Supplemental Material can be found at: http://jn.nutrition.org/content/suppl/2014/03/12/jn.113.18745 0.DCSupplemental.html
The Journal of Nutrition Nutrition and Disease
Bitter Gourd Inhibits the Development of Obesity-Associated Fatty Liver in C57BL/6 Mice Fed a High-Fat Diet1–3
Centers for 5Mitochondrial Biology & Medicine and 6Translational Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Frontier Institute of Science and Technology, XiÕan Jiaotong University, XiÕan, China; and 7Nestl´e Research Center Beijing, Beijing, China
Abstract Bitter gourd (BG) is a popular fruit in Asia with numerous well-known medicinal uses, including as an antidiabetic. In the current study, we aimed to explore the effects of BG on mitochondrial function during the development of obesityassociated fatty liver. C57BL/6 mice were divided into 4 experimental groups: mice fed a normal diet (control; included for reference only), mice fed a high-fat diet (HFD), and mice fed an HFD supplemented with freeze-dried BG powder through daily gavage at doses of 0.5 (HFD+0.5BG) and 5 (HFD+5BG) g/kg, respectively. After 16 wk, mice in the HFD+5BG group showed less body and tissue weight gain and less hyperglycemia and hyperlipidemia compared with those in the HFD group (P < 0.05). In both HFD+0.5BG and HFD+5BG groups, serum interleukin-6 concentration was lower than that in the HFD group (P < 0.02). The serum C-reactive protein concentration was lower in the HFD+5BG group compared with the HFD group (P < 0.04). An analysis of liver tissue revealed lower liver triglyceride and cholesterol concentrations in both HFD+0.5BG and HFD+5BG groups than in the HFD group (P < 0.01). The HFD+5BG group had less activation of the sterol regulatory element binding protein/fatty acid synthase (SREBP-1/FAS) pathway, greater superoxide dismutase activity, and less total protein and mitochondrial protein oxidation than did the HFD group (P < 0.05). Mitochondrial complex I, II, III, and V activity was greater in the HFD+0.5BG group than in the HFD group (P < 0.03). The HFD+5BG group only had greater complex V activity compared with the HFD group (P < 0.05). Mitochondrial dynamics regulators, including dynamin related protein 1 (DRP1) and mitofusin 1 (MFN1), as well as proapoptotic protein expression levels were restored by BG treatment (P < 0.02). Taken together, our results suggest that BG prevents inflammation and oxidative stress, modulates mitochondrial activity, suppresses apoptosis activation, and inhibits lipid accumulation during the development of fatty liver. J. Nutr. 144: 475–483, 2014.
Introduction Obesity and the associated metabolic pathologies, known as metabolic syndrome, have become the most prevalent worldwide epidemic diseases in recent decades. A recent national health survey conducted in mainland China revealed that 60 million people are obese and 200 million are overweight (1). The
1 Supported by the National Natural Science Foundation of China (81201023 and 31370844), the National "Twelfth Five-Year" Plan for Science & Technology Support (2012BAH30F03), the Fundamental Research Funds for the Central Universities, the 985 and 211 projects of XiÕan Jiaotong University, and Nestle´ Research Center, Switzerland. 2 Author disclosures: J. Xu, K. Cao, Y. Li, X. Zou, C. Chen, I. M.-Y. Szeto, Z. Dong, Y. Zhao, Y. Shi, J. Wang, J. Liu, and Z. Feng, no conflicts of interest. 3 Supplemental Tables 1–3 and Supplemental Figure 1 are available from the ÔÔOnline Supporting MaterialÕÕ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. 4 J.X. and K.C. contributed equally to this work. * To whom correspondence should be addressed. E-mail: [email protected]. cn (Z. Feng), [email protected] (J. Liu).
rising obesity epidemic is increasing the risk of nonalcoholic fatty liver disease (NAFLD),8 which is one of the most common chronic liver diseases in the world (2,3), particularly among children and young adults (4), and affects ;30% of all U.S. adults and 75–100% of obese or morbidly obese individuals (5). The liver is a functional tissue that controls the production of TGs and glucose for use by other tissues. The development of NAFLD indicates a disruption in lipid and glucose homeostasis, which is regulated by pathways such as lipogenesis and gluconeogenesis (6). Sterol regulatory element binding proteins 8 Abbreviations used: ACC-1, acetyl-CoA carboxylase 1; BG, bitter gourd; CRP, C-reactive protein; DRP1, dynamin related protein 1; FAS, FA synthase; GSH, reduced glutathione; HFD, high-fat diet; HFD+0.5BG, high-fat diet with daily oral gavage of 0.5 g/kg bitter gourd; HFD+5BG, high-fat diet with daily oral gavage of 5 g/kg bitter gourd; IRS-1, insulin receptor substrate 1; MFN1, mitofusin 1; NADH, reduced nicotinamide-adenine dinucleotide; NAFLD, nonalcoholic fatty liver disease; PTP-1B, protein-tyrosine phosphatase 1B; SOD, superoxide dismutase; SREBP, sterol regulatory element binding protein; TC, total cholesterol.
ã 2014 American Society for Nutrition. Manuscript received October 25, 2013. Initial review completed November 23, 2013. Revision accepted January 28, 2014. First published online February 12, 2014; doi:10.3945/jn.113.187450.
475
Downloaded from jn.nutrition.org at UNIVERSITY OF VIRGINIA CLAUDE MOORE HEALTH SCIENCES LIBRARY on June 26, 2014
Jie Xu,4,5 Ke Cao,4,5 Yuan Li,5 Xuan Zou,6 Cong Chen,5 Ignatius Man-Yau Szeto,7 Zhizhong Dong,7 Youyou Zhao,7 Yujie Shi,7 Junkuan Wang,7 Jiankang Liu,5* and Zhihui Feng5*
Materials and Methods Chemicals. Antibodies against b-actin were purchased from Sigma. Antibodies against tubulin, SREBP-1c, and mitofusin 1 (MFN1) were purchased from Santa Cruz Biotechnology. Antibodies against GAPDH, FA synthase (FAS), Bcl-2, Bcl-XL , and Bax were purchased from Cell Signaling Technology. An antibody against dynamin related protein 1 (DRP1) was purchased from BD. Antibodies against complexes I, II, III, IV, and V were purchased from Invitrogen. BG powder was purchased from Guangxi Zhennong Seed Industry. BG was prepared by freezedrying. Briefly, fresh BG was washed thoroughly with water and sliced into 15-mm fragments, which were homogenized by an electric juicer. The juice was frozen and then completely lyophilized by continuous freeze-drying. The yield of the BG extract was ;4.76%; the total amount of saponins was used as a quantity control (2.2%). Animals and treatments. Four-week-old male C57BL/6 mice were purchased from SLAC Laboratory Animal and were housed in a temperature (22–28°C)- and humidity (60%)-controlled room on a 12-h light/12-h dark cycle (light from 0800 to 2000), with food and water provided during the experiments. After 1 wk of acclimatization, the mice were randomly divided into 4 groups (n = 10 in each group), as follows: mice fed a normal diet (control; 12% kcal fat content), mice fed an HFD (45% kcal fat content), mice fed an HFD with daily oral gavage of 0.5 g/kg BG (HFD+0.5BG), and mice fed an HFD with daily oral gavage of 5 g/kg BG (HFD+5BG). The composition of the HFD is presented in Supplemental Table 1. Due to technological limitations, only 15 components in soluble BG were identified and presented in Supplemental Table 2. After 16 wk, the mice were deprived of feed overnight and killed by decapitation. All of the procedures were performed in accordance with the U.S. Public Health Services Guide for the Care and Use of Laboratory Animals, and all possible efforts were made to minimize the suffering and the number of animals used in this study. 476
Xu et al.
Oral-glucose-tolerance test. An oral-glucose-tolerance test (1 g/kg body weight) was performed after 16 wk of feeding and gavage. All mice were deprived of feed overnight before the oral-glucose-tolerance test. Blood was taken from the retrobulbar vein both before and 30, 60, 120, and 180 min after oral-glucose gavage. The plasma glucose concentration was determined by the glucose oxidation method. Sample preparation. After the mice were killed, liver tissue and visceral fat pads (including the perirenal and epididymal fat pads) were removed and weighed. Blood samples were obtained by cardiac puncture, and serum was separated by centrifugation (1100 3 g; 10 min). The concentrations of TGs, total cholesterol (TC), LDL cholesterol, and HDL cholesterol were analyzed by using an automated biochemistry analyzer (Hitachi) with biochemical analysis kits (R&D Systems). Serum concentrations of C-reactive protein (CRP), free FAs, insulin, and IL-6 were measured by using commercial ELISA kits according to the manufacturerÕs standards and protocols (R&D Systems). Minimum levels of detection and CVs for each kit assay are provided in detail in Supplemental Table 3. Histologic analysis of liver samples. Liver tissues were excised, washed with ice-cold PBS, and placed in 10% formalin. Several tissue sections (4–5-mm thick) were prepared, stained with hematoxylin and eosin for histopathologic analysis, and visualized with an Olympus BX71 microscope. Isolation of liver mitochondria. Mitochondria were isolated as previously described (21). Briefly, liver tissue was rinsed with saline, weighed, and placed in an ice-cold isolation buffer containing 0.25 mol/L sucrose, 10 mmol/L Tris, and 0.5 mmol/L EDTA at pH 7.4. The tissue was sheared, carefully minced, rinsed to remove residual blood, and homogenized in 2.5 volumes of isolation buffer. The volume of the homogenate was increased to 8 initial volumes with the isolation buffer and centrifuged at 1000 g for 10 min; the supernatant fraction was decanted and saved. The pellet was washed once with 2 volumes of isolation buffer, and the total supernatant fractions were combined and centrifuged at 10,000 g for 10 min. The mitochondrial pellet was washed twice by using isolation buffer, and the protein concentration was determined by using a bicinchoninic acid protein assay kit. Freshly isolated mitochondria were either used immediately for biochemical assays or stored at 280°C. Biochemical analysis. Small portions of liver tissue were collected and homogenized in ice-cold PBS. After centrifugation (1000 g; 10 min), the supernatant was collected for analysis. Concentrations of reduced glutathione (GSH), liver TGs, and TC, as well as glutathione S-transferase, Cu-Zn superoxide dismutase (SOD), and Mn SOD activity, were analyzed by using commercial clinical diagnosis kits according to the manufacturerÕs standards and protocols (Jiancheng). Protein carbonylation assay. Protein carbonyls in soluble proteins were assayed by using the Oxyblot protein oxidation detection kit (Cell Biolabs). Protein carbonyls were labeled with 2,4-dinitrophenylhydrazine and detected by Western blot. Assays for mitochondrial complex activity. The activities of reduced nicotinamide-adenine dinucleotide (NADH)-ubiquinone reductase (complex I), succinate-coenzyme Q oxidoreductase (complex II), and cytochrome c oxidase (complex IV) were measured spectrophotometrically by using conventional assays as previously described (22,23). Western blotting. The samples were lysed in Western and Immunoprecipitation lysis buffers (Beyotime). The lysates were homogenized, and the homogenates were centrifuged at 13,000 g for 15 min at 4°C. The supernatants were collected, and the protein concentrations were determined by using a bicinchoninic acid protein assay kit. Equal aliquots (20 mg) of protein were analyzed by Western blotting. Chemiluminescent detection was performed by using an ECL Western blotting detection kit (Thermo Scientific) and was quantified by scanning densitometry. Statistical analysis. Normal distribution was assessed by Shapiro-Wilk test (SPSS). Results are presented as means 6 SEMs. Because micronutrient
Downloaded from jn.nutrition.org at UNIVERSITY OF VIRGINIA CLAUDE MOORE HEALTH SCIENCES LIBRARY on June 26, 2014
(SREBPs) are a class of proteins that transcriptionally activate a cascade of enzymes that are required for the endogenous synthesis of cholesterol, FAs, TGs, and phospholipids (7). Among the 3 isoforms, SREBP-1c is the central regulator of lipid metabolism in vivo, and SREBP-1c activation contributes to hepatic lipogenesis and NAFLD (8,9). Studies have demonstrated that excessive lipid accumulation in the liver is associated with oxidative stress and mitochondrial dysfunction (10,11). In mice, a high-fat diet (HFD) induces the overproduction of reactive oxygen species in adipose tissue and liver, which precedes the onset of obesity and insulin resistance (12). In rats, hepatic mitochondrial dysfunction precedes the development of NAFLD and insulin resistance (13). Because several processes regulate mitochondrial function, the molecular basis of mitochondrial involvement in the development of NAFLD is unclear. However, the prominent role of mitochondrial dysfunction in NAFLD provides the basis for potential dietary and pharmacologic intervention. Momordica charantia, also known as bitter gourd (BG), is a climber plant that is distinguished by its bitter-tasting fruit. In recent decades, several hundred studies have been conducted using BG because of its popular medicinal uses. Among these properties, its antidiabetic activity is the most popular effect, and this is supported by biochemical experiments and animal models (14–17). Although several possible mechanisms, including b cell protection (18), the regulation of PPARa and PPARg (19), increasing skeletal muscle insulin-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation (20), and inhibiting protein-tyrosine phosphatase 1B (PTP-1B) (16), have been proposed for the antidiabetic activity of BG, studies of the effects of BG on mitochondrial dysfunction and NAFLD are limited. This study was designed to determine the effect of BG fruit extract on NAFLD and mitochondrial function in HFD-induced obese mice.
concentrations were not adjusted in the HFD, the control group in the study was included as a reference only. Statistical analysis was conducted by using data from only 3 HFD groups via 1-factor ANOVA followed by a least significant difference post hoc analysis. For all of the analyses, P values
The Journal of Nutrition Nutrition and Disease
Bitter Gourd Inhibits the Development of Obesity-Associated Fatty Liver in C57BL/6 Mice Fed a High-Fat Diet1–3
Centers for 5Mitochondrial Biology & Medicine and 6Translational Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Frontier Institute of Science and Technology, XiÕan Jiaotong University, XiÕan, China; and 7Nestl´e Research Center Beijing, Beijing, China
Abstract Bitter gourd (BG) is a popular fruit in Asia with numerous well-known medicinal uses, including as an antidiabetic. In the current study, we aimed to explore the effects of BG on mitochondrial function during the development of obesityassociated fatty liver. C57BL/6 mice were divided into 4 experimental groups: mice fed a normal diet (control; included for reference only), mice fed a high-fat diet (HFD), and mice fed an HFD supplemented with freeze-dried BG powder through daily gavage at doses of 0.5 (HFD+0.5BG) and 5 (HFD+5BG) g/kg, respectively. After 16 wk, mice in the HFD+5BG group showed less body and tissue weight gain and less hyperglycemia and hyperlipidemia compared with those in the HFD group (P < 0.05). In both HFD+0.5BG and HFD+5BG groups, serum interleukin-6 concentration was lower than that in the HFD group (P < 0.02). The serum C-reactive protein concentration was lower in the HFD+5BG group compared with the HFD group (P < 0.04). An analysis of liver tissue revealed lower liver triglyceride and cholesterol concentrations in both HFD+0.5BG and HFD+5BG groups than in the HFD group (P < 0.01). The HFD+5BG group had less activation of the sterol regulatory element binding protein/fatty acid synthase (SREBP-1/FAS) pathway, greater superoxide dismutase activity, and less total protein and mitochondrial protein oxidation than did the HFD group (P < 0.05). Mitochondrial complex I, II, III, and V activity was greater in the HFD+0.5BG group than in the HFD group (P < 0.03). The HFD+5BG group only had greater complex V activity compared with the HFD group (P < 0.05). Mitochondrial dynamics regulators, including dynamin related protein 1 (DRP1) and mitofusin 1 (MFN1), as well as proapoptotic protein expression levels were restored by BG treatment (P < 0.02). Taken together, our results suggest that BG prevents inflammation and oxidative stress, modulates mitochondrial activity, suppresses apoptosis activation, and inhibits lipid accumulation during the development of fatty liver. J. Nutr. 144: 475–483, 2014.
Introduction Obesity and the associated metabolic pathologies, known as metabolic syndrome, have become the most prevalent worldwide epidemic diseases in recent decades. A recent national health survey conducted in mainland China revealed that 60 million people are obese and 200 million are overweight (1). The
1 Supported by the National Natural Science Foundation of China (81201023 and 31370844), the National "Twelfth Five-Year" Plan for Science & Technology Support (2012BAH30F03), the Fundamental Research Funds for the Central Universities, the 985 and 211 projects of XiÕan Jiaotong University, and Nestle´ Research Center, Switzerland. 2 Author disclosures: J. Xu, K. Cao, Y. Li, X. Zou, C. Chen, I. M.-Y. Szeto, Z. Dong, Y. Zhao, Y. Shi, J. Wang, J. Liu, and Z. Feng, no conflicts of interest. 3 Supplemental Tables 1–3 and Supplemental Figure 1 are available from the ÔÔOnline Supporting MaterialÕÕ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. 4 J.X. and K.C. contributed equally to this work. * To whom correspondence should be addressed. E-mail: [email protected]. cn (Z. Feng), [email protected] (J. Liu).
rising obesity epidemic is increasing the risk of nonalcoholic fatty liver disease (NAFLD),8 which is one of the most common chronic liver diseases in the world (2,3), particularly among children and young adults (4), and affects ;30% of all U.S. adults and 75–100% of obese or morbidly obese individuals (5). The liver is a functional tissue that controls the production of TGs and glucose for use by other tissues. The development of NAFLD indicates a disruption in lipid and glucose homeostasis, which is regulated by pathways such as lipogenesis and gluconeogenesis (6). Sterol regulatory element binding proteins 8 Abbreviations used: ACC-1, acetyl-CoA carboxylase 1; BG, bitter gourd; CRP, C-reactive protein; DRP1, dynamin related protein 1; FAS, FA synthase; GSH, reduced glutathione; HFD, high-fat diet; HFD+0.5BG, high-fat diet with daily oral gavage of 0.5 g/kg bitter gourd; HFD+5BG, high-fat diet with daily oral gavage of 5 g/kg bitter gourd; IRS-1, insulin receptor substrate 1; MFN1, mitofusin 1; NADH, reduced nicotinamide-adenine dinucleotide; NAFLD, nonalcoholic fatty liver disease; PTP-1B, protein-tyrosine phosphatase 1B; SOD, superoxide dismutase; SREBP, sterol regulatory element binding protein; TC, total cholesterol.
ã 2014 American Society for Nutrition. Manuscript received October 25, 2013. Initial review completed November 23, 2013. Revision accepted January 28, 2014. First published online February 12, 2014; doi:10.3945/jn.113.187450.
475
Downloaded from jn.nutrition.org at UNIVERSITY OF VIRGINIA CLAUDE MOORE HEALTH SCIENCES LIBRARY on June 26, 2014
Jie Xu,4,5 Ke Cao,4,5 Yuan Li,5 Xuan Zou,6 Cong Chen,5 Ignatius Man-Yau Szeto,7 Zhizhong Dong,7 Youyou Zhao,7 Yujie Shi,7 Junkuan Wang,7 Jiankang Liu,5* and Zhihui Feng5*
Materials and Methods Chemicals. Antibodies against b-actin were purchased from Sigma. Antibodies against tubulin, SREBP-1c, and mitofusin 1 (MFN1) were purchased from Santa Cruz Biotechnology. Antibodies against GAPDH, FA synthase (FAS), Bcl-2, Bcl-XL , and Bax were purchased from Cell Signaling Technology. An antibody against dynamin related protein 1 (DRP1) was purchased from BD. Antibodies against complexes I, II, III, IV, and V were purchased from Invitrogen. BG powder was purchased from Guangxi Zhennong Seed Industry. BG was prepared by freezedrying. Briefly, fresh BG was washed thoroughly with water and sliced into 15-mm fragments, which were homogenized by an electric juicer. The juice was frozen and then completely lyophilized by continuous freeze-drying. The yield of the BG extract was ;4.76%; the total amount of saponins was used as a quantity control (2.2%). Animals and treatments. Four-week-old male C57BL/6 mice were purchased from SLAC Laboratory Animal and were housed in a temperature (22–28°C)- and humidity (60%)-controlled room on a 12-h light/12-h dark cycle (light from 0800 to 2000), with food and water provided during the experiments. After 1 wk of acclimatization, the mice were randomly divided into 4 groups (n = 10 in each group), as follows: mice fed a normal diet (control; 12% kcal fat content), mice fed an HFD (45% kcal fat content), mice fed an HFD with daily oral gavage of 0.5 g/kg BG (HFD+0.5BG), and mice fed an HFD with daily oral gavage of 5 g/kg BG (HFD+5BG). The composition of the HFD is presented in Supplemental Table 1. Due to technological limitations, only 15 components in soluble BG were identified and presented in Supplemental Table 2. After 16 wk, the mice were deprived of feed overnight and killed by decapitation. All of the procedures were performed in accordance with the U.S. Public Health Services Guide for the Care and Use of Laboratory Animals, and all possible efforts were made to minimize the suffering and the number of animals used in this study. 476
Xu et al.
Oral-glucose-tolerance test. An oral-glucose-tolerance test (1 g/kg body weight) was performed after 16 wk of feeding and gavage. All mice were deprived of feed overnight before the oral-glucose-tolerance test. Blood was taken from the retrobulbar vein both before and 30, 60, 120, and 180 min after oral-glucose gavage. The plasma glucose concentration was determined by the glucose oxidation method. Sample preparation. After the mice were killed, liver tissue and visceral fat pads (including the perirenal and epididymal fat pads) were removed and weighed. Blood samples were obtained by cardiac puncture, and serum was separated by centrifugation (1100 3 g; 10 min). The concentrations of TGs, total cholesterol (TC), LDL cholesterol, and HDL cholesterol were analyzed by using an automated biochemistry analyzer (Hitachi) with biochemical analysis kits (R&D Systems). Serum concentrations of C-reactive protein (CRP), free FAs, insulin, and IL-6 were measured by using commercial ELISA kits according to the manufacturerÕs standards and protocols (R&D Systems). Minimum levels of detection and CVs for each kit assay are provided in detail in Supplemental Table 3. Histologic analysis of liver samples. Liver tissues were excised, washed with ice-cold PBS, and placed in 10% formalin. Several tissue sections (4–5-mm thick) were prepared, stained with hematoxylin and eosin for histopathologic analysis, and visualized with an Olympus BX71 microscope. Isolation of liver mitochondria. Mitochondria were isolated as previously described (21). Briefly, liver tissue was rinsed with saline, weighed, and placed in an ice-cold isolation buffer containing 0.25 mol/L sucrose, 10 mmol/L Tris, and 0.5 mmol/L EDTA at pH 7.4. The tissue was sheared, carefully minced, rinsed to remove residual blood, and homogenized in 2.5 volumes of isolation buffer. The volume of the homogenate was increased to 8 initial volumes with the isolation buffer and centrifuged at 1000 g for 10 min; the supernatant fraction was decanted and saved. The pellet was washed once with 2 volumes of isolation buffer, and the total supernatant fractions were combined and centrifuged at 10,000 g for 10 min. The mitochondrial pellet was washed twice by using isolation buffer, and the protein concentration was determined by using a bicinchoninic acid protein assay kit. Freshly isolated mitochondria were either used immediately for biochemical assays or stored at 280°C. Biochemical analysis. Small portions of liver tissue were collected and homogenized in ice-cold PBS. After centrifugation (1000 g; 10 min), the supernatant was collected for analysis. Concentrations of reduced glutathione (GSH), liver TGs, and TC, as well as glutathione S-transferase, Cu-Zn superoxide dismutase (SOD), and Mn SOD activity, were analyzed by using commercial clinical diagnosis kits according to the manufacturerÕs standards and protocols (Jiancheng). Protein carbonylation assay. Protein carbonyls in soluble proteins were assayed by using the Oxyblot protein oxidation detection kit (Cell Biolabs). Protein carbonyls were labeled with 2,4-dinitrophenylhydrazine and detected by Western blot. Assays for mitochondrial complex activity. The activities of reduced nicotinamide-adenine dinucleotide (NADH)-ubiquinone reductase (complex I), succinate-coenzyme Q oxidoreductase (complex II), and cytochrome c oxidase (complex IV) were measured spectrophotometrically by using conventional assays as previously described (22,23). Western blotting. The samples were lysed in Western and Immunoprecipitation lysis buffers (Beyotime). The lysates were homogenized, and the homogenates were centrifuged at 13,000 g for 15 min at 4°C. The supernatants were collected, and the protein concentrations were determined by using a bicinchoninic acid protein assay kit. Equal aliquots (20 mg) of protein were analyzed by Western blotting. Chemiluminescent detection was performed by using an ECL Western blotting detection kit (Thermo Scientific) and was quantified by scanning densitometry. Statistical analysis. Normal distribution was assessed by Shapiro-Wilk test (SPSS). Results are presented as means 6 SEMs. Because micronutrient
Downloaded from jn.nutrition.org at UNIVERSITY OF VIRGINIA CLAUDE MOORE HEALTH SCIENCES LIBRARY on June 26, 2014
(SREBPs) are a class of proteins that transcriptionally activate a cascade of enzymes that are required for the endogenous synthesis of cholesterol, FAs, TGs, and phospholipids (7). Among the 3 isoforms, SREBP-1c is the central regulator of lipid metabolism in vivo, and SREBP-1c activation contributes to hepatic lipogenesis and NAFLD (8,9). Studies have demonstrated that excessive lipid accumulation in the liver is associated with oxidative stress and mitochondrial dysfunction (10,11). In mice, a high-fat diet (HFD) induces the overproduction of reactive oxygen species in adipose tissue and liver, which precedes the onset of obesity and insulin resistance (12). In rats, hepatic mitochondrial dysfunction precedes the development of NAFLD and insulin resistance (13). Because several processes regulate mitochondrial function, the molecular basis of mitochondrial involvement in the development of NAFLD is unclear. However, the prominent role of mitochondrial dysfunction in NAFLD provides the basis for potential dietary and pharmacologic intervention. Momordica charantia, also known as bitter gourd (BG), is a climber plant that is distinguished by its bitter-tasting fruit. In recent decades, several hundred studies have been conducted using BG because of its popular medicinal uses. Among these properties, its antidiabetic activity is the most popular effect, and this is supported by biochemical experiments and animal models (14–17). Although several possible mechanisms, including b cell protection (18), the regulation of PPARa and PPARg (19), increasing skeletal muscle insulin-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation (20), and inhibiting protein-tyrosine phosphatase 1B (PTP-1B) (16), have been proposed for the antidiabetic activity of BG, studies of the effects of BG on mitochondrial dysfunction and NAFLD are limited. This study was designed to determine the effect of BG fruit extract on NAFLD and mitochondrial function in HFD-induced obese mice.
concentrations were not adjusted in the HFD, the control group in the study was included as a reference only. Statistical analysis was conducted by using data from only 3 HFD groups via 1-factor ANOVA followed by a least significant difference post hoc analysis. For all of the analyses, P values
