Toxicology and Applied Pharmacology 207 (2005) 160 – 169 www.elsevier.com/locate/ytaap
Anti-diabetic action of Punica granatum flower extract: Activation of PPAR-g and identification of an active component Tom H.W. Huanga,1, Gang Penga,1, Bhavani P. Kotaa, George Q. Lia, Johji Yamaharab, Basil D. Roufogalisa, Yuhao Lia,* a
Herbal Medicines Research and Education Centre, Faculty of Pharmacy, A15, The University of Sydney, NSW 2006 Australia b Pharmafood Institute, Kyoto, Japan Received 1 October 2004; accepted 16 December 2004 Available online 16 February 2005
Abstract Peroxisome proliferator-activated receptor (PPAR)-g activators are widely used in the treatment of type 2 diabetes because they improve the sensitivity of insulin receptors. Punica granatum flower (PGF) has been used as an anti-diabetic medicine in Unani medicinal literature. The mechanism of actions is, however, unknown. In the current study, we demonstrated that 6-week oral administration of methanol extract from PGF (500 mg/kg, daily) inhibited glucose loading-induced increase of plasma glucose levels in Zucker diabetic fatty rats (ZDF), a genetic animal model for type 2 diabetes, whereas it did not inhibit the increase in Zucker lean rats (ZL). The treatment did not lower the plasma glucose levels in fasted ZDF and ZL rats. Furthermore, RT-PCR results demonstrated that the PGF extract treatment in ZDF rats enhanced cardiac PPAR-g mRNA expression and restored the down-regulated cardiac glucose transporter (GLUT)-4 (the insulin-dependent isoform of GLUTs) mRNA. These results suggest that the anti-diabetic activity of PGF extract may result from improved sensitivity of the insulin receptor. From the in vitro studies, we demonstrated that the PGF extract enhanced PPAR-g mRNA and protein expression and increased PPAR-g-dependent mRNA expression and activity of lipoprotein lipase in human THP-1-differentiated macrophage cells. Phytochemical investigation demonstrated that gallic acid in PGF extract is mostly responsible for this activity. Thus, our findings indicate that PPAR-g is a molecular target for PGF extract and its prominent component gallic acid, and provide a better understanding of the potential mechanism of the anti-diabetic action of PGF. D 2004 Elsevier Inc. All rights reserved. Keywords: Punica granatum; Gallic acid; Diabetes; Hyperglycemia; PPAR-g; Zucker diabetic fatty rats
Introduction Diabetes mellitus is a metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. It is well documented that chronic Abbreviations: BW, body weight; GA, gallic acid; GLUT, glucose transporter; LpL, lipoprotein lipase; OZR, obese Zucker rats; PGF, Punica granatum flowers; PPAR-g, peroxisome proliferator-activated receptor-g; PPREs, peroxisome proliferator response elements; RT-PCR, reverse transcriptase polymerase chain reaction; TZDs, thiazolidinediones; ZDF, Zucker diabetic fatty; ZL, Zucker lean. * Corresponding author. Fax: +61 2 9351 8638. E-mail address:
[email protected] (Y. Li). 1 These authors contributed equally. 0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.12.009
hyperglycemia of diabetes is associated with long-term damage, dysfunction, and eventually the failure of organs, especially the eyes, kidneys, nerves, heart, and blood vessels. Since antiquity, diabetes mellitus has been treated with plant medicines. Recent scientific investigation has confirmed the efficacy of many of these preparations, some of which are remarkably effective (Grover et al., 2002). Punica granatum (PG) Linn commonly known as pomegranate is a small tree belonging to the Punicaceae family. Pomegranate is grown in Iran, India, USA, and most near and far east countries. Pomegranate juice and wine have become increasingly popular because of the attribution of important biological actions to this plant (Schubert et al., 1999),
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including cardiovascular protection (Aviram et al., 2002). In traditional Ayurvedic medicine, all parts of PG are used for the treatment of various disorders. PG flowers (PGF) have been prescribed in Unani and Ayurvedic medicines for the treatment of diabetes (Jurjani, 1878; Majoosi, 1889). Recently, it has been reported that the aqueous ethanolic (50% v/v) extract from PGF showed hypoglycemic activity in a diabetic animal model (Jafri et al., 2000). The action mechanism for the anti-diabetic effects of PGF, however, is still unknown. The peroxisome proliferator-activated receptors (PPARs) form a subfamily of the nuclear receptor superfamily and three isoforms encoded by separate genes have been identified so far: PPAR-a (NR1C1), PPAR-h/y (also referred to as NUC1; NR1C2), and PPAR-g (NR1C3) (Gilde and Van Bilsen, 2003). PPARs are ligand-dependent transcription factors that regulate target gene expression by binding to specific peroxisome proliferator response elements (PPREs) in enhancer sites of regulated genes (Bishop-Bailey and Wray, 2003). PPARs are activators of key metabolic pathways that control fatty acid oxidation, adipocyte differentiation, and insulin sensitivity (Francis et al., 2003). For non-insulin-dependent diabetes mellitus (NIDDM) patients, insulin resistance develops as a result of pancreatic h-cell dysfunction and diminished ability to respond to circulating insulin (Dandona, 2002). Activation of PPAR-g by agonists such as thiazolidinediones (TZDs) helps improve endogenous insulin sensitivity (Houseknecht et al., 2002). In the current study, we investigate the effects and action mechanism of the methanol extract from PGF on hyperglycemia in Zucker diabetic fatty (ZDF) rats, a genetic model for obesity and NIDDM (Kasiske et al., 1992), and on receptor function in a cell line system. Furthermore, we identified the main components of PGF extract mostly responsible for the activity by using phytochemical methods.
Materials and methods Chemicals and reagents. Chemicals used in phytochemical procedures were of analytical grade. Anti-actin primary antibody, gallic acid (GA), GW1929, GW9662, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (Australia). Silica gel 60 was obtained from Merck. Extraction, partitioning, and fractionation of the extract from PGF, and isolation and identification of the active component. Dried PGF was extracted at room temperature three times with 5 volumes of methanol (W/V). The solvent was evaporated under reduced pressure below 50 8C to give dried methanolic extract (yield: 40%). The dried extract was partitioned between ethyl acetate (EtOAc) and water (1:1). The active EtOAc part was further fractionated with normal phase silica gel column chromatography using methanol– chloroform mobile phase into eight fractions (Fr A to Fr H,
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see Fig. 3a). The most active Fraction A was subjected to HPLC analysis using an acidic water–methanol mobile system in gradient mode. Alltech C18 (4.6 250 mm) column connected to a Shimadzu LC-10ATVP liquid chromatography pump was used to analyze the extract. Semi-preparative (10 Am, 10 250 mm, Alltech C18) column was used in isocratic mode to isolate the active fractions. Separation was achieved using a Shimadzu HPLC system. Finally, the isolated compound was characterized and quantified by NMR, LC/MS, GC/MS, and HPLC methods. Animals. All animal experimental procedures were approved by the Animal Ethics Committee of the University of Sydney, Australia. Male Zucker lean (ZL) rats (Fa/?) and ZDF rats (fa/fa) aged 13–15 weeks (Monash University Animal Services, Victoria, Australia) were housed in an airconditioned room at 23 F 1 8C with a 12-h light/dark cycle and were provided with standard food and water ad libitum. Animals were allowed free access to standard food and water for 1 week before starting the experiments. Extract administration and measurement of plasma glucose levels. Blood was taken from the orbital sinus of nonfasted and fasted ZL and ZDF rats under halothane anesthesia for determination of plasma glucose levels (as references for grouping of animals) before treatment (Week 0). Animals were divided into ZL control (receiving vehicle only), ZL PGF (receiving PGF extract), ZDF control (receiving vehicle only), and ZDF PGF (receiving PGF extract) groups (five rats each group). Body weight (BW) was measured twice a week and used for regulating the dose of the sample. Test sample (500 mg/kg, suspended in 5% acacia) or vehicle was given orally by gavage once daily for 6 weeks. To test the response of plasma glucose levels to exogenous glucose stimulation, vehicle or test sample was given after animals were fasted (18 h) at Week 5. One hour later, glucose (2 g/kg) was given orally. Plasma glucose levels were determined before (0 min), then 30 and 180 min after glucose loading. The kit used to determine plasma glucose was purchased commercially (Wako, Osaka, Japan). Tissue culture. The THP-1 human monocytic cell line was a kind gift from Dr. Asne Bauskin (St Vincent’s Hospital, Sydney, Australia). Cells were grown in RPMI 1640 containing l-glutamine supplemented with penicillin (100 U/ml)/streptomycin (100 Ag/ml), and 10% v/v fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% O2 at 37 8C (Invitrogen, Australia). The THP-1 monocytes were differentiated into macrophages by treatment with 1 Amol/l PMA for 2 days (Li et al., 2002). Gene expression analysis. After rats were sacrificed under halothane anesthesia (non-fasted condition) at Week 6, the heart was rapidly excised, and the left ventricle was frozen in
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liquid nitrogen and stored at 80 8C for study of mRNAs. Total mRNA was prepared separately from the left ventricle of individual rats and from THP-1-derived macrophage using TRIzol (Invitrogen, Australia). The relative levels of specific mRNAs were assessed by reverse transcriptase polymerase chain reaction (RT-PCR), as described previously (Abe et al., 2002). Single-stranded cDNA was synthesized from 1 Ag of total RNA using SuperScript II Rnase H Reverse Transcriptase as per instructions of the manufacturer (Invitrogen, Australia). PCR was performed on a thermocycler PTC-200 DNA engine (MJ Research Inc, MA, USA). The required cDNA was synthesized with the Platinum Pfx DNA Polymerase (Invitrogen, Australia). The genes examined were GLUT-4 (D28561; 449 bp; sense: 5V-AGGCACCCTCACTACCCTTT-3V and antisense: 5V- GA C A GA AGG GC AA C AG AA GC -3V), PPAR-g (NM013124; 416 bp; sense: 5V-TCATGACCAGGGAGTTCCTC-3V and antisense: 5V-TCAGCGACTGGGACTTTTCT-3V), and h-actin (NM031144; 228 bp; sense: 5V-AGCCATGTACGTAGCCATCC-3V and antisense: 5VCTCTCAGCTGTGGTGGTGAA-3V) from the left ventricle. The results shown are representative from a pool RNA population of 5 rats per group. For the THP-1-derived macrophage, the LpL and PPAR-g mRNA expression was determined as described previously (Li et al., 2002) with the cells treated for 2 days with positive control agonist (GW1929, 3 AM) and test samples. The PPAR-g antagonist GW9662 (1 AM) was added 1 h prior to addition of positive control or test samples. The genes of interest are LpL (NM000237, 428 bp; sense: 5V-CCCTAAGGACCCCTGAAGAC-3V and antisense: 5V-TGGATCGAGGCCAGTAATTC-3V), PPAR-g (L40904, 382 bp; sense: 5V-GAGCCCAAGTTTGAGTTTGC-3V and antisense: 5V-TGGAAGAAGGGAAATGTTGG-3V), and h-actin (NM001101, 629 bp; sense: 5V-GGAGTAACCAGGTCGTCCAA-3V and antisense: 5V-GAAGGTGCCCAGAATACCAA-3V). The PCR samples were electrophoresed on 3% agarose gels and stained with ethidium bromide. The gel images were digitally captured with a CCD camera and analyzed with the ImageJ 1.29 (NIH, USA). RT-PCR values are presented as a ratio of the specified gene signal in the selected linear amplification cycle divided by the h-actin signal. Protein extraction and semi-quantitative immunoblotting. Immunoblots were conducted as described previously (Li et al., 2002). The THP-1-derived macrophages were treated with vehicle, GW1929 (3 AM), and the test samples, and incubated for 48 h before lysing for 1 h. The PPAR-g antagonist GW9662 (1 AM) was added 1 h prior to addition of positive control or test samples. The protein contents were determined using the Bradford assay method (Bradford, 1976) and were loaded onto discontinuous gradient SDS–PAGE (10%) gels. After electrophoresis, the protein was transferred to PVDF membranes and blocked overnight. The membrane was incubated first with antihuman PPAR-g
rabbit polyclonal primary antibody (1:500 dilution; Santa Cruz Biotechnology, USA) then with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Promega, USA) for 60 min. Proteins were detected by chemiluminescence (Roche). The membranes were exposed to X-ray film (Kodak, USA) and developed using the SRX-101A Xray developer (Konica, Taiwan). Quantitation of the results was performed by scanning the X-ray film with Molecular Analyst software (version 2.1.2, Biorad, USA) followed by densitometry with the public domain software, NIH Image, version 1.62. To quantify the protein expression of PPAR-g, the membrane loadings were normalized with actin. The membranes were re-probed with anti-actin primary antibody (Sigma, Australia) after stripping with 0.2 M sodium hydroxide for 5 min. The membranes were washed, reincubated with the same secondary horseradish peroxidase antibody, and detected using the same procedure as described above. Determination of extracellular LpL activity. LpL activity was conducted as described previously (Li et al., 2002). THP-1 macrophages were cultured for 2 days with GW1929 (3 AM) or the test samples. The PPAR-g antagonist GW9662 (1 AM) was added 1 h prior to addition of positive control or test samples. Extracellular LpL activity was determined in the supernatants using the Confluolip kit according to manufacture’s instruction (Progen Biotechnik, Germany). Levels of LpL activity were expressed as percentages of basal values. Data analysis. All results are expressed as means F SEM. Data were analyzed by 1-factor analysis of variance (ANOVA). If a statistically significant effect was found, the Newman–Keuls test was performed to isolate the difference between the groups. P values less than 0.05 ( P b 0.05) were considered as indicative of significance.
Results PGF extract improves oral glucose tolerance in ZDF rats We first investigated the effect of PGF extract on plasma glucose levels in ZL and ZDF rats. As shown in Fig. 1, compared to ZL control, ZDF control showed slightly higher plasma glucose levels under fasted conditions (0 min), but the plasma glucose levels were much higher at 30 and 180 min after glucose loading, suggesting impaired glucose tolerance in ZDF rats. Five-week treatment with PGF extract inhibited the increase in glucose levels in ZDF rats at 30 and 180 min after glucose loading, whereas it had no effect in fasted ZDF rats. The treatment showed little effect on plasma glucose levels in fasted and glucose-loaded ZL rats. In contrast, 6-week administration of PGF extract did not significantly change body weight in ZL or ZDF rats
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Fig. 1. Plasma glucose response to oral glucose administration in Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats after 5-week treatment with P. granatum flower (PGF) extract. PGF extract (500 mg/kg, suspended in 5% acacia) or vehicle was administered by oral gavage once daily for 5 weeks. Glucose solution (2 g/kg) was given orally after the animals were fasted for 18 h. Plasma glucose levels were determined with a commercial kit, before and 30 and 180 min after glucose loading. All values are means F SEM (n = 5). *P b 0.05. Control (Con).
(Week 0: ZL control 268.8 F 11.4 g vs. ZL PGF 269.4 F 14.6 g; ZDF control 401.6 F 8.4 g vs. ZDF PGF 416.4 F 19.1 g. Week 6: ZL control 316.4 F 10.5 g vs. ZL PGF 321.2 F 12.3 g; ZDF control 426.4 F 6.4 g vs. ZDF PGF 434.0 F 15.2 g). PGF extract up-regulates cardiac PPAR-c mRNA expression and restores the decreased GLUT-4 expression in ZDF rats To help understand the mechanism by which PGF extract improves glucose metabolism in diabetes, we investigated cardiac expression of genes involved in cardiac glucose metabolism. The results showed that GLUT-4 mRNA expression was decreased in the hearts of ZDF rats, whereas PPAR-g mRNA was unchanged (Figs. 2a and b). Treatment with PGF extract for 6 weeks completely restored cardiac GLUT-4 mRNA expression and increased PPAR-g mRNA level in ZDF rats, but had no effect on these markers in ZL rats (Figs. 2a and b). PGF extract enhances PPAR-c mRNA and protein expressions in macrophage cell line As PPAR-g plays an important role in maintaining homeostasis of glucose metabolism in the body, we investigated the effect of PGF extract on PPAR-g protein expression in a macrophage cell line. GW1929 is a selective PPAR-g agonist (Brown et al., 1999). Although Davies et al. have demonstrated that GW1929 did not upregulate PPARg protein expression in rat hepatocytes
(Davies et al., 2002), Sung et al. have more recently demonstrated that GW1929 markedly enhanced PPARg mRNA expression in hepatocytes in early primary culture of rat hepatic stellate cells (Sung et al., 2004). In the present study, the results showed that GW1929 (1 AM) enhanced PPAR-g mRNA (Fig. 5a) and protein (Figs. 3b– d, 4b, and 5b) expressions in THP-1-derived macrophage cell line. The reason for the discrepancy between the hepatocyte results needs to be further clarified. Similar to GW1929, PGF extract also increased PPAR-g mRNA expression (Fig. 5a) and dose-dependently (10, 50, and 100 Ag/ml) enhanced PPAR-g protein expression (by 1.05-, 1.35-, and 1.45-fold, respectively) (Fig. 3b). Effects of fractions and fractionated components from PGF extract on PPAR-c protein expression in a cell line To clarify the components in the PGF extract responsible for induction of PPAR-g expression, we partitioned PGF extract into EtOAc and water parts, and investigated their effect on PPAR-g expression (see flow chart in Fig. 3a). The result showed that the EtOAc part had significant activity on PPAR-g in THP-1-derived macrophage, whereas the water part did not have a significant effect (Fig. 3c). Subsequently, we fractionated the EtOAc part into 8 fractions (Fr A to H, Fig. 3a) with normal-phase silica gel column chromatography and determined their activity. Fraction A was found to be the most active fraction among these (Fig. 3d). By using high performance liquid chromatography (HPLC), we isolated one main component (temporarily named Compound X: purity: N95%, 0.82% content in the PGF extract) from
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Fig. 2. Changes in cardiac glucose transporter (GLUT)-4 and peroxisome proliferator-activated receptor (PPAR)-g mRNA expression profile in animals. PGF extract (500 mg/kg, suspended in 5% acacia) or vehicle was orally administered once daily for 6 weeks. Total mRNAs were separately prepared from the individual left ventricle of the rats using the TRIzol method. (a) GLUT-4 and (b) PPAR-g. The relative levels of specific mRNAs were assessed by RT-PCR and the results were normalized to h-actin. Levels in LZR control were arbitrarily assigned a value of 1.0. All values are means F SEM (n = 5). *P b 0.05. Control (Con.).
Fraction A (see Fig. 4a). Compound X at concentrations of 5, 10, and 50 Ag/ml exhibited a 1.68-, 1.90-, and 2.32-fold increase of PPAR-g protein expression, respectively, whereas at the same concentrations the activities were 0.93-, 1.18-, and 1.36-fold for the remaining fraction lacking Compound X (Fraction A–Compound X) (Fig. 4b). Compound X was identified as GA by comparison with standard GA using HPLC and mass spectrometry (results not shown) and NMR (Fig. 4c) techniques. Both isolated and standard GA showed similar enhancing effects on PPAR-g mRNA and protein expressions, which were completely abolished by a selective PPAR-g antagonist GW-9662 (Leesnitzer et al., 2002) (Figs. 5a–b). PGF extract and GA enhance LpL mRNA expression and the enzyme activity in macrophage cell line To further investigate the effect of PGF extract and GA on PPAR-g-mediated transcription, we investigated the effect of PGF extract and GA on LpL mRNA expression and the enzyme activity in THP-1-derived macrophage cell line. The result showed that PGF extract (50 Ag/ml), GA (300 AM), and positive control GW1929 (3 AM) enhanced LpL mRNA expression and the enzyme activity in the same cell system (Figs. 6a and b). The enhancements were completely suppressed by the selective PPAR-g antagonist GW9662 (Figs. 6a and b).
Discussion The pathogenesis of type 2 diabetes is characterized by the failure of h-cell function to compensate for decreased insulin sensitivity, resulting in a progressive resistance of glucose metabolism to the action of insulin in multiple tissues (Stumvoll and Haring, 2002). One of the therapeutic approaches to improve the insulin resistance is to increase the expression of PPAR-g (Furnsinn and Waldhausl, 2002). Currently, insulin sensitizers or TZDs (pioglitazone and rosiglitazone) are used clinically to ameliorate insulin resistance by increasing insulin-stimulated glucose uptake in skeletal muscle and adipose tissue (Otto et al., 2002). There is substantial evidence of the beneficial effect of P. granatum (PG) extracts against hyperglycemia (Das et al., 2001; Jafri et al., 2000; Nogueira and Pereira, 1986; Zafar and Singh, 1990). However, all the studies to date have been carried out in normal, or chemical-induced diabetic (type 1) animals, or only in vitro. Furthermore, the action mechanism and principle(s) responsible for these effects are still elusive. Since only the flowering part of the plant has been recommended traditionally for the treatment of diabetes (Jurjani, 1878; Majoosi, 1889), the present study investigated the effect of PGF in an accepted type 2 diabetic animal model and in vitro models. Chronic treatment with PGF extract inhibited the increase of plasma glucose levels
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Fig. 3. Effects of test samples on PPAR-g protein expression in THP-1-derived macrophage cell line. (a) Flow chart of extraction, partitioning, and fractionation of the extract from PGF, and isolation of the active component; (b) PGF extract dose-dependently (10, 50, and 100 Ag/ml) enhanced PPAR-g protein expression. GW1929 (3 AM) was used as positive control. (c) The EtOAc part (50 Ag/ml) significantly enhanced PPAR-g protein expression, whereas the water part showed little effect. (d) At 50 Ag/ml, Fraction (Fr) A showed the most potent activity on PPAR-g protein expression. Protein contents were determined using the Bradford assay method with BSA as a standard. The protein extracts were subjected to immunoblotting specific to PPAR-g and quantified by normalizing with actin. Levels in control were arbitrarily assigned a value of 1.0. All values are mean fold increase F SEM (n = 5). *P b 0.05 vs. control (Con).
in glucose-loaded ZDF rats, whereas it neither affected fasted ZDF rats, nor lowered glucose levels in fasted and glucose-loaded ZL rats. The results clearly demonstrated that the response of plasma glucose levels to PGF treatment is different between normal and pathological (insulin insensitive) conditions. The results suggest that PGF extract lowers glucose levels by improving the sensitivity of insulin receptors in type 2 diabetes, rather than alternative mechanisms such as promoting secretion of insulin from h-cell or inhibiting absorption of glucose in the digestive tract. PPAR-g plays an important role in maintaining homeostasis of glucose metabolism in the body. Activation of PPAR-g to improve the sensitivity of insulin receptors is the predominant mechanism for the anti-diabetic efficacy of PPAR-g agonists (Willson et al., 2000). It has been reported that PPAR-g agonists upregulate human macrophage LpL expression (Li et al., 2002). We speculated that PGF extract improves glucose tolerance in ZDF rats via PPAR-g to improve the sensitivity of insulin receptors. To test this hypothesis, we investigated the effects of PGF extract on PPAR-g expression and LpL activity in various in vitro experiments. PGF extract dose-dependently
enhanced PPAR-g mRNA and protein expression in THP-1-derived macrophage cell line. This activity was completely abolished by a selective PPAR-g antagonist GW9662. The results suggest that the PGF extract stimulates the expression of the PPAR-g gene. The fact that the PGF extract-induced enhancements of LpL mRNA expression and the enzyme activity in THP-1differentiated macrophage cells were suppressed by a selective PPAR-g antagonist demonstrates that the transcription factor PPAR-g is also being stimulated by PGF extract to transcribe the LpL gene. Taking both the in vivo and in vitro results into consideration, our results suggest that the PGF extract is a PPAR-g activator which stimulates PPAR-g-mediated transcription of both the PPAR-g and LpL genes. To meet the high energy demands of the contracting muscle, the heart needs to produce a constant and plentiful supply of ATP. This energy is primarily produced by the metabolism of carbohydrates and fatty acids. Glucose is the principle carbohydrate metabolized by the heart. Glucose is taken up by the cardiomyocytes by GLUTs. The predominant GLUTs present in the heart are GLUT-4 and to a lesser extent GLUT-1 isoforms, in which GLUT-4 is insulin-
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Fig. 4. Effect of Compound (Comp) X on PPAR-g protein expression in THP-1-derived macrophage cell line. (a) Flow chart of isolation of Comp X from Fr A. (b) Comp X dose-dependently (5–50 Ag/ml) enhances PPAR-g protein expression, whereas the remaining fraction lacking Comp X (Fr A- Comp X) showed little effect. Protein contents were determined using the Bradford assay method with BSA as standard. The protein extracts were subjected to immunoblotting specific to PPAR-g and quantified by normalizing with actin. Levels in control were arbitrarily assigned a value of 1.0. All values are mean fold increase F SEM (n = 5). *P b 0.05 vs. control (Con.). (c) Chemical structure of GA and NMR results.
Fig. 5. GA- or Compound (Comp) X-induced enhancements of both PPAR-g mRNA (a) and protein (b) expressions were abolished by a selective PPAR-g antagonist GW9662. Total mRNAs were prepared from THP-1-differentiated macrophage cell pellets using TRIzol. The relative levels of specific mRNAs were assessed by RT-PCR. Results were normalized to h-actin. Protein contents were determined using the Bradford assay method with BSA as standard. The protein extracts were subjected to immunoblotting specific to PPAR-g and quantified by normalizing with actin. Levels in control were arbitrarily assigned a value of 1.0. All values are mean fold increase F SEM (n = 5) vs. the control (Con.) without GW9662, #P b 0.05; *P b 0.05.
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Fig. 6. Effects of PGF or GA on lipoprotein lipase (LpL) mRNA expression and the enzyme activity in THP-1-derived macrophage cell line. THP-1differentiated macrophage cells were cultured for 2 days with PGF, GA, or a positive control GW1929. A selective PPAR-g antagonist GW9662 was added 1 h prior to addition of the test samples. At the end of the incubation period, LPL mRNA (a) and activities (b) were determined by RT-PCR or Confluolip kit (Progen Biotechnik), respectively. The results for mRNA were normalized to h-actin and the levels in control were arbitrarily assigned a value of 1.0-fold increase. The levels of LpL activities were expressed as percentages of basal values. All values are mean F SEM (n = 5 for RT-PCR and n = 6 for LpL assay) vs. the control (Con) without GW9662, #P b 0.05; *P b 0.05.
dependent (Abel, 2004; Young et al., 1997). Hearts overexpressing human GLUT-4 glucose transporter had significantly higher rate of glucose uptake after perfusion than control hearts (Belke et al., 2001). In contrast, targeted disruption of GLUT-4 selectively in muscle causes a profound reduction in basal glucose transport and near absence of stimulation by insulin or contraction resulted, suggesting that GLUT-4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis (Zisman et al., 2000). A prominent change that occurs in the diabetic patient is a switch in cardiac energy metabolism, including decreases in glucose metabolism in the myocardium, which may contribute to cardiac dysfunction (Lopaschuk, 2002). Myocardial glucose transport has been shown to be defective in both diabetic humans and streptozotocin-induced diabetic animals (Stanley et al., 1999). In the streptozotocin-induced diabetic animal model, the hearts were found to have a decreased level of GLUT-4 content (Hall et al., 1995). Desrois et al. have reported that GLUT-4 protein level was 28% lower in the heart of GotoKakizaki rat, another model of type 2 diabetes, compared with its age-matched control. In isolated perfused hearts, insulin-stimulated (3)H-glucose uptake rate was decreased by 23% in male Goto-Kakizaki rat heart (Desrois et al., 2004). Insulin-stimulated glucose uptake was also reduced in cardiomyocytes from insulin-resistant type 2 diabetic db/ db mice (Carley et al., 2004). PPAR-g plays an important role in regulating GLUT-4 expression in the heart (Abel,
2004). A stimulatory effect of PPAR-g activation on GLUT4 gene expression has been described in adipocytes (Wu et al., 1998). Chronic oral administration of a novel PPAR-g ligand 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5acetic acid improved the reduction in insulin-stimulated glucose uptake in db/db mice (Carley et al., 2004). Continuous rosiglitazone treatment normalized GLUT-4 protein level in Zucker fatty rat (OZR) heart, leading to an improvement in cardiac glucose uptake during ischemia (Sidell et al., 2002). In the present study, our result demonstrated that cardiac expression of GLUT-4 mRNA in ZDF rats was decreased, although the expression of PPAR-g mRNA was unchanged. Treatment with PGF extract restored cardiac GLUT-4 mRNA in ZDF rats, accompanied by enhanced PPAR-g expression. Therefore, regulation of PPAR-g expression might be associated with restoration of GLUT-4 mRNA by PGF extract in ZDF rats. The results show that plasma glucose level increased greater in ZDF control than in ZL control after glucose meal suggests a decrease in glucose uptake by the tissues, for example, the myocardium of ZDF rats. Treatment with PGF extract significantly reduced the plasma glucose levels in ZDF rats after glucose challenge, demonstrating an improvement by PGF extract of glucose uptake by the tissues including the myocardium. This is consistent with the change in cardiac GLUT-4 mRNA expression. Although the extent of the contribution of myocardial glucose uptake following a glucose challenge to whole-body glucose
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regulation is still not known, our findings in the heart further strengthen the conclusion that the PGF extract activates PPAR-g, which is involved in improvement of impaired glucose tolerance in ZDF rats. Through bioassay guided fractionation experiments, the present study, for the first time, demonstrated that GA present in PGF extract is the constituent mostly responsible for the activation of PPAR-g and induction of PPAR-g in vitro. GA is a naturally abundant plant phenol, a known antioxidant and a component unit of tannins (Sanae et al., 2003). Various biological and pharmacological activities, including anti-inflammatory activity (Kroes et al., 1992), have been reported. Structurally, GA (3,4,5-trihydroxybenzoic acid) does not clearly concur with the structural characteristic common to TZDs, that is, a thiazolidinedione ring with divergent molecular moieties attached (Furnsinn and Waldhausl, 2002). The mechanism for GA to activate PPAR-g and to induce PPAR-g expression needs to be further clarified. Furthermore, the effect of GA in type 2 diabetic animals is still to be confirmed. The discovery of the PPAR-g activator property of PGF extract and GA may also help to explain their effects. It has been demonstrated that GA is capable of inducing endothelium-dependent contraction and inhibition of endothelium-dependent relaxation, which helps prevent vasodilative regulation and inflammatory disorders (Sanae et al., 2003). This is most likely a result of PPAR-g activation since rosiglitazone was shown to reduce the transcriptional coding of inducible endothelial nitric oxide synthase, an enzyme responsible for nitric oxide production (Song et al., 2004). Also, pioglitazone was shown to reduce proinflammatory cytokines, which could explain why pomegranate is used traditionally in Ayurvedic medicine for the treatment of colic and colitis (Schubert et al., 1999; Takagi et al., 2002). Lastly, pomegranate polyphenols were shown in humans and atherosclerotic apolipoprotein E-deficient mice to reduce the capacity of macrophages to oxidatively modify low density lipoprotein (LDL), due to their interaction with LDL to inhibit its oxidation by scavenging reactive oxygen species and reactive nitrogen species, delaying the development of atherosclerotic lesions (Aviram et al., 2002). This can be seen in experiments by Chawla et al. (2001), where the specific loss of PPAR-g activity in macrophages markedly increases atherosclerosis in LDL receptor knockout mice, and the presence of PPAR-g ligands helps control both macrophage-oxidized LDL scavenging and cholesterol efflux. The net effect of these pathways helps lipid removal from the artery wall (Nicholson et al., 2001). In conclusion, our findings have demonstrated a potential mechanism for the traditional anti-diabetic action of PGF through activation of PPAR-g. GA, a component widely distributed in anti-diabetic and anti-inflammatory herbal medicines, is shown to be mostly responsible for this activity in vitro. By applying the techniques of
modern science, we have moved a step closer in unraveling the mystery of the traditional use of PG and the mechanism(s) responsible for its role in the treatment of diabetes.
Acknowledgments We thank Drs. Andrew Cheung, Colin Duke, Van Hoan Tran, and Mr. Bruce Tattam (Mass Spectrometry Unit) for their suggestions and assistance in phytochemistry.
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