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Food and Nutritional Science Program, Department of Zoology, The University of Hong Kong, Hong Kong, The People’s Republic of China
2To whom correspondence should be addressed. E-mail: etsli{at}hkucc.hku.hk.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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coactivator-1 
(PGC-1) were determined in diet-induced obese (DIO) rats. Rats were fed high-fat (HF) diets to induce obesity, and the effect of BM was assessed at doses of 0.75, 1.0, or 1.25% (wt:wt). In a dose-response experiment, BM-supplemented rats had lower energy efficiency (g weight gained/kJ consumed), visceral fat mass, serum glucose, and insulin resistance index, but higher plasma norepinephrine than unsupplemented rats (P < 0.05). Hepatic and skeletal muscle triglyceride concentrations were lower in supplemented HF diet–fed rats than in unsupplemented HF diet–fed rats (P < 0.05). An HF diet supplemented with BM elevated activities of hepatic and muscle mitochondrial carnitine palmitoyl transferase-I (CPT-I) and acyl-CoA dehydrogenase (AD) (P < 0.05). In another experiment, BM (1.0 g/100 g) lowered visceral fat mass but increased serum adiponectin concentration in HF diet–fed rats (P < 0.05). In the final study, rats were fed the HF diet with 0, 1.0 or 1.25% BM. Both groups of BM-supplemented rats had higher uncoupling protein 1 in brown adipose tissue (P < 0.05) and uncoupling protein 3 in red gastrocnemius muscle (P < 0.05), measured by Western blotting and RT-PCR, than the controls. The expression of the transcription coactivator PGC-1 in both tissues was also significantly elevated in the BM-supplemented rats (P < 0.05). The present results suggest that decreased adiposity in BM-supplemented rats may result from lower metabolic efficiency, a consequence of increased lipid oxidation and mitochondrial uncoupling.
KEY WORDS: • bitter melon • carnitine palmitoyl transferase-I • acyl-CoA dehydrogenase • adiponectin • uncoupling proteins
Momordica charantia (family Cucurbitaceae), commonly known as karela, bitter gourd, balsam pear, or bitter melon (BM),3 is a popular vegetable that is widely grow in tropical areas. In addition to culinary usage, BM is also used in folklore medicine. Documentation of its pharmacological properties dates back to the 16th century (1). Although BM was found to possess antiviral, antibacterial, anti-HIV, anticancer, and immunomodulatory properties, attention has always been focused on its blood glucose–lowering effect (2). Such an effect was demonstrated in streptozotocin-induced diabetic (3) and diet-induced obese (DIO) rats (4). Despite the need for more information in randomized controlled trials, BM-induced decreases in blood glucose appear to be a recognized effect and no serious adverse effects on humans have been reported (5). The recent research focus has shifted to explore the potential antiobesity effect of BM.
We first reported slower weight gain and less adiposity in rats fed a high-fat (HF) diet supplemented with BM. The reduced fat content appears to be a generalized effect across different tissues including visceral fat, liver, and skeletal muscle. For instance, hepatic triglyceride (TG) level as well as steatosis scores of liver sections were significantly lower in DIO rats fed BM (6). The lower hepatic TG level agrees with results reported by others (7). Although the mechanism remains elusive, cell culture studies suggest the involvement of peroxisome proliferator-activated receptor (PPAR) activation (8) as well as inhibition of TG synthesis and apolipoprotein B secretion (9). Because the slower weight gain of BM rats is not caused by lower energy intake or apparent fat absorption (4), we hypothesize that involvement of uncoupling proteins (UCPs) might produce such a generalized effect on energy homeostasis.
In rodents, brown adipose tissue (BAT) is important for the regulation of thermogenesis (10). UCP in BAT could be induced by cold exposure as well as an HF diet (11). The UCP level can be influenced by many factors including sympathetic activation (12) and the adipocytokine, adiponectin (13). Many forms of UCPs exist and they can be found in tissues other than BAT. For instance, UCP3 also possesses the proton conductance property (14,15). It is lipid sensitive and plays a prominent role in fatty acid catabolism in skeletal muscle (16).
Accordingly, the present study aimed to examine the influence of BM supplementation on physiological and biochemical events associated with UCP expression.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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16 g powder was obtained from 1 kg fresh fruit. The freeze-dried powder was stored at –80°C. Experimental diet. The formulation of diets was based on the AIN-93G recommendation (17) and reported in detail in an earlier study (4). The low-fat (LF) diet contained 7 g corn oil/100 g diet. The HF diet contained 30 g fat/100 g diet, i.e., 7 g corn oil + 23 g Crisco shortening (Procter & Gamble). The additions of shortening and freeze-dried BM juice powder were at the expense of cornstarch.
Animals. All animal protocols were approved by the committee on the use of living animals in teaching and research, The University of Hong Kong. Male Sprague-Dawley rats, at 6 wk of age, were obtained from the animal unit of the Medicine Faculty. They were housed individually in rectangular hanging wire cages. The room was under a 12-h light:dark cycle, with lights on at 0700 h; the temperature was maintained at 22°C. Rats had free access to water and consumed laboratory food (Lab Diet-The Richmond Standard, PMI Nutrition International) until they were assigned to experimental diets.
Expt. 1. Rats (n = 35) were randomly assigned to one of the following diets for 7 wk: LF, HF, HF + 0.75% BM, HF + 1.0% BM, and HF + 1.25% BM. This dose-response experiment was designed primarily to examine the effect of BM supplementation on the activities of 2 mitochondrial enzymes crucial to lipid oxidation: carnitine palmitoyl transferase-I (CTP-I) and acyl-CoA dehydrogenase (AD). At the end of the feeding study, rats were deprived of food for 8 h before they were killed by decapitation.
Expt. 2. This experiment explored the relation between adiposity and serum adiponectin concentration. Rats (n = 39) were fed the LF or HF diet with or without freeze-dried BM juice powder (1.0%) for 6 wk.
Expt. 3.
The focus of this experiment was on the expression of UCP1, UCP3, and the transcriptional coactivator peroxisome proliferator-activated receptor- coactivator-1 (PGC-1). Because the primary focus was on the effect of BM supplementation in rats with substantial fat accumulation, the design differed slightly from the previous 2 experiments. Rats were fed an HF diet for 3 wk. At the beginning of wk 4, the rats (n = 8/group) were randomly assigned to HF, HF + 1.0% BM, or HF + 1.25% BM and fed their respective diets for an additional 6 wk. At the end of the experiment, rats were killed by decapitation. Plasma and serum were collected and stored at –80°C. Liver, skeletal muscle (soleus, red gastrocnemius, tibialis anterior), white adipose tissue, and BAT were exercised, snap-frozen in liquid nitrogen, and stored at –80°C.
Measurements. Plasma glucose was assayed immediately using a commercially available glucose oxidase kit (kit No. 510-A, Sigma Chemical). An ELISA kit was used to measure serum insulin (Mercodia rat insulin 10–1124-01, Mecordia AB) and adiponectin (Catalog # K1002–1, B-Bridge International). An insulin resistance index (IRI) was calculated as the product of insulin and glucose concentration (10–3 pmol insulin · mmol glucose · L–2) for each individual rat (18). Cytosolic heart-type fatty acid binding protein (H-FABP) was determined with a sandwich ELISA kit (HK403, HyCult Biotechnology). Plasma norepinephrine (NE) was measured with an HPLC-electrochemical method as described in detail earlier (6). The within-assay CV was 5.6% for NE. The between-assay CV was 8.1%. Recovery was 91.7%.
Tissue homogenization and preparation of mitochondrial fractions for enzyme assays. The mitochondrial fraction was isolated according to the method described by Cannon and Lindberg (19). The homogenate was centrifuged at 700 x g for 15 min, the supernatant retained, and the pellet was rehomogenized and centrifuged. The combined supernatant was centrifuged at 15,000 x g for 30 min (Eppendorf Centrifuge 5415 D). The resulting supernatant was used to measure FABP. The mitochondrial pellet was resuspended in 1 mL ice-cold 0.25 mol/L sucrose containing 1 mmol/L EDTA, 3 mmol/L Tris-HCl (pH 7.0), and 0.1% Triton X-100, to give a protein concentration of 20–25 g/L.
Assay of CPT-I activity. Total activity of CPT-I was measured in the direction of palmitoyl-CoA formation (20). The assay was based on measurement of the initial rate of total CoASH formed by the 5,5'-dithio-bis-(2-nitrobenzoic acid) reaction in the presence and absence of acyl-CoA. Rates were followed for 1–3 min at 25°C by monitoring changes in absorbance at 412 nm (UV-160A UV/visible spectrophotometer, Shimadzu). The molar extinction coefficient of 13,600 (mol/L)–1cm–1 for 5'-thio-2-nitrobenzoate was used for calculations. One unit of CPT activity was defined as the amount of enzyme catalyzing the release of 1 nmol CoASH/(min · mg protein).
Assay of acyl-CoA dehydrogenase (AD) activity. The method of Thorpe (21) was followed. The assay is based on the use of phenazine methosulfate to mediate the transfer of reducing equivalents to 2,6-dichlorophenolindophenol (DCPIP). Palmitoyl CoA served as substrates for the enzyme assays. The molar extinction coefficient of 21,000 (mol/L)–1cm–1 for DCPIP was used for calculations.
Western blot analysis. Mitochondrial protein from BAT and gastrocnemius muscle was prepared as previously described (22). Samples were centrifuged at 700 x g for 10 min to pellet the nuclei. Supernatants were centrifuged at 13,000 x g for 15 min to pellet the mitochondria. Mitochondria were resuspended in homogenization buffer without bovine serum albumin.
SDS-PAGE was carried out according to Laemmli (23). The protein concentration in the samples was measured using the Bradford Reagent (Bio-Rad). Mitochondrial protein (30 µg for BAT and 40 µg for gastrocnemius muscle) was loaded in each lane and separated on a 12.5% SDS-PAGE gel. Membranes were blocked with 5% (wt/v) nonfat dried milk powder in TBST buffer (50 mmol/L Tris-HCL, pH 7.4, 0.15 mol/L NaCl, 0.05% Tween 20) for 2 h. The membranes were then incubated with anti-UCP1 (AB 3038, Chemicon), anti-UCP3 (AB3046, Chemicon) or anti-tubulin (sc-5286, Santa Cruz) antibodies at a dilution of 1:1000, 1:2000, or 1:5000, respectively, for 2 h. -Tubulin was used as an internal control to verify equal loading of the protein. Horseradish peroxidase–conjugated secondary antibodies (sc-2004, sc-2061, Santa Cruz) were used. Membranes were developed using ECL Western blotting reagents (Amersham Biosciences) and the chemiluminescence was detected on HyperfilmTM ECLTM (Amersham Biosciences). The band intensities were measured with NIH Image, version 1.60.
RNA isolation and RT-PCR.
RNA was extracted by the TRIZOL reagent according to manufacturer’s protocol (Invitrogen). First-strand cDNAs were synthesized from 3 µg of total RNA using 200 U of M-MLV reverse transcriptase (28025–021, Invitrogen), oligo (dT) primers (18418–012, Invitrogen), dNTP mix (US77212, Amersham Biosciences), and RNaseOUT Recombinant Ribonuclease Inhibitor (10777–019, Invitrogen). The PCR was performed in the presence of Taq DNA polymerase with PCR buffer and MgCl2 (Bio-firm), dNTP mix (US77212, Amersham Biosciences), forward and reverse primers (Invitrogen). The PCR primers used were as follows: UCP1, forward, 5'-TGT CTT AGG GAC CAT CAC CA-3', and reverse, 5'-TGA CCT TCA CCA CCT CTG TG-3'; UCP3, forward, 5'-GAT GAC TCC GCC CTG TAA TG-3', and reverse, 5'-ATC TTC CCA ACA CCC TGC TG-3'; PGC-1, forward, 5'-AAC AAG CAC TTC GGT CAT CC-3', and reverse, 5'-AGA GCA AGA AGG CGA CAC AT-3'; ß-actin, forward, 5'-GGA AAT CGT GCG TGA CAT TA-3', and reverse, 5'-AGG AAG GAA GGC TGG AAG AG-3'.
Statistical methods. Data are expressed as means ± SEM. Analyses were carried out using SPSS for Windows, version 12.0. Data were analyzed by ANOVA (Expts. 1 and 3) followed by post hoc Duncan’s multiple range tests to determine treatment effect and compare differences among group means. For Expt. 2, 2-way ANOVA was used to determine the effect of fat and BM. In adiponectin analysis, visceral fat mass was used as a covariate to eliminate the effects due to the difference in visceral fat mass. Correlation analysis was performed to determine the relations between 2 variables. mRNA expression in Expt. 3 was analyzed with 1-way ANOVA using ß-actin as covariate. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Serum adiponectin and plasma NE. The plasma NE concentration of supplemented HF diet-fed rats was higher than that of the unsupplemented rats (Table 1, P < 0.05) and did not differ among rats administered different doses of BM.
Both dietary fat and BM supplementation affected serum adiponectin (Table 2). Visceral fat mass and serum adiponectin were positively correlated (r = 0.660–0.801, P < 0.05) in all groups other than the HF group (Fig. 1).
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FABP, CPT-I, and AD. Rats fed the HF diet had higher cytoplasmic FABP concentration in soleus, but not in red gastrocnemius (Table 3, P < 0.05). BM supplementation to the HF diet did not affect the FABP concentration in either type of muscle.
BM supplementation at a dose 
0.75% elevated the activities of hepatic CPT-I and AD (Table 3, P < 0.05), compared with the unsupplemented HF diet–fed rats. Hepatic CPT-I activity correlated positively with plasma NE (r2 = 0.24, n = 35, P < 0.01). Hepatic AD activity also correlated with the NE concentration (r2 = 0.325, n = 35, P < 0.01). Moreover, levels of hepatic TG correlated negatively with activities of CPT-I (r2 = 0.123, n = 35, P < 0.05) and AD in liver (r2 = 0.130, n = 35, P < 0.05).
BM supplementation to the HF diet elevated the activities of CPT-I and AD in soleus muscle (Table 3, P < 0.05). There was a positive relation between plasma NE and soleus AD activity (r2 = 0.155, P < 0.05). In red gastrocnemius, BM supplementation elevated the activities of CPT-I (P < 0.05), and had a dose-response effect. However, the activity of AD did not differ among the 5 groups.
Assays of CPT-I and AD were repeated using oleoyl CoA (18:1-CoA) and arachidonoyl CoA (20:4-CoA) as substrates. The results (data not shown) were similar to those presented in Table 3 when 16:0-CoA was used as substrate.
UCP1, UCP3, and PGC-1 expression.
Rats supplemented with BM had higher UCP1 mRNA in BAT and UCP3 mRNA in red gastrocnemius muscle (Fig. 2). Relative increases were 50% (Table 4). Protein levels of UCP1 and UCP3 were higher in both BM-supplemented groups by Western blot analysis (Fig. 3, P < 0.05).
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mRNA in BAT and red gastrocnemius were elevated in the BM-supplemented groups (Fig. 2, P < 0.05), although there was no dose-response effect (Table 4). Moreover, the PGC-1 mRNA level was positively correlated with the UCP1 mRNA level in BAT (r2 = 0.766, P < 0.001) as well as with the UCP3 mRNA level in red gastrocnemius (r2 = 0.438, P < 0.001). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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BM supplementation to the HF diet–fed rats lowered hepatic and muscle TG concentrations and improved insulin sensitivity (4,6). We are intrigued by the general reduction in TG level in major organs upon BM supplementation and thus proposed to explore several key candidates in the control of lipid oxidation and energy dissipation. The possibility that BM might influence lipid oxidation was first raised by Chao and Huang (8). An ethyl acetate BM extract significantly raised acyl CoA oxidase activity as well as its mRNA expression in H4llEC3, a PPAR-responsive murine hepatoma cell line. Thus, we measured the activities of 2 important oxidative enzymes, CPT-I and AD, in liver and skeletal muscle of HF diet–fed rats supplemented with different levels of BM. BM supplementation elevated the activities of both enzymes in liver, and gastrocnemius and soleus muscles (Table 3). At the level of 0.75% BM, the increases in CPT-I and AD activities ranged from 36 to 57% and 25 to 31%, respectively, compared with the unsupplemented rats. The reduction in lipid content of liver and gastrocnemius muscle among the BM-supplemented groups was 40%. The data suggest that BM supplementation enhanced mitochondrial transport of long-chain fatty acids, and the decreases in organ lipid content are associated with the enhancement in activity of AD, a key oxidative enzyme.
Because lipid oxidation could be influenced by substrate provision, plasma NE was also measured. Consistent with our earlier observation (6), plasma NE was elevated in BM-supplemented rats. Furthermore, plasma NE concentration correlated positively with activities of the hepatic and soleus oxidative enzymes but negatively with TG content. Hence, the activation of the sympathetic system might be an integral part of the mechanisms whereby BM exerts its effect on lipid catabolism.
The possibility that BM supplementation might influence the machinery for fatty acid transport into intracellular organelles was also explored by measuring cytoplasmic FABP (24,25). The FABP found in muscle belongs to the H-FABP. Physiological, nutritional, and pharmacological manipulations in fatty acid utilization are usually accompanied by changes in cytoplasmic FABP (26). Compared with rats fed an LF diet, feeding rats an HF diet led to varying increases in cytoplasmic FABP in skeletal muscle, with a significant increase in soleus but not gastrocnemius (Table 3). All 3 doses of BM, however, could not increase the FABP level. These data tend to suggest that the delivery of fatty acids may not be a limiting factor in rats fed an HF diet. Although dietary fat induced an increase in FABP in soleus muscle, the elevated capacity might be more than enough to meet substrate requirement upon BM supplementation. This argument is in keeping with the view that H-FABP is in relative excess and might play a permissive role in skeletal muscle fatty acid transport (27).
The increases in lipid oxidative enzyme activities in conjunction with reductions in tissue lipid content suggest the involvement of mitochondrial uncoupling-related energy dissipation because fuel oxidation is normally controlled by energy demand. The involvement of 2 UCPs, UCP1 and UCP3, was examined. In rodents, the role of BAT UCP1 in the dissipation of energy as heat via the proton conductance pathway is well established (28). The elevated level of UCP1 in BAT of BM-supplemented rats strongly supports the notion that mitochondrial coupling was reduced. The data are in keeping with the observations that rats overexpressing UCP1 are more resistant to weight gain (29). Elevated NE and PGC-1 levels in BM-supplemented rats might account for these changes. PGC-1 can interact with nuclear transcriptional factors and is a master regulator of mitochondrial biogenesis and oxidative phosphorylation (30). This transcription coactivator is stimulated by ß-agonists (31), whereas the latter are by themselves strong activators of UCP1 expression (32). Sympathetic activation and the strong correlation between mRNA levels of PGC-1 and UCP1 in BAT suggest a substantial effect of BM on thermogenesis, energy expenditure, and energy homeostasis.
Because skeletal muscle is a major organ that oxidizes lipid, the increase in UCP3 in gastrocnemius of BM-supplemented rats is of interest (Fig. 2). UCP3, a homologue of UCP1 found in skeletal muscle, was implicated in uncoupling activity (33). Although transgenic mice overexpressing UCP3 do gain less weight, have less epididymal fat, and are more glucose tolerant (34,35), UCP3 knockout mice are not more susceptible to obesity and type 2 diabetes (36). Whether UCP3 could mediate thermogenesis is still under debate, but a role in lipid metabolism is emerging. There is evidence that mitochondrial handling of fatty acids is a primary physiological function of UCP3 (16). To this end, it is pertinent to point out that in BM-supplemented rats, the PGC-1 mRNA level correlated positively with UCP3 mRNA expression.
Adipocytokines are implicated in fat metabolism and energy homeostasis. In particular, adiponectin bears a negative relation with adiposity (37) and it also has an insulin-sensitizing effect (38). Thus, it is not surprising to find a higher level of adiponectin in BM-supplemented rats (Table 2). In rats fed the LF diet, serum adiponectin correlated positively with visceral fat mass (Fig. 1A). Such a relation, however, did not exist among the HF rats, suggesting possible disruption in adiponectin secretion and/or metabolism. As the rate of weight gain in the HF rats was slowed by BM supplementation, the positive relation reappeared (Fig. 1C). Thus, BM might have some modulatory effect on white adipose tissue physiology. Adiponectin could influence UCP expression. Peripheral injection of adiponectin reduced adiposity and simultaneously upregulated UCP1 mRNA expression in BAT and UCP3 mRNA expression in skeletal muscle (39). The latter effect is likely to act via an increase in fatty acid oxidation (40). Together with a decrease in muscle TG concentration (Table 3), the evidence is supportive of the notion that BM probably exerts its antiobesity effect via integrated events involving lipid oxidation and energy uncoupling.
The present study reported for the first time the effect of BM supplementation on the expression of UCPs and the coactivator PGC-1 in rats. These results as well as those of others offer new insight into the biological effects of a common vegetable. The bioactive ingredient(s) in BM are capable of influencing cellular physiology through transcriptional changes in animals and cell culture studies. To further establish the beneficial effects of BM, more studies are required to understand the mechanisms of action. It will be useful to confirm the effect of BM on hepatic TG synthesis and secretion (9) as well as the involvement of PPARs (8) in animal studies. Although 0.75% appears to be the threshold dose, the dose response effect should be fine-tuned because larger amounts of BM did not increase enzyme activity or decrease the tissue TG concentration (Table 3). How the sympathetic system is activated is also an important and relevant question. Furthermore, the effectiveness, dosage, duration, biomarker, side effects, and safety of BM must be assessed in well-designed human trials.
In summary, BM appears to have great potential for use as a dietary adjunct in body weight management. In addition to the improvement in glucose tolerance, the lipid-lowering effect would be beneficial for obese patients with nonalcoholic steatohepatitis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AD, acyl-CoA dehydrogenase; BAT, brown adipose tissue; BM, bitter melon; CPT-I, carnitine palmitoyl transferase-I; DCPIP, 2,6-dichlorophenolindophenol; DIO, diet-induced obese; FABP, fatty acid binding protein; G, gastrocnemius; HF, high-fat; H-FABP, heart-type FABP; IRI, insulin resistance index; LF, low-fat; NE, norepinephrine; PGC-1, PPAR- coactivator-1 ; PPAR, peroxisome proliferator-activated receptor; TG, triglyceride; UCP, uncoupling protein.
Manuscript received 6 June 2005. Initial review completed 28 June 2005. Revision accepted 1 August 2005.
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