Eur J Nutr DOI 10.1007/s00394-015-0907-0
ORIGINAL CONTRIBUTION
Medium‑chain triglyceride ameliorates insulin resistance and inflammation in high fat diet‑induced obese mice Shanshan Geng1 · Weiwei Zhu1 · Chunfeng Xie1 · Xiaoting Li1 · Jieshu Wu1 · Zhaofeng Liang1 · Wei Xie1 · Jianyun Zhu1 · Cong Huang1 · Mingming Zhu1 · Rui Wu1 · Caiyun Zhong1
Received: 24 October 2014 / Accepted: 15 April 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose The aim of the present study was to investigate the in vivo effects of dietary medium-chain triglyceride (MCT) on inflammation and insulin resistance as well as the underlying potential molecular mechanisms in high fat diet-induced obese mice. Methods Male C57BL/6J mice (n = 24) were fed one of the following three diets for a period of 12 weeks: (1) a modified AIN-76 diet with 5 % corn oil (normal diet); (2) a high-fat control diet (17 % w/w lard and 3 % w/w corn oil, HFC); (3) an isocaloric high-fat diet supplemented with MCT (17 % w/w MCT and 3 % w/w corn oil, HF–MCT). Glucose metabolism was evaluated by fasting blood glucose levels and intraperitoneal glucose tolerance test. Insulin sensitivity was evaluated by fasting serum insulin levels and the index of homeostasis model assessment-insulin resistance. The levels of serum interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α were measured by ELISA, and hepatic activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways was determined using western blot analysis. Results Compared to HFC diet, consumption of HF-MCT did not induce body weight gain and white adipose tissue accumulation in mice. HFC-induced increases in serum fasting glucose and insulin levels as well as glucose intolerance were prevented by HF-MCT diet. Meanwhile, HFMCT resulted in significantly lower serum IL-6 level and Shanshan Geng and Weiwei Zhu have contributed equally to this work. * Caiyun Zhong [email protected] 1
Department of Nutrition and Food Safety, School of Public Health, Nanjing Medical University, Nanjing 211166, China
higher IL-10 level, and lower expression levels of inducible nitric oxide synthase and cyclooxygenase-2 protein in liver tissues when compared to HFC. In addition, HF-MCT attenuated HFC-triggered hepatic activation of NF-κB and p38 MAPK. Conclusions Our study demonstrated that MCT was efficacious in suppressing body fat accumulation, insulin resistance, inflammatory response, and NF-κB and p38 MAPK activation in high fat diet-fed mice. These data suggest that MCT may exert beneficial effects against high fat diet-induced insulin resistance and inflammation. Keywords Medium-chain triglyceride · Insulin resistance · Inflammation · NF-κB · p38 MAPK
Introduction Metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, and hypertension are major public health problems. Although the current pharmacological interventions for the treatment of obesity-related metabolic disorders are effective, these medications are often associated with adverse side effects. Therefore, lifestyle modifications remain essential components for the prevention and treatment of metabolic diseases. In this context, medium-chain triglyceride (MCT) exerts a variety of health benefits. In addition to their ability to reduce serum lipid levels [1], they decrease body fat and hepatic lipid accumulation and increase fatty acid oxidation in animals [2–5] and humans [6–8]; MCT consumption has been shown to reduce cardiovascular risk. Consumption of MCT oil at a level of approximately 18–24 g/d, as part of a weight loss diet, improves cardiovascular risk profile [9, 10]. It has also been reported that MCT is beneficial for the prevention
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of high fat diet-induced steatosis, alcoholic liver injury [1, 11], neurodegenerative disorders [12] and inflammatory diseases [13–16]. The pathophysiology of obesity and type 2 diabetes is characterized by low-grade chronic systemic and local inflammation. It is recognized that low-grade chronic inflammation elicited during obesity closely connects obesity to the development of glucose intolerance and insulin resistance [17]. This inflammatory state involved in the control of metabolic homeostasis has been reported in different organs, including adipose tissue, liver, endocrine pancreas, hypothalamus, and skeletal muscles [18]. Activation of pro-inflammatory signaling pathways has been shown to link inflammation with obesity and insulin resistance. The nuclear factor κB (NFκB) pathway and the mitogen-activated protein kinase (MAPK) pathways, which include the extracellular signal-regulated kinase (ERK), the Jun N-terminal kinase (JNK), and p38 MAPK, are among the key signal pathways in the regulation of inflammatory process. Through the transcription of various inflammatory cytokines and enzymes such as tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), NF-κB and MAPK pathways play crucial role in the development of inflammation as well as glucose intolerance and insulin resistance [19–21]. Previous studies have showed the anti-inflammatory effects of MCT. It has been reported that MCT diet resulted in suppressed inflammation and downregulated production of pro-inflammatory cytokines in various inflammation models, including interleukin-10-deficient spontaneous intestinal inflammation in mice [15], endotoxin-challenged mice [13], trinitrobenzene sulfonic acid-induced experimental colitis in rats [14], as well as intestinal inflammation in Caco-2 cells [16]. It is noted that inconsistent results for MCT effects on inflammation have been observed [22, 23]. Evidence has also showed the anti-diabetic properties of MCT in animals and humans. MCT-containing diet improves lipid metabolism and glucose intolerance in rats and diabetic patients [24, 25]. Nevertheless, previous studies almost exclusively depicted the effects of MCT on inflammation and glucose tolerance separately; little information is available so far with regard to the influence of MCT on both aspects of inflammatory response and insulin resistance simultaneously, an issue that needs to be clarified in the same context. Therefore, the present study investigated the effects of MCT on inflammation and glucose tolerance as well as the underlying potential molecular mechanisms in high fat diet-induced obese and insulinresistant C57BL/6 mice.
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Eur J Nutr Table 1 Compositions of experimental diets Ingredients (g/kg)
ND
HFC
HF–MCT
Casein DL-methionine Corn starch Sucrose Cellulose Corn oil Lard MCT oil Mineral mixture, AIN-76
200 3 150 500 50 50
200 3 111 370 50 30 170
200 3 111 400 20 30
35
42
170 42
Vitamin mixture, AIN-76 Choline bitartrate Cholesterol Tert-butyl hydroquinone Fat (% of energy)
10 2 0.01 11.5
12 2 10 0.04 37
12 2 10 0.04 39
Total energy (MJ/kg diet)
16.3
19.2
19.2
ND normal diet, HFC high-fat control diet, HF-MCT an isocaloric high-fat diet supplement with MCT
Materials and methods Animals and experimental protocol The animal experimental protocol was approved by the Institutional Animal Care & Use Committee (IACUC) of Nanjing Medical University (Protocol Number NJMU08092). Male C57BL/6J mice (5 weeks old) were purchased from the Animal Center of Nanjing Medical University (Nanjing, Jiangsu, China) and housed in a specific pathogenfree facility throughout the experimental period. Mice were maintained under controlled room temperature (22 ± 2 °C), 12-h light/dark cycles with ad libitum access to food and water. After 1-week acclimatization period, mice were randomly divided into three groups (n = 8 per group): a normal diet (ND), a high-fat control diet (HFC), and an isocaloric high-fat diet containing MCT (HF–MCT). The ND was a diet based on the AIN-76 standard rodent diet composition with 5 % corn oil. Both high-fat diets contained 200 g of fat per kilogram and 1 % cholesterol by weight. The HFC diet contained 170 g of lard and 30 g of corn oil which provides essential fatty acids. In HF-MCT diet, the lard was replaced by MCT. The MCT oil was purchased from Zhejiang Hailisheng Biotechnology Co. (Zhejiang, China), and the fatty acid compositions of MCT were 67 % caprylic acid (C8:0), 23 % capric acid (C10:0), and 10 % other MCT. The compositions of the experimental diets are shown in Table 1. The energy contents of both high-fat diets were 19.2 MJ/ kg, whereas that of the ND was 16.3 MJ/kg. All diets were given in the form of a pellet for 11 weeks.
Eur J Nutr
The diet consumption of mice was recorded daily, and body weight was monitored weekly. Food efficiency ratio (FER) was calculated by the following equation: FER = [body weight gain during experimental period (g)]/ [food intake during experimental period (g)]. At the end of 11-week feeding period, mice were anesthetized with diethyl ether after an overnight fasting for 16 h, and blood samples were collected. Serum was isolated by centrifugation at 3000×g for 10 min and stored at −80 °C for subsequent analysis. Liver tissues were collected, washed with phosphate-buffered saline, and frozen at −80 °C.
fat-free milk and incubated with anti-iNOS, anti-COX-2, anti-p-p65, anti-p65, anti-p50, anti-p-IKKβ, anti-IκBα, anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA), or anti-p-ERK1/2, anti-p-JNK, anti-p-p38, antilamin B (Bioss, Beijing, China), respectively. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies and visualized using enhanced chemiluminescence (ECL, Millipore, Billerica, MA, USA). The relative expression levels of target proteins to the controls were determined by densimetric analysis using ImageJ software.
ELISA measurements of serum cytokines and insulin
Statistical analysis
The concentrations of serum TNF-α, IL-6, and IL-10 were measured using cytokine-specific ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MI, USA). Similarly, the concentrations of serum insulin were determined using mouse insulin ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MI, USA).
Data were expressed as the mean ± standard deviation. The differences among groups were analyzed by one-way ANOVA and the least significant difference (LSD) using SPSS 13.0 program. A P value
ORIGINAL CONTRIBUTION
Medium‑chain triglyceride ameliorates insulin resistance and inflammation in high fat diet‑induced obese mice Shanshan Geng1 · Weiwei Zhu1 · Chunfeng Xie1 · Xiaoting Li1 · Jieshu Wu1 · Zhaofeng Liang1 · Wei Xie1 · Jianyun Zhu1 · Cong Huang1 · Mingming Zhu1 · Rui Wu1 · Caiyun Zhong1
Received: 24 October 2014 / Accepted: 15 April 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose The aim of the present study was to investigate the in vivo effects of dietary medium-chain triglyceride (MCT) on inflammation and insulin resistance as well as the underlying potential molecular mechanisms in high fat diet-induced obese mice. Methods Male C57BL/6J mice (n = 24) were fed one of the following three diets for a period of 12 weeks: (1) a modified AIN-76 diet with 5 % corn oil (normal diet); (2) a high-fat control diet (17 % w/w lard and 3 % w/w corn oil, HFC); (3) an isocaloric high-fat diet supplemented with MCT (17 % w/w MCT and 3 % w/w corn oil, HF–MCT). Glucose metabolism was evaluated by fasting blood glucose levels and intraperitoneal glucose tolerance test. Insulin sensitivity was evaluated by fasting serum insulin levels and the index of homeostasis model assessment-insulin resistance. The levels of serum interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α were measured by ELISA, and hepatic activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways was determined using western blot analysis. Results Compared to HFC diet, consumption of HF-MCT did not induce body weight gain and white adipose tissue accumulation in mice. HFC-induced increases in serum fasting glucose and insulin levels as well as glucose intolerance were prevented by HF-MCT diet. Meanwhile, HFMCT resulted in significantly lower serum IL-6 level and Shanshan Geng and Weiwei Zhu have contributed equally to this work. * Caiyun Zhong [email protected] 1
Department of Nutrition and Food Safety, School of Public Health, Nanjing Medical University, Nanjing 211166, China
higher IL-10 level, and lower expression levels of inducible nitric oxide synthase and cyclooxygenase-2 protein in liver tissues when compared to HFC. In addition, HF-MCT attenuated HFC-triggered hepatic activation of NF-κB and p38 MAPK. Conclusions Our study demonstrated that MCT was efficacious in suppressing body fat accumulation, insulin resistance, inflammatory response, and NF-κB and p38 MAPK activation in high fat diet-fed mice. These data suggest that MCT may exert beneficial effects against high fat diet-induced insulin resistance and inflammation. Keywords Medium-chain triglyceride · Insulin resistance · Inflammation · NF-κB · p38 MAPK
Introduction Metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, and hypertension are major public health problems. Although the current pharmacological interventions for the treatment of obesity-related metabolic disorders are effective, these medications are often associated with adverse side effects. Therefore, lifestyle modifications remain essential components for the prevention and treatment of metabolic diseases. In this context, medium-chain triglyceride (MCT) exerts a variety of health benefits. In addition to their ability to reduce serum lipid levels [1], they decrease body fat and hepatic lipid accumulation and increase fatty acid oxidation in animals [2–5] and humans [6–8]; MCT consumption has been shown to reduce cardiovascular risk. Consumption of MCT oil at a level of approximately 18–24 g/d, as part of a weight loss diet, improves cardiovascular risk profile [9, 10]. It has also been reported that MCT is beneficial for the prevention
13
of high fat diet-induced steatosis, alcoholic liver injury [1, 11], neurodegenerative disorders [12] and inflammatory diseases [13–16]. The pathophysiology of obesity and type 2 diabetes is characterized by low-grade chronic systemic and local inflammation. It is recognized that low-grade chronic inflammation elicited during obesity closely connects obesity to the development of glucose intolerance and insulin resistance [17]. This inflammatory state involved in the control of metabolic homeostasis has been reported in different organs, including adipose tissue, liver, endocrine pancreas, hypothalamus, and skeletal muscles [18]. Activation of pro-inflammatory signaling pathways has been shown to link inflammation with obesity and insulin resistance. The nuclear factor κB (NFκB) pathway and the mitogen-activated protein kinase (MAPK) pathways, which include the extracellular signal-regulated kinase (ERK), the Jun N-terminal kinase (JNK), and p38 MAPK, are among the key signal pathways in the regulation of inflammatory process. Through the transcription of various inflammatory cytokines and enzymes such as tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), NF-κB and MAPK pathways play crucial role in the development of inflammation as well as glucose intolerance and insulin resistance [19–21]. Previous studies have showed the anti-inflammatory effects of MCT. It has been reported that MCT diet resulted in suppressed inflammation and downregulated production of pro-inflammatory cytokines in various inflammation models, including interleukin-10-deficient spontaneous intestinal inflammation in mice [15], endotoxin-challenged mice [13], trinitrobenzene sulfonic acid-induced experimental colitis in rats [14], as well as intestinal inflammation in Caco-2 cells [16]. It is noted that inconsistent results for MCT effects on inflammation have been observed [22, 23]. Evidence has also showed the anti-diabetic properties of MCT in animals and humans. MCT-containing diet improves lipid metabolism and glucose intolerance in rats and diabetic patients [24, 25]. Nevertheless, previous studies almost exclusively depicted the effects of MCT on inflammation and glucose tolerance separately; little information is available so far with regard to the influence of MCT on both aspects of inflammatory response and insulin resistance simultaneously, an issue that needs to be clarified in the same context. Therefore, the present study investigated the effects of MCT on inflammation and glucose tolerance as well as the underlying potential molecular mechanisms in high fat diet-induced obese and insulinresistant C57BL/6 mice.
13
Eur J Nutr Table 1 Compositions of experimental diets Ingredients (g/kg)
ND
HFC
HF–MCT
Casein DL-methionine Corn starch Sucrose Cellulose Corn oil Lard MCT oil Mineral mixture, AIN-76
200 3 150 500 50 50
200 3 111 370 50 30 170
200 3 111 400 20 30
35
42
170 42
Vitamin mixture, AIN-76 Choline bitartrate Cholesterol Tert-butyl hydroquinone Fat (% of energy)
10 2 0.01 11.5
12 2 10 0.04 37
12 2 10 0.04 39
Total energy (MJ/kg diet)
16.3
19.2
19.2
ND normal diet, HFC high-fat control diet, HF-MCT an isocaloric high-fat diet supplement with MCT
Materials and methods Animals and experimental protocol The animal experimental protocol was approved by the Institutional Animal Care & Use Committee (IACUC) of Nanjing Medical University (Protocol Number NJMU08092). Male C57BL/6J mice (5 weeks old) were purchased from the Animal Center of Nanjing Medical University (Nanjing, Jiangsu, China) and housed in a specific pathogenfree facility throughout the experimental period. Mice were maintained under controlled room temperature (22 ± 2 °C), 12-h light/dark cycles with ad libitum access to food and water. After 1-week acclimatization period, mice were randomly divided into three groups (n = 8 per group): a normal diet (ND), a high-fat control diet (HFC), and an isocaloric high-fat diet containing MCT (HF–MCT). The ND was a diet based on the AIN-76 standard rodent diet composition with 5 % corn oil. Both high-fat diets contained 200 g of fat per kilogram and 1 % cholesterol by weight. The HFC diet contained 170 g of lard and 30 g of corn oil which provides essential fatty acids. In HF-MCT diet, the lard was replaced by MCT. The MCT oil was purchased from Zhejiang Hailisheng Biotechnology Co. (Zhejiang, China), and the fatty acid compositions of MCT were 67 % caprylic acid (C8:0), 23 % capric acid (C10:0), and 10 % other MCT. The compositions of the experimental diets are shown in Table 1. The energy contents of both high-fat diets were 19.2 MJ/ kg, whereas that of the ND was 16.3 MJ/kg. All diets were given in the form of a pellet for 11 weeks.
Eur J Nutr
The diet consumption of mice was recorded daily, and body weight was monitored weekly. Food efficiency ratio (FER) was calculated by the following equation: FER = [body weight gain during experimental period (g)]/ [food intake during experimental period (g)]. At the end of 11-week feeding period, mice were anesthetized with diethyl ether after an overnight fasting for 16 h, and blood samples were collected. Serum was isolated by centrifugation at 3000×g for 10 min and stored at −80 °C for subsequent analysis. Liver tissues were collected, washed with phosphate-buffered saline, and frozen at −80 °C.
fat-free milk and incubated with anti-iNOS, anti-COX-2, anti-p-p65, anti-p65, anti-p50, anti-p-IKKβ, anti-IκBα, anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA), or anti-p-ERK1/2, anti-p-JNK, anti-p-p38, antilamin B (Bioss, Beijing, China), respectively. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies and visualized using enhanced chemiluminescence (ECL, Millipore, Billerica, MA, USA). The relative expression levels of target proteins to the controls were determined by densimetric analysis using ImageJ software.
ELISA measurements of serum cytokines and insulin
Statistical analysis
The concentrations of serum TNF-α, IL-6, and IL-10 were measured using cytokine-specific ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MI, USA). Similarly, the concentrations of serum insulin were determined using mouse insulin ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MI, USA).
Data were expressed as the mean ± standard deviation. The differences among groups were analyzed by one-way ANOVA and the least significant difference (LSD) using SPSS 13.0 program. A P value
