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Anti-obesity and anti-insulin resistance effects of tomato vinegar beverage in diet-induced obese mice Kwon-Il Seo,†a Jin Lee,†a Ra-Yeong Choi,a Hae-In Lee,a Ju-Hye Lee,ab Yong-Ki Jeong,c Myung-Joo Kimd and Mi-Kyung Lee*a This study investigated the mechanism of processed tomato vinegar beverage (TVB)-mediated anti-obesity and anti-insulin resistance effects in high-fat diet (HF)-induced obese mice. Oral administration of TVB (14 mL kg1 body weight) to HF-fed mice for 6 weeks effectively reduced the body and visceral fat weight and significantly lowered plasma free fatty acid, triglyceride and hepatic triglyceride levels. TVB significantly increased fecal triglyceride excretion, both phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) and peroxisome proliferator-activated receptor (PPAR)a protein levels in the liver, which were associated with increased fatty acid b-oxidation and carnitine palmitoyltransferase activities in HF-fed mice. TVB improved glucose tolerance, hyperinsulinemia and
Received 21st February 2014 Accepted 14th April 2014
HOMA-IR levels in the HF + TVB group compared to the HF group. Additionally, TVB significantly increased glucokinase activity and decreased glucose-6-phosphatase activity in the liver, which
DOI: 10.1039/c4fo00135d www.rsc.org/foodfunction
enhanced glucose metabolism in obese mice. These results suggest that TVB prevents visceral obesity and insulin resistance via AMPK/PPARa-mediated fatty acid and glucose oxidation.
1. Introduction Obesity, which is currently one of most prevalent metabolic disorders, is the excessive accumulation of adipose tissue in the body. The progression of obesity causes type 2 diabetes mellitus, hyperlipidemia, cardiovascular disease, liver dysfunction, respiratory complications and digestive disturbances.1 Enlarged fat mass and dysregulation of lipid metabolism results in energy imbalance, which is a typical characteristic of obesity. Improving lipid metabolism is one of the most common strategies for treatment of obesity.2 In recent years, natural alternative anti-obesity agents in the form of beverages or tea have been used to treat obesity.3 Accordingly, many studies have been conducted to identify food materials and natural substances with inhibitory effects on obesity. Vinegar has long been consumed as a cooking ingredient and used as a folk medicine.4 Currently, various types of vinegar originating from different crops or fruits are consumed throughout the world, including in Korea and Japan.5 Vinegar has been reported to have antibacterial,6 cardiovascular
protective7 and antitumor effects.8 Tomatoes are a good source of potassium, folate, vitamin A, vitamin C and vitamin E that also contain useful phytochemicals, including carotenoids and polyphenols (a-, b-, g-carotene, lutein, lycopene) and avonoids.9,10 These components of tomatoes have been shown to be benecial for the cardiovascular function.11 Recently, the amount of farming area used for the production of tomatoes has increased, which has resulted in a surplus of fresh tomatoes. Surplus tomatoes are utilized in a variety of processedtomato products, including tomato soup, sauce, ketchup, paste, and juice. Although most tomatoes are commonly ingested in a fresh state, more than half are consumed in processed forms.12 We previously found that tomato vinegar inhibited lipid accumulation in 3T3-L1 cells and obese rats.13 Therefore, we developed tomato vinegar as a commercial beverage and investigated its anti-obesity properties in dietinduced obese mice, as well as the mechanisms underlying these effects.
2.
Materials and methods
2.1. Production of tomato vinegar beverage (TVB) a
Department of Food and Nutrition, Sunchon National University, Suncheon 540-950, Korea. E-mail: [email protected]; Fax: +82-61-752-3657; Tel: +82-61-750-3656
b
Research Institute of Basic Science, Sunchon National University, Suncheon 540-950, Korea
c
Department of Medical Bioscience, Dong-A University, Busan 604-714, Korea
d
Department of Hotel Cuisine, Suseong College, Daegu 706-022, Korea
† K. I. Seo and J. Lee contributed equally to this work.
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Tomato vinegar used in the manufacture of TVB was prepared by a two-stage fermentation process that included alcohol and acetic acid fermentation.13 Briey, mature tomatoes were separated from the stems, cut and then crushed in a mechanical juicer. Next, the crushed tomatoes and distilled water were mixed and fortied with an apple extract to obtain a solution of
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13 Bx. During the alcohol fermentation process, the tomato mixture was inoculated with Saccharomyces cerevisiae KCCM 34709 (5%, v/v) as a starter culture and then incubated at 30 C for 2 days. In the acetic acid fermentation process, the tomato wine was ltered and developed with Acetobacter sp. KCCM 40085 (10%, v/v), in a shaking incubator at 30 C and 200 rpm for 8 days. Consequently, tomato vinegar with a total acidity of 5.6% was obtained and stored at 4 C. TVB was produced by mixing 5% tomato vinegar and 10% oligosaccharides (CJ Cheiljedang, Corp., Seoul, Korea), which was then sterilized. TVB (100 mL) contained 0.22 g acetic acid, 85 mg lycopene, 130 mg total carotenoid and 1.37 mg total polyphenol. 2.2. Animals and diets Four-week-old male C57BL/6N mice were purchased from Orient Inc. (Seoul, Republic of Korea). All mice were fed with pellets of commercial chow for 1 week aer arrival. Aer the one week adaptation period, mice were randomly divided into three groups (n ¼ 10): normal diet-fed mice (NC), high-fat diet (40% calories from fat)-fed mice (HF), and HF and TVB administered mice (HF + TVB). TVB was administered to mice at a dose of 14 mL per kg per day using an oral feeding needle for 6 weeks, which corresponded to the daily amount of beverage intake by humans. The composition of the experimental diet was based on the AIN-93G semisynthetic diet.14 During the experiment, the mice had free access to food and water and their food consumption and weight gain were measured daily and weekly, respectively. At the end of the experimental period, mice were anesthetized with ethyl ether aer withholding food for 12 h and blood samples were collected into heparin-coated tubes from the inferior vena cava. White adipose tissues (epididymal, perirenal and abdominal) and liver tissues were collected and weighed immediately, aer which they were stored at 70 C until analysis. Feces was individually collected for 5 consecutive days and then completely dried by heating at 100 C. This study protocol was approved in strict accordance with the Sunchon National University guidelines for the care and use of laboratory animals (SCNU-IACUC-2012-1). 2.3. Blood glucose, plasma insulin level and homeostatic index of insulin resistance The blood glucose concentration was monitored in blood drawn from the tail vein using a glucometer (GlucoDr supersensor, Allmeicus, Korea) aer a 6 h fast. The plasma insulin level was determined using commercially available quantitative sandwich enzyme immunoassay kits (Crystal Chem Inc., IL, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated according to the homeostasis of assessment, as follows: HOMA-IR ¼ fasting glucose (mmol L1) fasting insulin (mIU mL1)/22.5. 2.4. Histological analysis For histological analysis, the liver and epididymal adipose tissues were xed in a buffer solution containing 10% formalin, aer which they were paraffin-embedded. Next, 4 mm sections were prepared and stained with hematoxylin and eosin. The
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stained areas were observed using an optical microscope (Olympus, Japan) at a 200 magnication. 2.5. Plasma, hepatic and fecal lipids The plasma concentrations of free fatty acid (FFA) (Wako Chemicals, Richmond, VA), total cholesterol (TC), HDL-cholesterol (HDL-C) and triglycerides (Asan Diagnostics, Seoul, Korea) were determined using commercial kits. The hepatic and fecal lipid was extracted as previously described,15 while the cholesterol and triglyceride contents were analyzed using the same enzymatic kit for plasma analysis. 2.6. Lipid metabolic enzyme activities The fatty acid b-oxidation (b-oxidation) activity was measured spectrophotometrically by monitoring the reduction of NAD+ to NADH in the presence of palmitoyl-CoA as described by Lazarow16 with slight modication. Carnitine palmitoyltransferase (CPT) was assayed spectrophotometrically by following the release of CoA-SH from palmitoyl-CoA using the general thiol reagent 5,50 dithiobis (2-nitrobenzoate) (DTNB) as described by Bieber et al.17 with slight modication. Fatty acid synthase (FAS) activity was determined using a spectrophotometric assay based on the malonyl-CoA-dependent oxidation of NADPH, where one unit of enzyme activity represented the oxidation of 1 nmol of NADPH per minute at 37 C.18 Phosphatidate phosphohydrolase (PAP) activity was determined spectrophotometrically as previously described and the results were expressed as mmol min1 mg1 protein.18 Glucose-6-phosphate dehydrogenase (G6PD) activity was determined as previously described.18 The reaction mixture contained 55 mM Tris-HCl buffer (pH 7.3), 3.3 mM MgCl2, 240 mM NADP+, 4 mM glucose-6-phosphate and the cytosolic enzyme. The activity was then measured based on the reduction of 1 mol NADP+ per min at 340 nm using a spectrophotometer. 2.7. Glucose metabolic enzyme activities Glucokinase (GK) activity was determined using a spectrophotometric continuous assay as previously described,19 in which glucose-6-phosphate formation was coupled to its oxidation by glucose-6-phosphate dehydrogenase and NAD+ at 37 C. Glucose-6-phosphatase (G6Pase) activity was determined as previously described19 using a reaction mixture that contained 97 mM sodium Hepes, pH 6.5, 26.5 mM glucose-6-phosphate, 1.8 mM EDTA, 2 mM NADP+, 0.6 unit mutarotase and 0.6 unit glucose dehydrogenase. 2.8. Western blotting The liver was homogenized at 4 C in lysis buffer and then centrifuged at 12 000 g and 4 C for 20 min, aer which the supernatants were used for western blot analyses. The total protein concentrations were determined using the Bradford method. The protein samples (50 mg) were separated on a 10% sodium dodecylsulfate–polyacrylamide gel and transferred onto nitrocellulose membranes (Whatman, Dassel, Germany), which were incubated overnight at 4 C with antibodies for AMPK (Cell Signaling, Danvers, MA, USA), p-AMPK (Cell Signaling), ACC
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(Cell Signaling), p-ACC (Cell Signaling), PPARa (Santa Cruz Biotechnology, California, USA) or b-actin (Sigma, Saint Louis, MO, USA). Next, the membranes were incubated for 2 h with a secondary antibody (Amersham, Buckinghamshire, UK). The protein bands were then visualized using the ECL reagent (Santa Cruz Biotechnology, California, USA), followed by a brief exposure using an automated detection system (LAS 4000, Fujilm, Tokyo, Japan). The amount of protein was quantied by densitometric analysis using the Multi Gauge program (Version 3.0, Fujilm). 2.9. Statistical analysis All data are presented as the mean S.E. The data were assessed by a Student's t-test using the Social Science Soware (SPSS) program (Chicago, IL). A p-value < 0.05 was considered to indicate statistical signicance.
3.
Results
3.1. Effect on body weight and fat pad weight TVB effectively suppressed the HF-induced body weight gain from the rst week (Fig. 1A). At the end of the experiment, the body weight gain was reduced by 42% in the HF + TVB group compared to the HF group (Fig. 1B) with no change in food intake (Fig. 1C). However, TVB signicantly reduced the food efficiency ratio increased by HF, too close to that of the NC group (data not shown). The decrease in the body weight in the HF + TVB group was investigated to determine if it was due to the fat pad weight (Fig. 1E–F). The HF induced an increase in white adipose tissue weights (epididymal, perirenal and abdominal); however, the fat pad weight was signicantly reduced in the HF + TVB group compared to the HF group. The total visceral
Fig. 2 Effects of TVB on the hepatic morphology H & E (A), hepatic lipid contents (B) and fecal lipid contents (C) in diet-induced obese mice. Mean S.E values are significantly different between groups according to the Student's t-test. **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ###p < 0.001, HF versus HF + TVB. Yellow arrows indicate lipid droplets (200 magnification).
fat weight in mice administered with TVB decreased signicantly by 29% compared to the HF control group. Thus, TVB has powerful anti-visceral obesity effects in high-fat diet induced obese mice.
Fig. 1 Effects of TVB on changes in body weight (A), body weight gain (B), food intake (C), adipocyte morphology H & E stain (D, 200 magnification) and visceral fat weights (E & F) in diet-induced obese mice. Mean S.E values are significantly different between groups according to the Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ##p < 0.01; ###p < 0.001, HF versus HF + TVB.
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Effects of TVB on plasma lipid levels in diet-induced obese
micea
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NC FFA (mmol L1) TG (mmol L1) TC (mmol L1) HDL-C (mmol L1) HTRb (%)
0.69 1.38 2.58 2.02 70.94
0.06 0.11 0.08 0.06 1.83
HF
HF + TVB
0.80 0.08 1.72 0.08* 4.45 0.15*** 2.25 0.14 50.07 3.10***
0.57 0.05# 1.39 0.08# 4.32 0.9 2.63 0.07# 60.64 1.37#
Mean SE (n ¼ 10) values are signicantly different between groups according to the Student's t-test. *p < 0.05; ***p < 0.001, NC versus HF. #p < 0.05, HF versus HF + TVB. b HTR ¼ HDL-cholesterol/total cholesterol 100. a
3.2. Effect on the hepatic and adipocyte morphology The hepatic fat globule accumulation and adipocyte size were increased in the HF group compared to the NC group. However, mice treated with TVB showed markedly reduced lipid droplet and adipocyte sizes compared to the HF group (Fig. 1D and 2A). 3.3. Effect on lipid contents in plasma, liver and feces TVB signicantly decreased the plasma free fatty acid and triglyceride levels and the hepatic triglyceride level, while it increased the plasma HDL-C and HDL-C/TC ratio compared to the HF group (Table 1 and Fig. 2B). Conversely, fecal triglyceride excretion was increased by TVB administration in diet-induced obese mice, while the cholesterol level did not differ between groups (Fig. 2C). 3.4. Effect on insulin resistance factors in plasma The HF caused an increase in the plasma insulin level, which resulted in an increased HOMA-IR level (Fig. 3C and D). Although TVB did not signicantly improve glucose levels, it signicantly reduced the insulin level compared to the NC group (Fig. 3B). Thus, TVB has potential anti-insulin resistance activity in diet-induced obese mice. In addition, glucose tolerance, which is an indirect insulin sensitivity index, was improved by TVB administration in the HF + TVB group compared to the HF group (Fig. 3A).
Effects of TVB on the glucose tolerance test (A), blood glucose level (B), insulin level (C) and HOMA-IR level (D) in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test: **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ##p < 0.01; ###p < 0.001, HF versus HF + TVB. Fig. 3
3.5. Effect on lipid-regulating enzyme activities and protein expressions in liver The HF + TVB group showed signicantly elevated b-oxidation activity and CPT activity (Fig. 4). The activities of the lipogenic enzymes FAS, G6PD and PAP did not differ between the HF and
Effects of TVB on hepatic lipid metabolic enzyme activities in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ###p < 0.001, HF versus HF + TVB. Fig. 4
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Effects of TVB on hepatic AMPK, ACC and PPARa protein expressions in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test: **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ##p < 0.01; ###p < 0.001, HF versus HF + TVB. The AMPK, p-AMPK, ACC, p-ACC and PPARa protein values are expressed in arbitrary units (AU). The b-actin protein band was used to confirm equal loading and to normalize the data. The level in each group was related to an assigned value of 1 in the NC. Fig. 5
Effects of TVB on hepatic glucose metabolic enzyme activities in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test. *p < 0.05; **p < 0.01, NC versus HF. #p < 0.05; ###p < 0.001, HF versus HF + TVB.
Fig. 6
HF + TVB groups. Therefore, to elucidate the molecular mechanism of lipid oxidation by TVB, we measured the protein levels of the AMPK pathway and PPARa. TVB signicantly increased both the phosphorylated AMPK and ACC levels, as well as the PPARa protein levels in the liver (Fig. 5). 3.6. Effect on glucose-regulating enzyme activities in liver The activity of GK, which is involved in glycolysis, was signicantly lower in the HF group than the NC group, but mice administered with TVB in HF recovered to levels similar to those of the NC group. Conversely, the activity of G6Pase, which is involved in the process of gluconeogenesis, was signicantly lower in the HF + TVB group than in the HF group. Thus, TVB intervention occurred via an increase in the GK/G6Pase ratio in obese mice (Fig. 6).
4. Discussion This study demonstrated that TVB had potent anti-obesity and anti-insulin resistance effects on HF-induced obese mice. It has
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been reported that a HF led to weight gain and hepatic fat accumulation. These results revealed that the TVB signicantly suppressed body weight gain and visceral fat mass compared to the HF control mice without changing food intake. The difference in the body weight was apparent within the rst week of TVB treatment and persisted till the end of the study. Our previous study showed that tomato vinegar suppressed lipid accumulation in 3T3-L1 cells and an animal model;13 however, the mechanism was uncertain. In recent years, there has been an increase in the number of natural alternative foods or beverages with specic health benets.20 Therefore, we developed TVB as a functional beverage, which effectively reduced body fat mass (e.g., perirenal, abdominal and epididymal white adipose tissues), hyperlipidemia, hepatic lipid accumulation and insulin resistance, while increasing fecal lipid excretion. The TVB group showed signicantly greater triglyceride excretion (1.5 fold greater) than the HF group, which likely contributed to the lower body weight and plasma lipid levels in dietinduced obese mice. Recent studies showed that AMPK activation plays an important role in the regulation of body weight, systemic glucose homeostasis, lipid metabolism and mitochondrial biogenesis. In the liver, AMPK is a key master switch associated with regulation of glucose and lipid metabolism.21,22 Once AMPK is activated, it inactivates ACC and increases mitochondrial fatty acid oxidation.2 Yamashita et al.23 recently suggested that acetic acid, which is a primary component of vinegar, suppressed body fat accumulation. Acetic acid is absorbed immediately aer oral administration and metabolized to acetyl-CoA with the production of AMP in the liver, which results in an increased AMP–ATP ratio and subsequent phosphorylation of AMPK.24–26 Consequently, genes involved in lipogenesis are down-regulated via AMPK, resulting in a decreased body weight in an obese animal model.23 Many studies have also shown that the HF inhibited AMPK phosphorylation in C57BL/6 mice.27–29 In this study, we conrmed that the p-AMPK level was signicantly reduced by HF; however, TVB restored AMPK phosphorylation and inhibited ACC activation, which reduced the production of malonyl-CoA and inhibited CPT. CPT regulates acyl-CoA inow and b-oxidation in the mitochondrial outer membrane, which is a rate-limiting step for fatty acid oxidation.30 In this study, TVB signicantly increased CPT activity compared to the HF group while upregulating p-AMPK and p-ACC. These ndings are consistent with those of Choi et al.31 who reported that the anti-obesity effect of a green tomato extract was mediated by AMPK phosphorylation in C57BL/6 mice. Conversely, it has also been reported that AMPK decreases fatty acid synthesis by reducing the SREBP-1c expression.2 In the present study, TVB did not affect the activity of lipogenic enzymes, such as FAS, G6PD and PAP in high-fat fed mice. Therefore, the anti-obesity effect of TVB may be mediated by fatty acid oxidation rather than inhibition of lipid biogenesis. PPARs are members of the nuclear receptor superfamily that play a major role in regulating lipid homeostasis and metabolic disease.32 PPARa is most abundantly expressed in the liver, where it decreases the circulating triglyceride, while increasing
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HDL-cholesterol levels.33 We found that a HF signicantly reduced the PPARa protein expression level, which was restored by TVB. Kim et al.34 recently suggested that 13-oxo-9,11-octadecadienoic acid derived from tomato juice acts as a potent PPARa agonist in obese diabetic mice. In the absence of PPARa, animals are more susceptible to hepatic steatosis.35 PPARa induces fatty acid uptake, b-oxidation, apolipoprotein expression and triglyceride metabolism.36 It has been reported that AMPK promotes transcription of PPAR.37 Kondo et al.4 suggested that acetic acid leads to PPARa gene expression through AMPK phosphorylation. Interestingly, our results revealed that although AMPK and PPARa protein levels were signicantly lower in the HF control group than the NC group, fatty acid oxidation enzyme activity was higher, indicating that elevated fatty acid oxidation was the primary response to HF load to control lipid homeostasis. In this study, TVB recovered the p-AMPK and PPARa levels, resulting in increased fatty acid oxidation activity. Additionally, the fecal triglyceride level was signicantly higher in the HF group compared to the NC group, while TVB promoted more fecal excretion. The dysregulation of lipid metabolism not only leads to hepatic steatosis but also insulin resistance.38 Insulin resistance is characterized by the reduced response of target tissues, such as the skeletal muscle, the liver and adipose tissue to insulin, resulting in compensatory hyperinsulinemia.39 Our results showed that TVB signicantly reduced plasma insulin levels and the FFA concentration in obese mice. Increased plasma FFA plays a critical role in exacerbation of insulin resistance.40 HF has been shown to induce hepatic fat accumulation, which could decrease insulin sensitivity and produce glucose.41 It has been suggested that vinegar reduced the glucose response to a carbohydrate load in subjects with insulin resistance and healthy adults.42 Therefore, we measured the HOMA-IR level as an insulin resistance biomarker and found that HF induced insulin resistance and impaired glucose tolerance; however, TVB signicantly improved HOMA-IR and postprandial glucose levels with decreased plasma insulin levels, which suggested that TVB improves insulin sensitivity in obese mice. Finally, to elucidate the effect of TVB on glucose metabolism in the liver, we measured the GK and G6Pase activities, which are important enzymes to control glucose utilization and production.43 In this study, a HF signicantly inhibited GK activity, which was consistent with the results of previous studies.43 Additionally, TVB signicantly increased GK activity and decreased G6Pase activity, resulting in an increased GK/G6Pase ratio compared to the HF group. Thus, TVB effectively stimulated glucose utilization and inhibited gluconeogenesis in the liver of obese mice.
5.
Conclusions
The results of this study demonstrated that TVB not only reduced fat accumulation, but also insulin resistance in HFinduced obese mice, and these changes were mediated by AMPK and PPARa up-regulation. These ndings suggest that TVB can be used as a functional beverage that regulates body weight.
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Abbreviations ACC p-ACC AMPK p-AMPK boxidation CPT FAS FFA GK G6Pase G6PD HDL-C HF HOMA-IR
Acetyl-CoA carboxylase phosphorylated ACC AMP-activated protein kinase phosphorylated AMPK Fatty acid b-oxidation
Carnitine palmitoyltransferase Fatty acid synthase Free fatty acid Glucokinase Glucose-6-phosphatase Glucose-6-phosphate dehydrogenase HDL-cholesterol High-fat diet Homeostasis model assessment of insulin resistance MDA Malondialdehyde NAD Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide, reduced form NC Normal control PAP Phosphatidate phosphohydrolase PPARa Peroxisome proliferator-activated receptor alpha SREBP-1c Sterol regulatory element binding transcription factor 1c TC Total-cholesterol TG Triglyceride TVB Tomato vinegar beverage
Acknowledgements This research was supported by a Grant (no. 610003-03-3HD120) from the Technology Development Program for Agriculture and Forestry food and Fisheries of the Republic of Korea.
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Anti-obesity and anti-insulin resistance effects of tomato vinegar beverage in diet-induced obese mice Kwon-Il Seo,†a Jin Lee,†a Ra-Yeong Choi,a Hae-In Lee,a Ju-Hye Lee,ab Yong-Ki Jeong,c Myung-Joo Kimd and Mi-Kyung Lee*a This study investigated the mechanism of processed tomato vinegar beverage (TVB)-mediated anti-obesity and anti-insulin resistance effects in high-fat diet (HF)-induced obese mice. Oral administration of TVB (14 mL kg1 body weight) to HF-fed mice for 6 weeks effectively reduced the body and visceral fat weight and significantly lowered plasma free fatty acid, triglyceride and hepatic triglyceride levels. TVB significantly increased fecal triglyceride excretion, both phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) and peroxisome proliferator-activated receptor (PPAR)a protein levels in the liver, which were associated with increased fatty acid b-oxidation and carnitine palmitoyltransferase activities in HF-fed mice. TVB improved glucose tolerance, hyperinsulinemia and
Received 21st February 2014 Accepted 14th April 2014
HOMA-IR levels in the HF + TVB group compared to the HF group. Additionally, TVB significantly increased glucokinase activity and decreased glucose-6-phosphatase activity in the liver, which
DOI: 10.1039/c4fo00135d www.rsc.org/foodfunction
enhanced glucose metabolism in obese mice. These results suggest that TVB prevents visceral obesity and insulin resistance via AMPK/PPARa-mediated fatty acid and glucose oxidation.
1. Introduction Obesity, which is currently one of most prevalent metabolic disorders, is the excessive accumulation of adipose tissue in the body. The progression of obesity causes type 2 diabetes mellitus, hyperlipidemia, cardiovascular disease, liver dysfunction, respiratory complications and digestive disturbances.1 Enlarged fat mass and dysregulation of lipid metabolism results in energy imbalance, which is a typical characteristic of obesity. Improving lipid metabolism is one of the most common strategies for treatment of obesity.2 In recent years, natural alternative anti-obesity agents in the form of beverages or tea have been used to treat obesity.3 Accordingly, many studies have been conducted to identify food materials and natural substances with inhibitory effects on obesity. Vinegar has long been consumed as a cooking ingredient and used as a folk medicine.4 Currently, various types of vinegar originating from different crops or fruits are consumed throughout the world, including in Korea and Japan.5 Vinegar has been reported to have antibacterial,6 cardiovascular
protective7 and antitumor effects.8 Tomatoes are a good source of potassium, folate, vitamin A, vitamin C and vitamin E that also contain useful phytochemicals, including carotenoids and polyphenols (a-, b-, g-carotene, lutein, lycopene) and avonoids.9,10 These components of tomatoes have been shown to be benecial for the cardiovascular function.11 Recently, the amount of farming area used for the production of tomatoes has increased, which has resulted in a surplus of fresh tomatoes. Surplus tomatoes are utilized in a variety of processedtomato products, including tomato soup, sauce, ketchup, paste, and juice. Although most tomatoes are commonly ingested in a fresh state, more than half are consumed in processed forms.12 We previously found that tomato vinegar inhibited lipid accumulation in 3T3-L1 cells and obese rats.13 Therefore, we developed tomato vinegar as a commercial beverage and investigated its anti-obesity properties in dietinduced obese mice, as well as the mechanisms underlying these effects.
2.
Materials and methods
2.1. Production of tomato vinegar beverage (TVB) a
Department of Food and Nutrition, Sunchon National University, Suncheon 540-950, Korea. E-mail: [email protected]; Fax: +82-61-752-3657; Tel: +82-61-750-3656
b
Research Institute of Basic Science, Sunchon National University, Suncheon 540-950, Korea
c
Department of Medical Bioscience, Dong-A University, Busan 604-714, Korea
d
Department of Hotel Cuisine, Suseong College, Daegu 706-022, Korea
† K. I. Seo and J. Lee contributed equally to this work.
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Tomato vinegar used in the manufacture of TVB was prepared by a two-stage fermentation process that included alcohol and acetic acid fermentation.13 Briey, mature tomatoes were separated from the stems, cut and then crushed in a mechanical juicer. Next, the crushed tomatoes and distilled water were mixed and fortied with an apple extract to obtain a solution of
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13 Bx. During the alcohol fermentation process, the tomato mixture was inoculated with Saccharomyces cerevisiae KCCM 34709 (5%, v/v) as a starter culture and then incubated at 30 C for 2 days. In the acetic acid fermentation process, the tomato wine was ltered and developed with Acetobacter sp. KCCM 40085 (10%, v/v), in a shaking incubator at 30 C and 200 rpm for 8 days. Consequently, tomato vinegar with a total acidity of 5.6% was obtained and stored at 4 C. TVB was produced by mixing 5% tomato vinegar and 10% oligosaccharides (CJ Cheiljedang, Corp., Seoul, Korea), which was then sterilized. TVB (100 mL) contained 0.22 g acetic acid, 85 mg lycopene, 130 mg total carotenoid and 1.37 mg total polyphenol. 2.2. Animals and diets Four-week-old male C57BL/6N mice were purchased from Orient Inc. (Seoul, Republic of Korea). All mice were fed with pellets of commercial chow for 1 week aer arrival. Aer the one week adaptation period, mice were randomly divided into three groups (n ¼ 10): normal diet-fed mice (NC), high-fat diet (40% calories from fat)-fed mice (HF), and HF and TVB administered mice (HF + TVB). TVB was administered to mice at a dose of 14 mL per kg per day using an oral feeding needle for 6 weeks, which corresponded to the daily amount of beverage intake by humans. The composition of the experimental diet was based on the AIN-93G semisynthetic diet.14 During the experiment, the mice had free access to food and water and their food consumption and weight gain were measured daily and weekly, respectively. At the end of the experimental period, mice were anesthetized with ethyl ether aer withholding food for 12 h and blood samples were collected into heparin-coated tubes from the inferior vena cava. White adipose tissues (epididymal, perirenal and abdominal) and liver tissues were collected and weighed immediately, aer which they were stored at 70 C until analysis. Feces was individually collected for 5 consecutive days and then completely dried by heating at 100 C. This study protocol was approved in strict accordance with the Sunchon National University guidelines for the care and use of laboratory animals (SCNU-IACUC-2012-1). 2.3. Blood glucose, plasma insulin level and homeostatic index of insulin resistance The blood glucose concentration was monitored in blood drawn from the tail vein using a glucometer (GlucoDr supersensor, Allmeicus, Korea) aer a 6 h fast. The plasma insulin level was determined using commercially available quantitative sandwich enzyme immunoassay kits (Crystal Chem Inc., IL, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated according to the homeostasis of assessment, as follows: HOMA-IR ¼ fasting glucose (mmol L1) fasting insulin (mIU mL1)/22.5. 2.4. Histological analysis For histological analysis, the liver and epididymal adipose tissues were xed in a buffer solution containing 10% formalin, aer which they were paraffin-embedded. Next, 4 mm sections were prepared and stained with hematoxylin and eosin. The
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stained areas were observed using an optical microscope (Olympus, Japan) at a 200 magnication. 2.5. Plasma, hepatic and fecal lipids The plasma concentrations of free fatty acid (FFA) (Wako Chemicals, Richmond, VA), total cholesterol (TC), HDL-cholesterol (HDL-C) and triglycerides (Asan Diagnostics, Seoul, Korea) were determined using commercial kits. The hepatic and fecal lipid was extracted as previously described,15 while the cholesterol and triglyceride contents were analyzed using the same enzymatic kit for plasma analysis. 2.6. Lipid metabolic enzyme activities The fatty acid b-oxidation (b-oxidation) activity was measured spectrophotometrically by monitoring the reduction of NAD+ to NADH in the presence of palmitoyl-CoA as described by Lazarow16 with slight modication. Carnitine palmitoyltransferase (CPT) was assayed spectrophotometrically by following the release of CoA-SH from palmitoyl-CoA using the general thiol reagent 5,50 dithiobis (2-nitrobenzoate) (DTNB) as described by Bieber et al.17 with slight modication. Fatty acid synthase (FAS) activity was determined using a spectrophotometric assay based on the malonyl-CoA-dependent oxidation of NADPH, where one unit of enzyme activity represented the oxidation of 1 nmol of NADPH per minute at 37 C.18 Phosphatidate phosphohydrolase (PAP) activity was determined spectrophotometrically as previously described and the results were expressed as mmol min1 mg1 protein.18 Glucose-6-phosphate dehydrogenase (G6PD) activity was determined as previously described.18 The reaction mixture contained 55 mM Tris-HCl buffer (pH 7.3), 3.3 mM MgCl2, 240 mM NADP+, 4 mM glucose-6-phosphate and the cytosolic enzyme. The activity was then measured based on the reduction of 1 mol NADP+ per min at 340 nm using a spectrophotometer. 2.7. Glucose metabolic enzyme activities Glucokinase (GK) activity was determined using a spectrophotometric continuous assay as previously described,19 in which glucose-6-phosphate formation was coupled to its oxidation by glucose-6-phosphate dehydrogenase and NAD+ at 37 C. Glucose-6-phosphatase (G6Pase) activity was determined as previously described19 using a reaction mixture that contained 97 mM sodium Hepes, pH 6.5, 26.5 mM glucose-6-phosphate, 1.8 mM EDTA, 2 mM NADP+, 0.6 unit mutarotase and 0.6 unit glucose dehydrogenase. 2.8. Western blotting The liver was homogenized at 4 C in lysis buffer and then centrifuged at 12 000 g and 4 C for 20 min, aer which the supernatants were used for western blot analyses. The total protein concentrations were determined using the Bradford method. The protein samples (50 mg) were separated on a 10% sodium dodecylsulfate–polyacrylamide gel and transferred onto nitrocellulose membranes (Whatman, Dassel, Germany), which were incubated overnight at 4 C with antibodies for AMPK (Cell Signaling, Danvers, MA, USA), p-AMPK (Cell Signaling), ACC
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(Cell Signaling), p-ACC (Cell Signaling), PPARa (Santa Cruz Biotechnology, California, USA) or b-actin (Sigma, Saint Louis, MO, USA). Next, the membranes were incubated for 2 h with a secondary antibody (Amersham, Buckinghamshire, UK). The protein bands were then visualized using the ECL reagent (Santa Cruz Biotechnology, California, USA), followed by a brief exposure using an automated detection system (LAS 4000, Fujilm, Tokyo, Japan). The amount of protein was quantied by densitometric analysis using the Multi Gauge program (Version 3.0, Fujilm). 2.9. Statistical analysis All data are presented as the mean S.E. The data were assessed by a Student's t-test using the Social Science Soware (SPSS) program (Chicago, IL). A p-value < 0.05 was considered to indicate statistical signicance.
3.
Results
3.1. Effect on body weight and fat pad weight TVB effectively suppressed the HF-induced body weight gain from the rst week (Fig. 1A). At the end of the experiment, the body weight gain was reduced by 42% in the HF + TVB group compared to the HF group (Fig. 1B) with no change in food intake (Fig. 1C). However, TVB signicantly reduced the food efficiency ratio increased by HF, too close to that of the NC group (data not shown). The decrease in the body weight in the HF + TVB group was investigated to determine if it was due to the fat pad weight (Fig. 1E–F). The HF induced an increase in white adipose tissue weights (epididymal, perirenal and abdominal); however, the fat pad weight was signicantly reduced in the HF + TVB group compared to the HF group. The total visceral
Fig. 2 Effects of TVB on the hepatic morphology H & E (A), hepatic lipid contents (B) and fecal lipid contents (C) in diet-induced obese mice. Mean S.E values are significantly different between groups according to the Student's t-test. **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ###p < 0.001, HF versus HF + TVB. Yellow arrows indicate lipid droplets (200 magnification).
fat weight in mice administered with TVB decreased signicantly by 29% compared to the HF control group. Thus, TVB has powerful anti-visceral obesity effects in high-fat diet induced obese mice.
Fig. 1 Effects of TVB on changes in body weight (A), body weight gain (B), food intake (C), adipocyte morphology H & E stain (D, 200 magnification) and visceral fat weights (E & F) in diet-induced obese mice. Mean S.E values are significantly different between groups according to the Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ##p < 0.01; ###p < 0.001, HF versus HF + TVB.
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Effects of TVB on plasma lipid levels in diet-induced obese
micea
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NC FFA (mmol L1) TG (mmol L1) TC (mmol L1) HDL-C (mmol L1) HTRb (%)
0.69 1.38 2.58 2.02 70.94
0.06 0.11 0.08 0.06 1.83
HF
HF + TVB
0.80 0.08 1.72 0.08* 4.45 0.15*** 2.25 0.14 50.07 3.10***
0.57 0.05# 1.39 0.08# 4.32 0.9 2.63 0.07# 60.64 1.37#
Mean SE (n ¼ 10) values are signicantly different between groups according to the Student's t-test. *p < 0.05; ***p < 0.001, NC versus HF. #p < 0.05, HF versus HF + TVB. b HTR ¼ HDL-cholesterol/total cholesterol 100. a
3.2. Effect on the hepatic and adipocyte morphology The hepatic fat globule accumulation and adipocyte size were increased in the HF group compared to the NC group. However, mice treated with TVB showed markedly reduced lipid droplet and adipocyte sizes compared to the HF group (Fig. 1D and 2A). 3.3. Effect on lipid contents in plasma, liver and feces TVB signicantly decreased the plasma free fatty acid and triglyceride levels and the hepatic triglyceride level, while it increased the plasma HDL-C and HDL-C/TC ratio compared to the HF group (Table 1 and Fig. 2B). Conversely, fecal triglyceride excretion was increased by TVB administration in diet-induced obese mice, while the cholesterol level did not differ between groups (Fig. 2C). 3.4. Effect on insulin resistance factors in plasma The HF caused an increase in the plasma insulin level, which resulted in an increased HOMA-IR level (Fig. 3C and D). Although TVB did not signicantly improve glucose levels, it signicantly reduced the insulin level compared to the NC group (Fig. 3B). Thus, TVB has potential anti-insulin resistance activity in diet-induced obese mice. In addition, glucose tolerance, which is an indirect insulin sensitivity index, was improved by TVB administration in the HF + TVB group compared to the HF group (Fig. 3A).
Effects of TVB on the glucose tolerance test (A), blood glucose level (B), insulin level (C) and HOMA-IR level (D) in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test: **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ##p < 0.01; ###p < 0.001, HF versus HF + TVB. Fig. 3
3.5. Effect on lipid-regulating enzyme activities and protein expressions in liver The HF + TVB group showed signicantly elevated b-oxidation activity and CPT activity (Fig. 4). The activities of the lipogenic enzymes FAS, G6PD and PAP did not differ between the HF and
Effects of TVB on hepatic lipid metabolic enzyme activities in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ###p < 0.001, HF versus HF + TVB. Fig. 4
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Effects of TVB on hepatic AMPK, ACC and PPARa protein expressions in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test: **p < 0.01; ***p < 0.001, NC versus HF. #p < 0.05; ##p < 0.01; ###p < 0.001, HF versus HF + TVB. The AMPK, p-AMPK, ACC, p-ACC and PPARa protein values are expressed in arbitrary units (AU). The b-actin protein band was used to confirm equal loading and to normalize the data. The level in each group was related to an assigned value of 1 in the NC. Fig. 5
Effects of TVB on hepatic glucose metabolic enzyme activities in diet-induced obese mice. Mean SE values are significantly different between groups according to the Student's t-test. *p < 0.05; **p < 0.01, NC versus HF. #p < 0.05; ###p < 0.001, HF versus HF + TVB.
Fig. 6
HF + TVB groups. Therefore, to elucidate the molecular mechanism of lipid oxidation by TVB, we measured the protein levels of the AMPK pathway and PPARa. TVB signicantly increased both the phosphorylated AMPK and ACC levels, as well as the PPARa protein levels in the liver (Fig. 5). 3.6. Effect on glucose-regulating enzyme activities in liver The activity of GK, which is involved in glycolysis, was signicantly lower in the HF group than the NC group, but mice administered with TVB in HF recovered to levels similar to those of the NC group. Conversely, the activity of G6Pase, which is involved in the process of gluconeogenesis, was signicantly lower in the HF + TVB group than in the HF group. Thus, TVB intervention occurred via an increase in the GK/G6Pase ratio in obese mice (Fig. 6).
4. Discussion This study demonstrated that TVB had potent anti-obesity and anti-insulin resistance effects on HF-induced obese mice. It has
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been reported that a HF led to weight gain and hepatic fat accumulation. These results revealed that the TVB signicantly suppressed body weight gain and visceral fat mass compared to the HF control mice without changing food intake. The difference in the body weight was apparent within the rst week of TVB treatment and persisted till the end of the study. Our previous study showed that tomato vinegar suppressed lipid accumulation in 3T3-L1 cells and an animal model;13 however, the mechanism was uncertain. In recent years, there has been an increase in the number of natural alternative foods or beverages with specic health benets.20 Therefore, we developed TVB as a functional beverage, which effectively reduced body fat mass (e.g., perirenal, abdominal and epididymal white adipose tissues), hyperlipidemia, hepatic lipid accumulation and insulin resistance, while increasing fecal lipid excretion. The TVB group showed signicantly greater triglyceride excretion (1.5 fold greater) than the HF group, which likely contributed to the lower body weight and plasma lipid levels in dietinduced obese mice. Recent studies showed that AMPK activation plays an important role in the regulation of body weight, systemic glucose homeostasis, lipid metabolism and mitochondrial biogenesis. In the liver, AMPK is a key master switch associated with regulation of glucose and lipid metabolism.21,22 Once AMPK is activated, it inactivates ACC and increases mitochondrial fatty acid oxidation.2 Yamashita et al.23 recently suggested that acetic acid, which is a primary component of vinegar, suppressed body fat accumulation. Acetic acid is absorbed immediately aer oral administration and metabolized to acetyl-CoA with the production of AMP in the liver, which results in an increased AMP–ATP ratio and subsequent phosphorylation of AMPK.24–26 Consequently, genes involved in lipogenesis are down-regulated via AMPK, resulting in a decreased body weight in an obese animal model.23 Many studies have also shown that the HF inhibited AMPK phosphorylation in C57BL/6 mice.27–29 In this study, we conrmed that the p-AMPK level was signicantly reduced by HF; however, TVB restored AMPK phosphorylation and inhibited ACC activation, which reduced the production of malonyl-CoA and inhibited CPT. CPT regulates acyl-CoA inow and b-oxidation in the mitochondrial outer membrane, which is a rate-limiting step for fatty acid oxidation.30 In this study, TVB signicantly increased CPT activity compared to the HF group while upregulating p-AMPK and p-ACC. These ndings are consistent with those of Choi et al.31 who reported that the anti-obesity effect of a green tomato extract was mediated by AMPK phosphorylation in C57BL/6 mice. Conversely, it has also been reported that AMPK decreases fatty acid synthesis by reducing the SREBP-1c expression.2 In the present study, TVB did not affect the activity of lipogenic enzymes, such as FAS, G6PD and PAP in high-fat fed mice. Therefore, the anti-obesity effect of TVB may be mediated by fatty acid oxidation rather than inhibition of lipid biogenesis. PPARs are members of the nuclear receptor superfamily that play a major role in regulating lipid homeostasis and metabolic disease.32 PPARa is most abundantly expressed in the liver, where it decreases the circulating triglyceride, while increasing
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HDL-cholesterol levels.33 We found that a HF signicantly reduced the PPARa protein expression level, which was restored by TVB. Kim et al.34 recently suggested that 13-oxo-9,11-octadecadienoic acid derived from tomato juice acts as a potent PPARa agonist in obese diabetic mice. In the absence of PPARa, animals are more susceptible to hepatic steatosis.35 PPARa induces fatty acid uptake, b-oxidation, apolipoprotein expression and triglyceride metabolism.36 It has been reported that AMPK promotes transcription of PPAR.37 Kondo et al.4 suggested that acetic acid leads to PPARa gene expression through AMPK phosphorylation. Interestingly, our results revealed that although AMPK and PPARa protein levels were signicantly lower in the HF control group than the NC group, fatty acid oxidation enzyme activity was higher, indicating that elevated fatty acid oxidation was the primary response to HF load to control lipid homeostasis. In this study, TVB recovered the p-AMPK and PPARa levels, resulting in increased fatty acid oxidation activity. Additionally, the fecal triglyceride level was signicantly higher in the HF group compared to the NC group, while TVB promoted more fecal excretion. The dysregulation of lipid metabolism not only leads to hepatic steatosis but also insulin resistance.38 Insulin resistance is characterized by the reduced response of target tissues, such as the skeletal muscle, the liver and adipose tissue to insulin, resulting in compensatory hyperinsulinemia.39 Our results showed that TVB signicantly reduced plasma insulin levels and the FFA concentration in obese mice. Increased plasma FFA plays a critical role in exacerbation of insulin resistance.40 HF has been shown to induce hepatic fat accumulation, which could decrease insulin sensitivity and produce glucose.41 It has been suggested that vinegar reduced the glucose response to a carbohydrate load in subjects with insulin resistance and healthy adults.42 Therefore, we measured the HOMA-IR level as an insulin resistance biomarker and found that HF induced insulin resistance and impaired glucose tolerance; however, TVB signicantly improved HOMA-IR and postprandial glucose levels with decreased plasma insulin levels, which suggested that TVB improves insulin sensitivity in obese mice. Finally, to elucidate the effect of TVB on glucose metabolism in the liver, we measured the GK and G6Pase activities, which are important enzymes to control glucose utilization and production.43 In this study, a HF signicantly inhibited GK activity, which was consistent with the results of previous studies.43 Additionally, TVB signicantly increased GK activity and decreased G6Pase activity, resulting in an increased GK/G6Pase ratio compared to the HF group. Thus, TVB effectively stimulated glucose utilization and inhibited gluconeogenesis in the liver of obese mice.
5.
Conclusions
The results of this study demonstrated that TVB not only reduced fat accumulation, but also insulin resistance in HFinduced obese mice, and these changes were mediated by AMPK and PPARa up-regulation. These ndings suggest that TVB can be used as a functional beverage that regulates body weight.
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Abbreviations ACC p-ACC AMPK p-AMPK boxidation CPT FAS FFA GK G6Pase G6PD HDL-C HF HOMA-IR
Acetyl-CoA carboxylase phosphorylated ACC AMP-activated protein kinase phosphorylated AMPK Fatty acid b-oxidation
Carnitine palmitoyltransferase Fatty acid synthase Free fatty acid Glucokinase Glucose-6-phosphatase Glucose-6-phosphate dehydrogenase HDL-cholesterol High-fat diet Homeostasis model assessment of insulin resistance MDA Malondialdehyde NAD Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide, reduced form NC Normal control PAP Phosphatidate phosphohydrolase PPARa Peroxisome proliferator-activated receptor alpha SREBP-1c Sterol regulatory element binding transcription factor 1c TC Total-cholesterol TG Triglyceride TVB Tomato vinegar beverage
Acknowledgements This research was supported by a Grant (no. 610003-03-3HD120) from the Technology Development Program for Agriculture and Forestry food and Fisheries of the Republic of Korea.
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