Peroxisome Proliferator-Activated Receptor δ-Agonist, GW501516, Ameliorates Insulin Resistance, Improves Dyslipidaemia in Monosodium l-Glutamate Metabolic Syndrome Mice

© 2008 The Authors Doi: 10.1111/j.1742-7843.2008.00268.x Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 240–246

Peroxisome Proliferator-Activated Receptor δ-Agonist, GW501516, Ameliorates Insulin Resistance, Improves Dyslipidaemia in Monosodium l-Glutamate Metabolic Syndrome Mice Blackwell Publishing Ltd

Wei Chen, Li-Li Wang, Hong-Ying Liu, Long Long and Song Li Beijing Institute of Pharmacology and Toxicology, Beijing, China (Received December 8, 2007; Accepted January 29, 2008) Abstract: We evaluated the effects of GW501516, a specific peroxisome proliferator-activated receptor β/δ (PPARδ) agonist in metabolic syndrome mice, obtained by perinatal injection of monosodium l-glutamate, to investigate the efficacy of GW501516 against metabolic syndrome and the effectiveness of PPAR δ activation as therapeutic target for metabolic syndrome. After 14 days treatment, GW501516 effectively improved the glucose intolerance, normalized the fasted blood glucose, and increased the serum high-density lipoprotein cholesterol (HDL-C) level. Postprandial blood glucose, serum insulin, leptin, free fatty acid (FFA) levels, and total cholesterol/HDL-C ratio were also significantly decreased. Moreover, semiquantitative reverse transcription–polymerase chain reaction results indicated that the above phenotypes might be due to (i) enhancement of fatty acid oxidation in muscle, adipose tissue and the liver; (ii) improvement of insulin-stimulated glucose transportation in skeletal muscle and adipose tissue; and (iii) reduced local glucocorticoid synthesis. Therefore, GW501516 could significantly ameliorate dyslipidaemia and insulin resistance in monosodium l-glutamate mice and activation of PPARδ could be envisioned as a useful strategy against human metabolic syndrome and related diseases.

Obesity and the interrelated disorders of the metabolic syndrome have become an epidemic and serious worldwide public health issue [1,2]. Metabolic syndrome is characterized by multiple closely related disorders, such as abdominal obesity, insulin resistance, hypertension, dyslipidaemia, atherosclerosis and so on. Until now, the existing approaches for metabolic syndrome treatment, single drug or combination of different drugs (e.g. statins, fibrates, angiotensin-converting enzyme inhibitors), have not been ideal, partially due to the limitation of the therapeutic efficacy and the accompanying side effects. As the pathophysiology of metabolic syndrome revealed, the dysfunction of adipocyte, excess accumulation of intraabdominal fat, and ectopic fat deposition are responsible for the pathogenesis of metabolic syndrome [3–5]. Thus, rectifying the dysfunction of adipose tissue and related metabolic disorders could be an effective therapeutic strategy against metabolic syndrome. The peroxisome proliferator-activated receptors (PPARs) are transcription factors, belonging to the nuclear hormone receptor superfamily. Composed of three isotypes, α, γ and β/δ (hereafter refered to as δ), PPARs regulate genes involved in glucose and lipids metabolism. PPARα, the major target of the marketed fibrates class of lipid-lowering drugs, primarily expressed in the liver, where it regulates genes involved in fatty acid catabolism [6,7]. PPARγ, the major target of the thiazolidinediones class of insulin sensitizers, highly was expressed in adipose tissue, where it regulates adipogenesis Author for correspondence: Li-Li Wang, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China (fax +86-106693-1250, e-mail [email protected]).

and improves insulin sensitivity [8]. PPARδ extensively expressed in metabolically active tissues, including skeletal muscle, adipose tissue and its roles in metabolic syndrome, and type 2 diabetes have also come to light [9–12]. Recently, several studies have demonstrated the critical roles of PPAR δ in preventing obesity, alleviating hyperglycaemia, improving insulin sensitivity and correcting dyslipidaemia [13,14]. Constitutively activation of PPARδ can increase serum HDL-C [15], considerably raise the number of oxidative myofibres [16], and relieve atherosclerotic lesion progression in obese dyslipidaemic rhesus monkeys [17]. GW501516 can prevent and ameliorate diet-induced and genetic obesity, respectively, decrease the plasma glucose and blood insulin levels in ob/ob (genetically obese) and db/db mice (genetic diabetes) [18]. However, PPARδ knockout mice exhibited glucose intolerance and are not responsive to treatment with GW501516 [11,18,19]. Accordingly, pharmacological activations of PPARδ could be an ideal therapeutic approaches to rectify some aspects of metabolic syndrome [11,13,20]. Monosodium l-glutamate (MSG) mice, a mouse model for obesity, are prepared by neonatal injection of MSG, which damaged more than 80% arcuate nucleus of the hypothalamus (ANH) [21–23]. Neuroendocrine and metabolic dysfunctions are typical phenotypes observed in MSG mice. Low plasma levels of growth hormone and insulin-like growth factor-1 contributed to the stunting and decreased lean body mass. Skeleton development atrophy and retardation are common manifestations of adult animals. Four- to five-month-old MSG mice exhibit the main features of metabolic syndrome (i.e. excess abdominal fat accumulation, hyperinsulinaemia,

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Table 1. Effects of PPARδ agonist on body shape of normal, control model and GW501516-treated MSG mice. Normal Waist circumference (cm) Body length (cm) LEE’ indexes Liver/body weight (%) Visceral fat/body weight (%)

9.52 ± 1.31 11.65 ± 0.55 288.91 ± 11.28 5.99 ± 1.07

Model

GW501516 1

11.37 ± 0.96 10.61 ± 0.171 331.67 ± 20.492 2.78 ± 0.652 29.8 ± 2.53

11.27 ± 0.59 10.71 ± 0.55 328.24 ± 11.42 3.32 ± 0.60 31.4 ± 4.34

LEE’ indexes = Body weight1/3(g)/ body length (cm). Values are mean ± S.D. of eight to nine animals per group. 1P < 0.05, 2P < 0.01 versus normal.

hypercorticosteronaemia, hypometabolic rate, impaired glucose tolerance, and so on) [24 –26], which more accurately reflect the common manifestations of the human metabolic syndrome. In the current study, GW501516 was used as a probe compound to validate the efficacy of PPARδ activation as therapeutic target for metabolic syndrome in MSG mice. The changes in insulin sensitivity, serum insulin and leptin levels, glucose and lipid metabolism, and the mRNA encoding genes involve in glucose and lipids metabolism as well as glucocorticoids, local synthesis were observed. Materials and Methods Compounds. GW501516 was synthesized by a new drug design centre of our institute, and the purity and structure were confirmed by high-performance liquid chromatography, mass spectrometry and 1 H-nuclear magnetic resonance. Animal and experimental protocols. The animals were obtained from the experimental animal centre of Beijing Medical Science Academy (Beijing, China) and maintained in an air-conditioned room under controlled illumination (a 12-hr light:dark cycle), temperature (23 ± 1°) and humidity of 40– 60%, and they had free access to standard rodent chow and tap water throughout the experimental period. MSG-induced obese mice preparation. Neonatal imprinting control region mice were injected subcutaneously with 4 mg/g body weight monosodium l-glutamate (Sigma, St. Louis, MO, USA) (MSG mice) or equivalent volume of saline water (normal control, normal) within the first 8 days of life. Pups were weaned on the 21st day of life and kept under normal condition for another 5 months. Then, the MSG mice were assigned to two different groups, that is, model control (model) and GW501516-treated MSG mice (GW) (n = 10/ group, male and female in half), based on fasted blood glucose, total cholesterol (TC), triglyceride (TG) levels and initial body weight. Normal and model group were gavaged with vehicle (i.e. dimethyl sulfoxide and 0.5% carboxymethylcellulose), GW group were gavaged with 10 mg/kg GW501516 once a day for 2 weeks [18]. GW501516 was dissolved in dimethyl sulfoxide and suspended in 0.5% carboxymethyl cellulose solution. Body weight and food intake were recorded every other day during the treatment period. At the end of the experimental period, blood samples were collected for immediate assessment of serum biochemical parameters. The liver, gastrocnemius muscle (SK), interscapular brown adipose (iBAT) and epididymal white adipose tissue (eWAT) were excised and rapidly frozen in liquid nitrogen for subsequent RNA extraction and reverse transcription– polymerase chain reaction (RT-PCR) analysis. All animal experiments were conducted in accordance with the correspondent guidelines of the Animal Experimentation Ethics

Committee of Beijing Institute of Pharmacology and Toxicology for animal care, handling and termination. Glucose tolerance testing and analysis of blood samples. Oral glucosetolerance test (OGTT) was performed by gavage a glucose bolus (2 g/kg of body weight) after an overnight fast, and blood glucose levels were determined at 0, 30, 60 and 120 min. using the One Touch Ultra Meter (Johnson & Johnson, Milpitas, CA, USA) through the tail tip. The area under the glucose curves (AUC0~2 hr) generated from data collected during the OGTT were calculated. Serum TC, TG and free fatty acid (FFA) levels were measured using enzymatic colorimetric methods according to the manufacturer’s instructions (Rongsheng Biotech, Shanghai, China). Serum insulin and leptin concentrations were assayed by using commercial mice radioimmunoassay kits respectively (Linco Research, St. Charles, MO, USA). Extraction of total RNA and semiquantitative RT-PCR. Total RNA from the liver, eWAT, iBAT and SK of the mice were prepared using the Trizol RNA preparation kit following the manufacturer’s recommended procedures (Gibco-BRL, Grand Island, NY, USA). For the semiquantitative RT-PCR analysis of genes expression, total RNA (0.5 μg) was reverse transcribed and subsequently amplified by PCR using the RNA PCR Kit Version 1.1 (TaKaRa Biotechnology Co. Ltd., Dalian, China) in a thermocycler (Mastercycler, Eppendorf, Hamburg, Germany). The expression results of specific mRNAs are always presented relative to the expression of the control gene (β-actin). Statistical analysis. All values are expressed as mean ± S.D. Statistical analyses were performed using one-way anova with SPSS to compare the experimental groups (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.

Results Five-month-old MSG mice are apple-shaped, stunted and obese with excess deposition of fat in the abdomen and subcutaneous as previously reported [23,27]. After a 14-day GW501516 treatment (10 mg/kg/day), no significant changes were observed on body length, body weight, liver/body weight ratio, and fat/body weight ratio (table 1, fig. 1A and B). The effect of GW501516 on insulin sensitivity. Although the 5-month MSG mice exhibited definite insulin resistance [28,29] (fig. 2), the fasted blood glucose level did not increase significantly (table 2). Thus, we monitored the postprandial blood glucose level during the treatment. From day 7 on, GW501516 significantly suppressed the increase of postprandial blood glucose (fig. 3) in MSG mice. Moreover, as displayed in fig. 2 and table 2 (OGTT-AUC0~2 hr), MSG

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Fig. 3. PPARδ agonist prevented postprandial hyperglycaemia in MSG-treated mice. Postprandial blood glucose was measured after 2 hr of light cycling. Values are mean ± S.D. of six animals per group. **P < 0.01 versus normal; †P < 0.05 versus model.

n = 8–9). Furthermore, fasting insulin level was markedly reduced in GW group (fig. 4A). The effects of GW501516 on lipid profiles and serum leptin. Fig. 1. Effects of PPARδ agonist on body weight. (A) Body weight before (day 0) and after (day 14) treatment. (B) Net body weight change during administration period, which was calculated for individual mice and then averaged. Values are mean ± S.D. of eight to nine animals per group. **P < 0.01 versus normal.

mice showed obvious hyperglycaemia at all test points after glucose load and the AUC0~2 hr of the glucose response during the OGTT was significantly increased than the normal control (AUC0~2 hr, normal versus model, P < 0.01, n = 8–9). However, after GW501516 treatment, the glucose intolerance tendency was obviously improved (model versus GW, P < 0.01,

We next sought to determine whether GW501516 affected serum lipids levels in MSG mice. Consistent with a previous study [23], when compared to model controls, GW501516 significantly increased HDL-C and total cholesterol levels (51% and 24%, respectively), and the ratio of TC/HDL-C was also obviously decreased (P < 0.05) (table 2). In parallel with the increase of HDL-C, FFA was nearly normalized (table 2). But unexpectedly, there is also a little increase in serum TG, although non-significant. As revealed in fig. 4, serum leptin concentration in MSG mice was seven times higher than that of the normal control. However, GW501516 significantly corrected the hyperleptinaemia (P < 0.05) (fig. 4B). RT-PCR/gene expression results.

Fig. 2. PPARδ agonist improved insulin resistance in MSG-treated mice. Mice were fasted overnight and then subjected to oral glucose tolerance test (OGTT). Values are mean ± S.D. of eight to nine animals per group. *P < 0.05 versus normal; †P < 0.05 versus model.

To evaluate the mechanism of GW501516, the insulinsensitive target organs (i.e. the liver, skeletal muscle and adipose tissue) were chosen to detect the levels of mRNA encoding genes involved in fatty acid oxidation, energy expenditure and glucose metabolism. After treatment for 14 days, GW501516 increased mRNA levels of acyl-CoA oxidase (ACO) in eWAT, iBAT and the liver, carnitine parmitoyl transferase 1b (CPT1b) and uncoupling protein 2 (UCP2) in eWAT, iBAT and the SK, uncoupling protein 3 (UCP3) in the SK, and modest increased lipoprotein lipase in eWAT and iBAT. At the same time, GW501516 also increased the expression level of glucose transporter 4 (GLUT4) mRNA in eWAT and iBAT, decreased the mRNA expression level of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme in gluconeogenesis in the liver and SK. Furthermore, decreased expression of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) were found in eWAT, iBAT and the liver (fig. 5).

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Table 2. Effects of PPARδ agonist on serum lipid concentrations and OGTT-AUC0~2 hr. Normal –1

TG (mmol ) FFA (mmol–1) TC (mmol–1) HDL-C (mmol–1) TC/HDL-C FBG (12 hr) (mmol–1) OGTT-AUC0~2 hr (mmol hr l–1)

1.28 ± 0.28 2.37 ± 0.19 2.5 ± 0.39 2.03 ± 0.80 1.22 ± 0.06 3.66 ± 1.44 6.69 ± 1.60

Model

GW501516 1

2.16 ± 0.53 3.42 ± 0.771 4.52 ± 1.261 2.64 ± 0.66 1.59 ± 0.111 4.06 ± 0.46 22.06 ± 7.921

2.45 ± 0.76 2.66 ± 0.272 5.62 ± 1.062 4.15 ± 0.773 1.33 ± 0.142 3.68 ± 1.76 8.96 ± 1.683

Five-month-old MSG mice and control littermates (normal control) were treated with GW501516 (10 mg/kg) or vehicle respectively for 14 days. Values are mean ± S.D. of eight to nine animals per group. 1 P < 0.01 versus normal; 2P < 0.05, 3P < 0.01 versus model.

Discussion In this study, we observed the effects of administration of GW501516 to MSG mice that exhibits most of the features (the abnormalities mainly including abdominal obesity, blood sugar abnormalities, and lipid abnormalities in the present

Fig. 4. PPARδ agonist decreased serum insulin and leptin levels in MSG-treated mice. (A) Serum insulin. (B) Serum leptin. Values are mean ± S.D. of eight to nine animals per group. **P < 0.01 versus normal; †P < 0.05 versus model.

experimental animal) associated with mass spectrometry. The results indicated that activation of PPARδ by GW501516 could enhance insulin sensitivity and partially improve the dyslipidaemia and other disorders associated with metabolic syndrome. The underlying mechanism of insulin resistance was attenuated insulin-mediated glucose utilization and/or impaired insulin-dependent down-regulation of mainly hepatic glucose production. Insulin binding to plasma membranes was decreased in MSG rats and lower content of GLUT4 protein was also found in adipocytes from MSG rats [30]. Compared to vehicle-treated MSG mice, GW501516 decreased the postprandial hyperglycaemia, hyperinsulinaemia and improved glucose intolerance. Postprandial hyperglycaemia represents failure of first phase insulin secretion that is seen in the impaired glucose state, and the impaired OGTT reveals dysfunction of β-cell secretion and peripheral insulin action. Further studies revealed that GW501516 up-regulated the gene expression of GLUT4 in adipose tissue and reduced the gene expression of PEPCK in liver, a key enzyme for the gluconeogenesis. The results indicated that increased glucose disposal rate and decreased hepatic glucose production constituted the main pathway of GW501516 in improving insulin resistance. Whether glucose transporter in skeletal muscle or other mechanisms also involved in this effect still needs to be clarified. Therefore, our study is in agreement with previous results in genetically obese (ob/ob) mice and rhesus monkey [31], showing that GW501516 obviously improved the diabetic conditions and insulin sensitivity in MSG mice. Leptin, the first discovered adipo-hormone secreted by adipocyte, possesses a pivotal role in regulating appetite and adiposity, affecting the synthesis and secretion of adiponectin, as well. Now, it is clear that leptin resistance in hypothalamic neurons also participates in the pathogenesis of the metabolic syndrome, and hyperleptinaemia itself had also been shown to elicit oxidative stress and vascular inflammation. Neonatal administration of MSG damages the ANH, the anatomic site for leptin action on fat mobilization [21,32], and the serum biochemical analysis further showed that MSG mice are marked hyperleptinaemic. The fact that GW501516 obviously decreased serum leptin level (fig. 4B) could be

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Fig. 5. Transcription regulation of GW501516 on the expression of representative genes. Total RNA was isolated from the liver, iBAT, eWAT and SK, which were then subjected to semiquantitative RT-PCR to detect the mRNA levels of the indicated genes. Results are expressed as relative expression levels normalized to the expression of the model (WAT and BAT) or normal (liver and SK) control group. β-Actin was used as internal positive control. (A) Representative agarose gel figure. (B) Quantification of the results in fig. 5A. Values are mean ± S.D. of 8 animals per group. *P < 0.05, **P < 0.01 versus normal; †P < 0.05, ‡P < 0.01 versus model.

related to its improvement in the adipocytokine production and the metabolism of adipose tissue. This may also contribute to the relievement of insulin resistance in MSG mice. The fact that FFAs mediated insulin resistance and impaired insulin secretion are recognized as one of the key events in the pathogenesis of type II diabetes and metabolic syndrome [33,34]. Upon activation, PPARδ significantly promotes lipid consumption and thereby reduces the levels of FFAs in the circulation of MSG mice. At the same time, serum HDL-C level was significantly elevated, consistent with the results in other mice models, the obese rhesus monkeys and healthy volunteers [15,18,31]. Serum TG and TC were

abnormally elevated in MSG mice, which might be related to the significantly higher incorporation of glucose into lipids and the inhibited lipolysis by increased corticosterone level [30]. Quite unexpectedly, serum TG and TC were also increased in the GW501516-treated group, similar to the diet-induced obese (DIO) mice [18], which needs to be further clarified in further studies. However, in the MSG mice, the ratio of TC/HDL-C was significant decreased, suggesting that GW501516 increased the partition of cholesterol into high-density lipoprotein. Recently, glucocorticoid (GC) received considerable attention in metabolic syndrome studies [27,35]. Clinical data

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have shown that higher GC levels are linked to the development of metabolic syndrome. It has been suggested that the excessive GC concentration in local tissues especially liver and adipose tissue could promote visceral obesity. The changes of corticosterone levels play an important role in hormonol and metabolic adaptation of MSG obese rats. Intracellular GC levels are regulated by 11βHSD1, the key enzyme locally regenerates active GCs (corticosterone in rodents and cortisol in human beings) from inert 11keto forms (11-dehydrocorticosterone and cortisone) [34]. 11βHSD1 thereby modulates local GC effects by regulating ligand supply to the GC receptors. GW501516 down-regulates the increased expression of 11βHSD1 in MSG mice liver and adipose tissues (fig. 5), indicating that GW501516 could diminish higher concentrations of GC caused by obesity. At the same time, this effect may also partially account for the beneficial role of GW501516 in improving the thermoregulatory anomaly, glucose intolerance and related lipid disorders, for the existence of an inverse relation between plasma GC level and insulin binding [30]. To further elucidate the mechanism of GW501516, we measured mRNA levels of some representative genes involved in fatty acids β-oxidation, glucose utilization and energy homeostasis in the liver, adipose and skeletal muscle tissues. Consistent with previous findings [18], activation of the nuclear receptor PPARβ promotes fatty acids catabolism in skeletal muscle and adipose tissue by up-regulation of fatty acid uptake, β-oxidation (CPT1b, ACO) and burning (UCP2, UCP3). Uncoupling proteins (UCP), inducing adaptive thermogenesis by promoting uncoupling of oxidative phosphorylation from ATP synthesis, are essential for energy metabolism regulation. At the same time, lipoprotein lipase mRNA expression in adipose tissue was also up-regulated, a major enzyme of lipoprotein metabolism, tethered to endothelial cells, hydrolyzes the TG of chylomicron and very low density lipoprotein. It is worth noticing that activation of PPARα and PPARγ may also participate in the regulation of the above genes [6,36]. Therefore, in order to determine the specificity, we measured both PPAR isoforms in liver and adipose tissue and found that the expression of PPARα and PPARγ genes were not affected (data not shown), and consequently, the above-observed regulations to genes are attributed to PPARδ. Weight loss has been shown to improve insulin-mediated glucose disposal and was generally acknowledged as an improvement of metabolic syndrome treatment. It has been reported that GW501516 prevents weight gain in the ob/ob mice and DIO mice [18], but not in the db/db mice and ZUCKER fa/fa obese rat [31]. In our study, GW501516 did not affect the body weight of MSG mice when compared to that of control model, the reason may be due to the treatment period not being long enough, or maybe due to the dosage used not being adequate to produce pharmacological effects on body weight in the MSG mice, or maybe that GW501516 is not as effective in mice that had already gained body weight. In conclusion, the present study demonstrates that GW501516, a specific PPARδ agonist significantly corrected

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