by resembling the upper body weight segment of a normally distributed human population characterized by metabolic syndrome.
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RESEARCH DESIGN AND METHODS |
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Upon arrival to the animal unit, rats were housed individually at controlled temperature conditions and in a 12-h light/dark cycle. After 1 week of acclimatization (day 0), animals were randomized into stratified groups to ensure equal body weight means. At day 0, animals were randomized (n = 32 in each group) to eat either a high-fat diet (4.41 kcal/g [carbohydrate 51.4% kcal, fat 31.8%, protein 16.8%], diet no. 12266B; Research Diets, New Brunswick, NJ) or a low-fat diet (4.41 kcal/g [carbohydrate 72.6% kcal, fat 10.6%, protein 16.8%], diet no. D12489; Research Diets). Measurements of food and water intake were initialized and continued every weekday throughout the study. Body weights were measured three times weekly and on the day of the oral glucose tolerance test (OGTT).
The experimental diets were administered in a schedule-fed regimen such that food was removed for a 7-h period during the light phase (from 0800 to 1500), ensuring that animals had not eaten 46 h before drug administration. From previous experience with feeding experiments on rats, we know that an acclimatization period of no less than 10 days should be allowed if novel food is introduced. At day 15, each diet group was further subdivided into four treatment groups (n = 8 in each group): vehicle (0.2% CMC + 0.4% Tween-80 [wt/vol] in NaCl), pioglitazone (30 mg · kg-1 · day-1), fenofibrate (100 mg · kg-1 · day-1), and ragaglitazar (3 mg · kg-1 · day-1). Drugs were dosed twice daily for a total of 32 days (days 1646). On day 41, animals were subjected to an OGTT. Six days later, on day 47, animals were killed in a semistarved state (80% of ad libitum food intake). In the morning period, animals were anesthetized by CO2 inhalation and orbital blood samples were collected before decapitation and tissue dissection. Blood and tissue sampling were carried out in a room adjacent to the permanent stable to ensure the lowest possible level of stress. Fat depot analyses were carried out by removing mesenterial, retroperitoneal, epididymal, and subcutaneous inguinal fat.
OGTT.
This test was carried out at 1400 h in the afternoon on day 41. Animals were mildly fasting (without food since 0700 h). Blood samples were taken as tail vein droplets and blood glucose measured on a conventional Glucometer at time points 0, 15, 30, 60, and 120 min after oral administration of 1 g/kg glucose (using 1 g/ml dH2O). The oral glucose load was given as gavage.
Blood sampling and plasma measurements.
On the day animals were killed, orbital blood was collected in three tubes: Vacutainer-EDTA, VacutainerEDTA + 1% NaF, and VacutainerEDTA + Aprotinin (750 KIU). Blood samples were subjected to analyses for HbA1c, total cholesterol, and glycerol. These variables were all measured using standard enzyme assay kits on a fully automated analyzer (Hitachi). Plasma triacyl glycerol levels were measured by the GPO-Trinder triglyceride kit (Sigma). Samples taken in VacuatinerEDTA + 1% NaF were used for analyses of plasma nonesterified free fatty acids using a acyl-CoA oxidasebased colorimetric kit (NEFA-C; Wako, Osaka, Japan).
Plasma leptin, insulin, C-peptide, GLP-1(7-37), adiponectin, and glucagon were assayed using immunologically based assay kits (ELISA or RIA; Linco Research Immunoassay, St. Charles, MO).
Hepatic and adipose tissue gene expression.
Small samples of liver and inguinal fat were submerged in RNAlater at room temperature and stored in RNAlater at -20°C until use. Total RNA was extracted in TRIzol (Life Technologies, Gaithersburg, MD). Subsequent cDNA synthesis was carried out using standard procedures based on Gibcos reverse transcriptase Superscript II and random primers. The PCR step used two or more primer sets per reaction and incorporated hot nucleotides during the PCR. The PCR was run between 16 and 25 cycles and analyzed on a 6% sequencing gel. Bands were quantified using a Phosphor Imager and the results expressed as a ratio of the gene in question to the internal standard.
The internal standards used depended on the gene being analyzed and the number of amplification cycles. In order of increasing cycles, the following internal standards were used: Tubulin, 36B4 (acidic ribosomal phosphoprotein), EF1-a, G6PDH, and TBP (up to 18, 18, 22, 24, and 25 cycles, respectively).
To test for drug-specific actions in target tissues, the following rat transcripts were analyzed: 1) liver: PEPCK and fatty acyl-CoA oxidase; 2) adipose tissues: aP2, leptin, adiponectin, lipoprotein lipase (LPL), and uncoupling protein-1 (UCP-1); compounds: ragaglitazar (also known as NNC 610029, DRF(-)2725) and pioglitazone (AD-4833, Actos) were both synthesized at Novo Nordisk (Måløv, Denmark). Fenofibrate was purchased from Sigma.
Statistical evaluation.
All results are presented as means ± SE, unless otherwise stated. Data were analyzed for main effects by a two-factor ANOVA (diet times drug treatment). When a significant main effect was observed, data within each diet group were analyzed by a one-factor ANOVA followed by Fisher or Bonferroni post hoc analyses, as appropriate (n = 8 per group). All data were digitized using Excel 5.0 as database software, and statistical analysis were performed using Statview or Graph Pad software.
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RESULTS |
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Food intake.
To assess the underlying cause of diet- and pharmacologically induced alterations of body weight, food intake was assessed daily. Diets were equicaloric, such that the daily gravimetrically determined consumption of food accurately reflected the caloric consumption.
Cumulated food intake from day 16 to 44 is shown in Fig. 2A. Two-factor ANOVA revealed that drug treatment (F3,54 = 9.110; P < 0.0001) but not diet (F1,54 = 3.893; P = 0.06) exerted main effects on cumulated food intake of the entire treatment period. Interaction between drug treatment and diet was not observed for cumulated food intake between days 16 and 44. Cumulated food intake over the first 2 weeks of drug treatment was markedly elevated in pioglitazone-treated rats (Fig. 2A). Cumulated food intake for high-fatfed fenofibrate-treated rats was significantly lower than that for vehicle-treated rats eating the same diet, as well as for low-fatfed fenofibrate-treated rats, clearly indicating that the anorectic action of fenofibrate is diet dependent (Fig. 3).
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As most experimental rat diets are rather dry and require moistening to allow proper mastication, virtually all orexigenic treatments are accompanied by moderate increases of fluid intake. We therefore assessed daily water intake in all groups. Water intake was affected to a lesser degree than food intake (Table 3). However, when assessing cumulated water intake from day 16 to 44, significant main effects of both drug and diet were clearly observed (drug F3,45 = 4.201, P = 0.011; diet F1,45 = 8.043, P = 0.007; two-factor ANOVA) but without interaction (F3,45 = 0.073; P = 0.97). Thus, high-fatfed rats drank slightly more than low-fatfed animals, and the orexigenic pioglitazone treatment resulted in elevated water intake in both low-fatfed (694 ± 26 vs. 599 ± 22 ml, n = 68; P = 0.02) and high-fatfed (752 ± 46 vs. 654 ± 31 ml, n = 68; P = 0.02) rats. None of the other drug treatments gave rise to elevated water intake.
OGTT.
On day 41, all animals were subjected to an OGTT. Two-factor ANOVA revealed that both diet (F1,54 = 7.637; P = 0.008) and drug treatment (F3,54 = 16.185; P < 0.0001) exerted significant main effects on the OGTT without interaction between diet and drug treatment (F3,54 = 0.823; P = 0.5). The diet-dependent effect was seen, with high-fatfed animals displaying poorer OGTT scores (Table 4). Both pioglitazone and ragaglitazar significantly reduced the area under the curve in both low-fatand high-fatfed animals, thereby eliminating the diet-dependent effect on glucose tolerance (Table 4). Fenofibrate treatment had no impact on OGTT scores.
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In the rat, PPAR agonists are potent stimulators of hepatic ß-oxidation, but the extent to which PPAR
and dual activators stimulate such catabolism of nonesterified fatty acids is unknown. Plasma concentrations of ketone bodies were used as an indirect measure of hepatic ß-oxidation (Table 6). The main effects of drug treatment and diet on plasma total ketone bodies were analyzed using two-factor ANOVA, revealing a significant effect of drug treatment (F3,54 = 3.857; P = 0.014) but not of diet (F1,54 = 1.444; P = 0.24), and there was no drug times diet interaction (F3,54 = 0.526; P = 0.67). Interestingly, plasma levels of 3-hydroxybutyrate were neither affected by drug treatment (F3,54 = 2.654; P = 0.06) nor by diet (F1,54 = 1.485; P = 0.23), whereas plasma levels of acetoacetate were markedly affected by drug treatment (F3,54 = 20.130; P < 0.0001). Diet had no impact on plasma concentrations of acetoacetate (F1,54 = 0.888; P = 0.35), and no interaction between drug and diet was observed (F3,54 = 0.647; P = 0.59).
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Two-factor ANOVA revealed the main effects of both diet (F1,55 = 12.059; P = 0.001) and drug treatment (F3,55 = 8.005; P = 0.0002) on plasma leptin levels. No interaction between diet and drug treatment was observed (F3,55 = 1.970; P = 0.13). In low-fatfed rats, fenofibrate treatment gave rise to significantly lower levels of leptin. Interestingly, the dose of pioglitazone used elevated leptin to levels significantly higher than those seen in any other high-fatfed group (Table 7).
Finally, plasma levels of adiponectin were measured. Main effects of both diet (F1,56 = 13.483; P = 0.0005) and drug treatment (F3,56 = 66.1919; P < 0.0001) were observed without interaction between the two variables. In vehicle-treated rats, high-fat feeding had no impact on plasma adiponectin concentrations, but in both pioglitazone- and ragaglitazar-treated groups, high-fatfed rats displayed lower adiponectin levels compared with low-fatfed groups. Also, it was quite clear that treatment with either of the PPAR activators pioglitazone or ragaglitazar significantly increased adiponectin levels compared with vehicle-treated animals fed the same diet. Fenofibrate treatment had no impact on plasma adiponectin levels in any of the diet groups.
In vehicle-treated rats, plasma adiponectin levels were not correlated to plasma levels of leptin (regression line: Y = 2.8 + 0.05X; R2 = 0.046), irrespective of diet (Fig. 3). Interestingly, pioglitazone treatment induced a marked change such that plasma adiponectin levels became negatively correlated to plasma leptin levels (regression line: Y = 14.60.46X; R2 = 0.615). None of the other drug treatments introduced such correlations, indicating that a specific PPAR action was absent in the concomitant presence of PPAR
stimulation.
Hepatic and adipose tissue gene expression.
To validate drug actions in the liver, we assessed hepatic expression of fatty acyl-CoA oxidase and PEPCK as markers of PPAR activation and insulin sensitivity, respectively (Table 8). In addition, drug actions were assessed in inguinal adipose tissue by measurements of aP2 expression and by measurement of interscapular brown adipose tissue UCP-1 expression (Table 8).
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Similarly, both PPAR activators stimulated white adipose tissue expression of the adipocyte marker aP2 (Table 8). Expression of other adipocyte-specific genes was also examined in epididymal fat sampled on day 47. Thus, two-factor ANOVA revealed main effects of both diet (F1,55 = 17.811; P < 0.0001) and drug treatment (F3,55 = 10.043; P < 0.0001) on leptin expression, which was significantly lower in low-fatfed animals. Treatment with pioglitazone significantly lowered leptin expression in both low-fatand high-fatfed animals (Table 8). Expression of adiponectin was also analyzed by two-factor ANOVA, and the main effects of both diet (F1,52 = 9.609; P = 0.005) and drug treatment (F3,52 = 7.042; P = 0.0005) were observed. A significant interaction between the two treatment modalities was also observed (F3,52 = 9.018; P = 0.0002), and it was reflected by a high-fat feedingdependent increase of pioglitazone-stimulated adiponectin mRNA expression (Table 7). In low-fatfed rats, drug treatment had no impact on adiponectin expression. The main effect of drug treatment on adipocyte expression of LPL mRNA was observed with the two-factor ANOVA (F3,54 = 4.186; P = 0.009). Diet had no main impact on LPL expression (F1,54 = 0.132; P = 0.72), and no interaction between the two treatment modilaties was seen. Ragaglitazar markedly increased LPL expression in both low-fatand high-fatfed rats, while pioglitazone had no impact on the expression of LPL.
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DISCUSSION |
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The currently observed actions of pioglitazone emphasize that increased adipogenesis is not simply a result of increased storing capacity of triacylglycerol, but that it also arises from an increased urge to ingest energy. A previous study using the PPAR agonists BRL49653 similarly showed increased feeding over 1 week of drug administration but with no apparent effect on total body weight (11). A number of reports have shown that PPAR
induced food intake in rodents with deficient leptin receptors, but there was a lack of effect on feeding in normal rats (13,14,1921). Obviously, neither fa/fa rats nor db/db mice constitute optimal animal models for the study of feeding behavior because data are bound to be skewed by absence of key components of normal energy homeostasis. Also, in some of the earlier studies, PPAR
agonists were administered as part of the food, potentially affecting texture and palatability and resulting in altered feeding patterns.
The orexigenic action of pioglitazone was independent of diet, as cumulated food intakes in both low-fatand high-fatfed rats were markedly elevated. Pioglitazone-treated rats were both heavier and fatter than other drug-treated groups, but both plasma leptin and leptin mRNA levels were comparable to those observed in ragaglitazar-treated rats displaying normal food intake throughout the treatment period. Agonists of PPAR have been shown to inhibit adipocyte leptin synthesis (11), suggesting that pioglitazone treatment actually increases sensitivity to leptin. The apparent altered sensitivity to plasma leptin concentrations of PPAR
-treated rats emphasizes why animals with deficient leptin signaling should be avoided in experimental analysis of the potential impact of these compounds on energy homeostasis.
In fa/fa Zucker rats, pioglitazone has no impact on whole-body energy expenditure (22), but these measures may not have been properly adjusted for the expected increase of thermogenesis associated with excess energy intake. Thus, it is likely that adjustment for food intake yield lowers measures of energy expenditure per consumed calorie. Both pioglitazone and ragaglitazar markedly improved feed efficiency, with or without a concomitant increase of caloric intake, emphasizing that positive energy balance associated with PPAR activation is not solely ascribed to the orexigenic actions of these compounds. Thus, PPAR
may favor positive energy balance by a combined action of enhanced food intake and decreased energy expenditure.
Treatment with fibrates is associated with decreased body weight gain and reduced adiposity in animal models of diet-induced obesity and in fa/fa Zucker rats (14,15,26). A few clinical studies have shown decreased body weight, fat mass, and waist circumference in fibrate-treated humans but no apparent effect on insulin sensitivity, which is in line with our current observations (27). Fenofibrate-mediated negative impact on energy homeostasis is most pronounced in high-fatfed animals (15) and was reflected by a marked reduction of food intake in high-fatfed animals in the present study. Data on cumulated caloric intake have not been published for PPAR-/- mice, which display a nonsignificant lowering of daily food intake that reflects a higher feed efficiency (28). This is in line with the current observations of markedly reduced feed efficiency in high-fatfed fenofibrate-treated rats, which is likely to be the result of the combined actions of reduced feeding and increased resting energy expenditure. In a recent study, Mancini et al. (15) demonstrated that fenofibrate markedly enhances hepatic mitochondrial and peroxisomal palmiotyldependent oxygen consumption, leading to a slight increase in resting metabolic rate. Thus, amelioration of the metabolic syndrome markers associated with fibrate treatment may actually be accounted for by the anorectic effects of these compounds.
Increased hepatic fatty acid oxidation is significantly elevated in fenofibrate-treated animals, as evidenced by marked upregulation of hepatic acyl CoA oxidase and carnitine palmiotyl transferase expression (14). The currently observed hepatomegali and upregulated acyl-CoA oxidase expression supports a profound fenofibrate-induced peroxisome proliferation. Despite a moderate induction of hepatic acyl-CoA oxidase expression, treatment with the dual activator ragaglitazar had no impact on liver size. Fenofibrate treatment activated formation of anorectic signals markedly enhanced by intake of dietary fats, but neither free fatty acids nor circulating triacylglycerol levels were directly accountable for this anorectic action, as these parameters were similarly decreased in both pioglitazone- and ragaglitazar-treated rats. However, it is tempting to speculate that fibrate-induced intracellular accumulation of long-chain fatty acids within hepatocytes may constitute a nutritive signal curtailing further intake of nutrients. Hepatic fatty acid oxidation constitutes an important metabolic variable in the control of feeding, and much experimental evidence points toward hepatic ketone body formation comprising at least some of the measured variables (29). PPAR activation constitutes a major stimulus to hepatic expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMG-CoAS), a key enzyme in ketogenesis (30). Furthermore, the anorectic actions of systemically administered 3-hydroxybutyrate are abolished by selective hepatic vagotomy (31), but the brain may also detect circulating ketone bodies because direct intracerebroventricular injections of 3-hydroxybutyrate induce hypophagia (32). However, our observations suggest that acetoacetate rather than 3-hydroxybutyrate serves an anorectic role, as this ketone body was selectively elevated in fenofibrate-treated rats. The underlying mechanism mediating information about hepatocyte fatty acid oxidation to afferent terminals of hepatic vagus nerve is unknown. Lipoprivic feeding induced by intraportal infusion of mercaptoacetate depolarizes hepatocytes and enhances the discharge rates of the hepatic vagus branch (33,34). Thus, it is possible that the enhanced lipid oxidation induced by fibrates and other activators of PPAR
mediate anorexia via their stimulatory action of the hepatic vagus branch.
The apparent lack of pharmacological effects on feeding seen in both diet groups treated with the dual PPAR/
activator ragaglitazar may be explained in several ways. Based on the results of the OGTT and measures of plasma insulin concentrations, it was evident that equipotent insulin-sensitizing doses of pioglitazone and ragaglitazar were used. Also, the comparable induction of brown adipose tissue UCP-1 and white adipose tissue aP2 provides evidence that equiefficacious PPAR
-activating doses were used. It is therefore tempting to speculate that the PPAR
component of ragaglitazar counterbalances PPAR
-induced feeding. However, the absence of marked activation of hepatic peroxisomes and lipid oxidation suggests that lack of orexigenic actions may not simply be due to PPAR
activation. Interestingly, plasma concentrations of adiponectin were significantly higher in ragaglitazar-treated rats than in pioglitazone-treated rats (Table 8). Adiponectin is an anti-inflammatory protein, synthesized and released from adipocytes upon PPAR
stimulation and in response to weight reduction (3537). In mice, the globular head domain of adiponectin decreases weight gain via enhanced muscular lipid oxidation (38).
In conclusion, we have shown that selective PPAR activation is associated with weight gain due to both increased food intake and improved feed efficiency, whereas selective PPAR
activation leads to opposite effects. This observation calls for increased awareness of possible disadvantageous metabolic consequences for type 2 diabetic patients subject to long-term clinical use of selective PPAR
agonists. The actions were independent of dietary fat content; therefore, caloric restriction of type 2 diabetic patients treated with selective PPAR
agonists is of utmost importance. Dual activators of both PPAR
and -
appear less devastating for metabolic control, as they are devoid of orexigenic actions. Thus, it seems likely that this class of compounds may constitute therapeutically more interesting drugs, as they induce favorable metabolic control of glucose homeostasis accompanied by a lower degree of body fat accumulation.
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ACKNOWLEDGMENTS |
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Part of this study has appeared in abstract form [Larsen PJ, Jensen PB, Larsen LK, Vrang N, Sørensen RV, Wassermann K: Ragaglitazar prevents intra-abdominal fat accumulation and is devoid of orexigenic and adipogenic effects in diet-induced obese rats (Abstract). Diabetes 51 (Suppl. 2):A140, 2002].
Address correspondence and reprint requests to Dr. Philip J. Larsen, Rheoscience, Glerupvej 2, 2610 Rødovre, Denmark. E-mail: pjl{at}rheoscience.com
Received for publication May 5, 2003 and accepted in revised form June 10, 2003
LPL, lipoprotein lipase; OGTT, oral glucose tolerance test; PPAR, peroxisome proliferatoractivated receptor; UCP-1, uncoupling protein-1
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REFERENCES |
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