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Dietary intake of medium- and long-chain triacylglycerols ameliorates insulin resistance in rats fed a high-fat diet
Nutrition 28 (2012) 92–97
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Basic nutritional investigation
Dietary intake of medium- and long-chain triacylglycerols ameliorates insulin resistance in rats fed a high-fat diet Shin Terada Ph.D. *, Sayuri Yamamoto M.Sc., Seiji Sekine Ph.D., Toshiaki Aoyama Ph.D., R.D. Central Research Laboratory, The Nisshin OilliO Group, Ltd., Yokosuka City, Kanagawa Prefecture, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2011 Accepted 30 April 2011
Objective: Excessive accumulation of visceral fat is strongly associated with insulin resistance. The present investigation examined the effects of dietary intake of medium- and long-chain triacylglycerols (MLCTs), which have been shown to induce significantly lower visceral fat accumulation in rats and humans, on high-fat diet–induced obesity and insulin resistance in rats. These effects were then compared with those observed in long-chain triacylglycerol (LCT)-fed rats. Methods: After an 8-wk feeding of a high-fat diet, which induced severe whole-body insulin resistance, male Sprague-Dawley rats were fed a standard diet containing LCTs or MLCTs for 6 wk. After the dietary treatment, an oral glucose tolerance test was performed. Results: Although body weight and total intra-abdominal fat mass did not differ between the two groups, mesenteric fat weight in the MLCT-fed group was significantly lower than that in the LCT group (P < 0.05). The increase in plasma insulin concentrations, but not in glucose, after glucose administration (area under the curve) was significantly smaller in the MLCT group than in the LCT group (P < 0.01) and was significantly associated with mesenteric fat weight (P < 0.05). MLCT-fed rats had significantly higher plasma adiponectin concentrations compared with LCT rats (P < 0.05). Adiponectin concentrations were negatively correlated with the area under the curve for plasma insulin (P < 0.05) and tended to be inversely related to mesenteric fat weight (P ¼ 0.08). Conclusion: These results suggest that dietary intake of MLCTs may improve insulin resistance in rats fed a high-fat diet, at least in part through increased adiponectin concentrations caused by a lower mesenteric fat mass. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Medium-chain fatty acids Insulin resistance High-fat diet Adiponectin Rat
Introduction Over the past three decades, there has been an explosive increase in the prevalence of type 2 diabetes [1]. Insulin resistance is an early and key defect associated with type 2 diabetes [2] and is postulated to be caused by an excessive accumulation of visceral fat [3,4]. The typical Western diet, which is very high in fat and sucrose, is considered a major factor in the visceral fat accumulation and the development of insulin resistance. Foods that are designed to decrease visceral fat mass may help to prevent the development of diet-induced insulin resistance and diabetes. Compared with long-chain fatty acids (LCFAs), mediumchain fatty acids (MCFAs; composed of chains of 8–12 carbon chains) have several unique nutritional and physiologic * Corresponding author. Tel.: þ81-46-837-2438; fax: þ81-46-837-2471. E-mail address: [email protected] (S. Terada). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.04.008
properties. Although LCFAs are absorbed through the intestinal lymphatic ducts and are transported as chylomicrons to the systemic circulation, it is well documented that MCFAs are absorbed through the portal system and are easily oxidized in the liver because the intramitochondrial transport of MCFAs does not require carnitine palmitoyltransferase, a rate-limiting enzyme of mitochondrial b-oxidation [5–8]. These characteristics make medium-chain triacylglycerols (MCTs), composed exclusively of MCFAs, a useful dietary treatment for preventing and reversing obesity and lifestyle-related disease [5]. Many investigators have reported the body weight- and body fat–decreasing effects of MCTs in animal studies [9–11], and we previously demonstrated that sufficiently calorie-controlled diets containing 10 g of MCT per day can induce significant decreases in body weight and fat mass in human subjects [12]. In addition, there has been accumulating evidence from animal and human studies that MCTs improve insulin sensitivity and glucose tolerance in diabetic and non-diabetic states [13–16].
S. Terada et al. / Nutrition 28 (2012) 92–97
Our previous study also demonstrated that rats fed a diet containing 20% MCTs for 8 wk had lower visceral fat weight accompanied by better glucose tolerance compared with rats fed log-chain triacylglycerols (LCTs) [17]. Although MCTs may be a valuable tool for preventing or alleviating insulin resistance and diabetes, it is not feasible to substitute MCTs for LCTs in the diet for long-term therapy because their lower smoking point and greater tendency to bubble limit the daily use of MCTs as cooking oil. A cooking oil containing medium- and long-chain fatty acids in the same glycerol molecule (MLCT) prepared by random interesterification is commercially available and widely used in Japan [18,19]. MLCT has overcome the disadvantages encountered with MCTs and can easily replace LCTs in the daily diet. Despite the lower MCFA content (w13%), this edible oil has also been reported to increase energy expenditure [20,21] and to cause a significantly lower visceral fat accumulation in humans and rats compared with LCT oil [21–26]. These results have led us to hypothesize that MLCT, instead of MCT, could be a useful dietary treatment to ameliorate insulin resistance, which is associated with visceral fat accumulation. In this context, the present study evaluated the effects of a dietary intake of MLCT on high-fat diet–induced insulin resistance in rats. Materials and methods Materials The MLCT and LCT (rapeseed oil) oils were obtained from The Nisshin OilliO Group (Tokyo, Japan). Their fatty acid compositions, as measured by gas chromatography, are listed in Table 1. Treatment of animals Four-week-old male Sprague-Dawley rats (70–90 g of body weight) were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan) and fed a high-fat (28% wt/wt) diet (n ¼ 31) for 8 wk. The high-fat diet contained the following ingredients (grams per kilogram): sucrose, 347.286; casein, 293.4; lard, 180; rapeseed oil, 100; methionine, 5; AIN-93 vitamin mix, 22; AIN-93G mineral mix, 51; choline bitartrate, 1.3; and tert-butyl hydroquinone, 0.014 [27–29]. The rats were provided with the diets and water ad libitum. Feeding of the high-fat diet for 4 to 8 wk has been shown to cause severe whole-body insulin resistance in rats [27–29]. The rats were separated into two groups matched for body weight, plasma glucose, and insulin concentrations after 8 wk on the high-fat diet. Each group of rats was allowed free access to the experimental diet containing LCTs (n ¼ 15) or MLCTs (n ¼ 16) for 6 wk. The composition of the diet is presented in Table 2. Both diets were based on the AIN-93 formula with the modification that sucrose was decreased from 100 to 40 g/kg of diet [24]. The energy content of the high-fat diet was 5.2 kcal/g, whereas that of the experimental diet was 4.0 kcal/g. Food intake and body weight were recorded every second day. All rats were treated in accordance with the guidelines established by the Japanese Society of Nutrition and Food Science (Law No. 105 and Notification No. 6 of the Japanese Government).
Table 1 Fatty acid compositions of test lipids Fatty acid*
LCT
MLCT
8:0 10:0 16:0 18:0 18:1 18:2 18:3 Others
ND ND 5.6 2.4 62.9 18.3 6.6 4.2
8.3 2.7 2.8 1.2 57.5 18.1 7.2 2.2
LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols; ND, not detected * Numbers are carbon atoms:double bonds.
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Table 2 Composition of experimental diets Ingredients
Dietary group (g/kg diet) LCT
MLCT
Cornstarch Casein Alpha-cornstarch Sucrose LCT oil MLCT oil Cellulose powder Mineral mix (AIN-93M) Vitamin mix (AIN-93) L-Cystine Choline bitartrate tert-Butyl hydroquinone
457.5 200 132 40 70 – 50 35 10 3 2.5 0.014
457.5 200 132 40 – 70 50 35 10 3 2.5 0.014
LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols
Oral glucose tolerance test After 6 wk of dietary treatment, an oral glucose tolerance test (OGTT) was performed. After overnight (16-h) fasting, glucose (1.5 g/kg of body weight) was orally administered using a stainless-steel gavage needle with a ball diameter. Blood samples were collected into capillary tubes from the tail vein immediately before and 30, 60, and 120 min after glucose administration. The capillary tubes were then centrifuged, and plasma samples were stored at 80 C until analysis. The areas under the curve (AUCs) for plasma glucose and insulin were calculated with the trapezoidal rule.
Tissue collection At the end of the feeding period, rats were sacrificed under anesthesia with diethyl ether without fasting. Blood samples were collected by cardiac puncture, and the triceps muscle and liver were then removed and weighed. After the muscle and liver dissections were completed, epididymal, mesenteric, and retroperitoneal fat pads were removed and weighed. Liver and muscle samples were frozen in liquid N2 and stored at 80 C until analysis. Analytical procedure Plasma glucose, free fatty acid, and triacylglycerol concentrations were determined using kits (Glucose C2 Test Wako, NEFA-C Test Wako, and Triglyceride E Test Wako, respectively) obtained from Wako Pure Chemical (Osaka, Japan). Plasma concentrations of insulin, adiponectin, resistin, and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (insulin, Mercodia AB, Uppsala, Sweden; adiponectin, Otsuka Pharmaceutical, Tokyo, Japan; resistin, BioVendor, Karasek, Czech Republic; MCP-1: Bender MedSystems, San Diego, CA, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as the product of the fasting serum glucose and insulin levels divided by a constant, assuming that rats have an average HOMA-IR of 1, analogous to the assumptions applied in the development of HOMA-IR in humans [30]. The equation was as follows: HOMA-IR ¼ (fasting glucose level [mg/dL] fasting insulin level [mU/mL]/2430 [31]. Muscle and liver triacylglycerol concentrations were determined by extracting total lipids from frozen triceps muscle and liver with chloroformmethanol (2:1 vol/vol), as described by Folch et al. [32], separating the chloroform and methanol water phases, removing phospholipids, and further processing the sample using Frayn and Maycock’s [33] modification of the method of Denton and Randle [34]. Triacylglycerols were then quantified spectrophotometrically as glycerol using an assay kit (Triglyceride E TEST Wako).
Statistical analysis The data are presented as mean standard error of the mean. In the experiment that involved OGTT, two-way analysis of variance (Jandel Sigma Stat, San Jose, CA, USA) was performed to examine the effects of time and dietary conditions. For the other experiments, statistical analysis was performed using the Student t test (Jandel Sigma Stat). Statistical significance was defined as P < 0.05.
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Results Body weight, food intake, intra-abdominal fat, and liver weights
Table 3 Body weight, food intake, food efficacy, and liver and intra-abdominal fat weights in LCT and MLCT rat groups LCT
Figure 1 shows the body weight change in rats. During the 8wk feeding of a high-fat diet, body weight progressively increased. Although body weights slightly increased in response to the LCT and MLCT diets, no significant differences in the final body weight and body weight gain were observed between the two groups (Table 3). Food intake and food efficiency were not significantly different between the LCT and MLCT groups (Table 3). Although epididymal, retroperitoneal, and total intra-abdominal fat weights did not significantly differ between the two groups, the mesenteric fat weight, expressed relative to body weight, in the MLCT group was significantly lower than that observed in the LCT group (P < 0.05; Table 3). There was no significant difference in liver weight between the two groups (Table 3). Oral glucose tolerance test The plasma glucose and insulin concentrations before glucose administration (after 16-h fasting) were not significantly different between the LCT and MLCT groups (Table 4, Fig. 2), resulting in similar values for HOMA-IR in the two groups (Table 4). Plasma glucose levels during the OGTT and the AUC for plasma glucose were not significantly different between the two groups (Fig. 2A, B). Two-way analysis of variance revealed that dietary treatment had a significant effect on plasma insulin levels during the OGTT, resulting in significantly lower plasma insulin concentrations in the MLCT group (P < 0.001; Fig. 2C). The increase in plasma insulin concentrations (plasma insulin AUC) in the MLCT group was also significantly smaller than that observed in the LCT group (Fig. 2D). As shown in Figure 3, there was a significant correlation between plasma insulin AUC and mesenteric fat weight (r ¼ 0.420, P < 0.05). No such correlation was observed with the other adipose tissues examined (data not shown). Blood lipid and adipocytokine concentrations and their relation to the OGTT No significant differences in plasma free fatty acid and triacylglycerol concentrations were observed between the LCT and
Initial body weight (g) Final body weight (g) Body weight gain (g) Food intake (g/d) Food efficiency (%)* Liver weight (g/100 g BW) Epididymal fat (g/100 g BW) Retroperitoneal fat (g/100 g BW) Mesenteric fat (g/100 g BW) Total intra-abdominal fat (g/100 g BW)
459 519 60 19.0 7.8 3.78 2.81 3.85 2.22 8.87
MLCT
10 10 7 0.4 0.8 0.08 0.13 0.14 0.10 0.34
459 516 58 18.4 7.8 3.71 2.81 3.93 1.86 8.60
9 10 5 0.5 0.6 0.06 0.11 0.14 0.10y 0.32
BW, body weight; LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols Values are presented as mean SEM * Equal to weight gain (grams)/food intake (grams) 100. y Significant difference from the LCT group at P < 0.05. The initial body weight was measured at the beginning of feeding a diet containing LCT or MLCT.
MLCT groups (Table 4). There were also no significant differences in plasma resistin and MCP-1 concentrations between the two groups (Table 4). In contrast, plasma adiponectin concentrations were significantly higher in the MLCT than the LCT group (P < 0.05). As shown in Figure 4A, plasma adiponectin concentrations tended to be inversely correlated with mesenteric fat mass, although the relation did not reach statistical significance (P ¼ 0.08). There was a significant negative correlation between adiponectin concentrations and plasma insulin AUC (r ¼ 0.420, P < 0.05; Fig. 4B). Muscle and liver triacylglycerol concentrations There were no significant differences in muscle and liver triacylglycerol concentrations between the LCT and MLCT groups (Table 5). No significant correlation was observed between plasma insulin AUC and liver/muscle triacylglycerol content (data not shown). Discussion To examine the effects of dietary MLCT consumption on glucose tolerance and insulin sensitivity, we performed the OGTT after 6-wk feeding of meals containing MLCTs or LCTs in rats previously fed a high-fat diet, which is an animal model frequently used to induce obesity and severe insulin resistance [27–29]. In consequence, we found that rats fed MLCTs had
Table 4 Plasma glucose, FFA, triacylglycerol, insulin, and adipocytokine concentrations in the fasting state LCT Glucose (mg/dL) FFA (mEq/L) Triacylglycerol (mg/dL) Insulin (ng/mL) MCP-1 (ng/mL) Resistin (ng/mL) Adiponectin (mg/mL) HOMA-IR Fig. 1. Body weight of rats during feedings of high-fat and experimental diets. The final data points represent final values the day before sacrifice. Values are presented as mean SEM. LCT, long-chain triacylglycerols; MLCT, medium- and long-chain triacylglycerols.
107 1.53 265 1.61 7.7 23.0 1.87 2.0
MLCT
4 0.07 22 0.23 0.4 0.6 0.09 0.3
107 1.52 258 1.70 7.8 23.4 2.12 2.2
3 0.06 17 0.27 0.5 0.9 0.07* 0.4
FFA, free fatty acid; HOMA-IR, homeostasis model assessment of insulin resistance; LCT, long-chain triacylglycerols (rapeseed oil); MCP-1, monocyte chemoattractant protein-1; MLCT, medium- and long-chain triacylglycerols Values are presented as mean SEM * Significant difference from the LCT group at P < 0.05.
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Fig. 2. Plasma glucose (A) and insulin (C) responses after oral glucose administration (1.5 g/kg of body weight) in rats fed an MLCT- or LCT-containing diet for 6 wk. The AUCs for plasma glucose (B) and insulin (D) during the 120-min period after oral glucose administration was calculated in accordance with the trapezoidal rule. Values are presented as mean SEM. **P < 0.01 and ***P < 0.001, significant differences from LCT. AUC, area under the curve; LCT, long-chain triacylglycerols; MLCT, medium- and longchain triacylglycerols.
a markedly blunted insulin response to a glucose load compared with the LCT-fed group (Fig. 2C, D). These results suggest that MLCTs compared with LCTs could be a valuable dietary intervention to alleviate excess calorie-induced insulin resistance.
Fig. 3. Correlation between the AUC of plasma insulin during the oral glucose tolerance test and mesenteric fat mass in rats fed an LCT- or MLCT-containing diet for 6 wk. AUC, area under the curve; BW, body weight; LCT, long-chain triacylglycerols; MLCT, medium- and long-chain triacylglycerols.
Previous studies have shown that MCT exerts favorable effects on insulin resistance and glucose intolerance in rodents [13,16, 17]. In those studies [13,16,17], a relatively larger amount of MCT (w200 g/kg of diet in animals), which consisted exclusively of MCFAs, was administered. In the present investigation, the MLCT-containing diet, in which the total MCFA content was only w0.8% (wt/wt), ameliorated insulin resistance in rats fed a high-fat diet (Fig. 2B, D), suggesting that even a small amount of MCFA is enough to prevent or alleviate insulin resistance in rats. However, because MLCTs consist of MCFAs and LCFAs in the same glycerol molecule by random interesterification [18,19], we could not rule out the possibility that the unique structure, not MCFA itself, might be responsible for its beneficial effects. Further studies will be required to elucidate the mechanism by which MLCT improves excess calorie-induced insulin resistance. Our results showed that rats fed an MLCT diet had significantly lower mesenteric fat mass than the LCT rats (Table 3). This result confirmed our previous findings observed in non-obese rats [21,23–25] and might provide further evidence that MLCT intake has a small but significant effect on visceral fat accumulation even in excess calorie-induced obese rats. It is well documented that an increase in visceral fat is associated with the development of insulin resistance [3,4]. Kim et al. [29] reported that calorie restriction, leptin injection, and fish oil feeding, which are well known to decrease visceral fat mass, completely prevented high-fat diet–induced insulin resistance in rats,
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Fig. 4. Correlation between plasma adiponectin concentrations and the AUC of plasma insulin during the oral glucose tolerance test (A) or mesenteric fat mass (B) in rats fed an LCT- or MLCT-containing diet for 6 wk. AUC, area under the curve; LCT, long-chain triacylglycerols; MLCT, medium- and long-chain triacylglycerols.
suggesting that an intervention that prevents or reverses increased visceral fat accumulation should prevent or reverse insulin resistance [29]. In the present investigation, lower mesenteric fat mass in the MLCT-fed group (Table 3) was accompanied by an improvement of insulin resistance (Fig 2B, D). Furthermore, a significant correlation was observed between plasma insulin AUC during the OGTT and mesenteric fat mass (Fig. 3). It is therefore plausible that the decreased mesenteric fat mass is associated with the MLCT-induced improvement of insulin resistance in rats fed a high-fat diet. In support of this possibility is the recent observation by Catalano et al. [35] that mesenteric fat is a better predictor of insulin sensitivity than other abdominal depots in rats. The concept that adipocytokines, bioactive proteins secreted from adipocytes especially in the visceral compartment, regulate energy metabolism and insulin sensitivity in peripheral tissues is now widely accepted [4,36]. Of these adipocytokines, adiponectin has recently attracted much attention because of its antidiabetic and antiatherogenic effects and is expected to be a novel therapeutic tool for diabetes and the metabolic syndrome [37]. Indeed, a decrease in the circulating levels of adiponectin by genetic and environmental factors causing obesity has been shown to contribute to the development of diabetes and metabolic syndromes. In contrast, Takeuchi et al. [17] reported that MCT intake suppresses adipocyte hypertrophy, leading to higher serum adiponectin concentrations in rats. In our study, plasma adiponectin concentrations were significantly higher in the MLCT than in the LCT group (Table 4), although resistin and MCP-1, which have been suggested to be involved in abdominal obesity-induced insulin resistance [38,39], did not significantly differ between the two groups (Table 4). In addition, plasma adiponectin concentrations tended to be negatively associated with mesenteric fat mass (Fig. 4A). Taken together, the beneficial
Table 5 Triacylglycerol concentrations in liver and skeletal muscle
Liver triacylglycerol (mg/g) Muscle triacylglycerol (mg/g)
LCT
MLCT
47.9 3.2 4.1 0.5
44.8 3.0 4.6 0.7
LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols Values are presented as mean SEM
effects of MLCT on plasma adiponectin levels could be attributed to a decrease in the adipocyte size in mesenteric fat tissue. Furthermore, a significant inverse correlation was observed between plasma adiponectin concentrations and insulin AUC (Fig. 4B). These results may provide evidence that dietary intake of MLCT improves high-fat diet–induced insulin resistance, at least in part through increased adiponectin concentrations owing to a lower mesenteric fat mass. There has been interest in the possibility that increased triacylglycerol content is involved in insulin resistance in skeletal muscle [40], which accounts for w80% of insulin-stimulated glucose disposal in human [41]. It has been shown that longterm treatment with adiponectin increases fatty acid oxidation by the activation of adenosine monophosphate–activated protein kinase (AMPK) and decreases triacylglycerol concentrations in skeletal muscle, resulting in increased insulin sensitivity [41]. However, studies from the laboratory of Holloszy have indicated that increased muscle triacylglycerol concentrations do not always account for insulin resistance in skeletal muscle [29]. Our results also showed that no significant difference in muscle triacylglycerol content was observed between the LCT and MLCT groups (Table 5). It is therefore unlikely that higher plasma adiponectin levels in the MLCT group improved insulin resistance through a decrease in muscle triacylglycerol concentrations. Even acute activation of AMPK by adiponectin has been shown to potentiate insulin-stimulated glucose uptake in cultured myotubes in vitro [42]. Although we did not evaluate AMPK- and insulin-signaling pathways and glucose transport in skeletal muscle, the activation of AMPK by increased plasma adiponectin concentrations in MLCT-fed rats may have increased the sensitivity of muscle glucose transport to insulin independently of triacylglycerol concentrations, resulting in a blunted plasma insulin response during the OGTT (Fig. 2C, D). An intake of MCTs at a relatively high level has been shown to increase liver triacylglycerol concentrations in rats because of an increased lipogenic gene expression [21]. Turner et al. [16] reported that MCT intake induces steatosis and insulin resistance in the liver, despite decreasing adiposity and preservation of insulin action in muscle and adipose tissue. In the present study, liver triacylglycerol concentrations in the MLCT group were comparable to those observed in the LCT group (Table 5). Furthermore, HOMA-IR, which is thought to largely reflect insulin resistance in the liver [43], was not significantly different
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between the two groups (Table 4). It is therefore unlikely that a dietary intake of MLCT worsens hepatic insulin resistance induced by a high-fat diet. Conclusion A dietary intake of MLCT was found to have favorable effects on insulin resistance in rats induced by long-term feeding of a high-fat diet. The mechanism responsible for this phenomenon appears to be, at least in part, higher plasma adiponectin concentrations cause by a decreased mesenteric fat mass. Acknowledgments The authors thank Yumiko Ishikawa and Shihoko Kudo for their conscientious assistance with the animal care. References [1] Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27:1047–53. [2] Martin BC, Warram JH, Krolewski AS, Bergman RN, Soeldner JS, Kahn CR. Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet 1992;340:925–9. [3] Kissebah AH. Insulin resistance in visceral obesity. Int J Obes 1991;15: 109–15. [4] Matsuzawa Y. The role of fat topology in the risk of disease. Int J Obes 2008;32:S83–92. [5] Bach AC, Babayan VK. Medium-chain triglycerides: an update. Am J Clin Nutr 1982;36:950–62. [6] Chow BP, Shaffer EA, Parsons HG. Absorption of triglycerides in the absence of lipase. Can J Physiol Pharmacol 1990;68:519–23. [7] Greenberger NJ, Skillman TG. Medium-chain triglycerides. N Engl J Med 1969;280:1045–58. [8] Zurier RB, Campbell RG, Hashim SA, Van Itallie TB. Enrichment of depot fat with odd and even numbered medium chain fatty acids. Am J Physiol 1967;212:291–4. [9] Lavau MM, Hashim SA. Effect of medium chain triglyceride on lipogenesis and body fat in the rat. J Nutr 1978;108:613–20. [10] Bray GA, Lee M, Bray TL. Weight gain of rats fed medium-chain triglycerides is less than rats fed long-chain triglycerides. Int J Obes 1980;4:27–32. [11] Crozier G, Bois-Joyeux B, Chanez M, Girard J, Peret J. Metabolic effects induced by long-term feeding of medium-chain triglycerides in the rat. Metabolism 1987;36:807–14. [12] Tsuji H, Kasai M, Takeuchi H, Nakamura M, Okazaki M, Kondo K. Dietary medium-chain triacylglycerols suppress accumulation of body fat in a double-blind, controlled trial in healthy men and women. J Nutr 2001;131:2853–9. [13] Han J, Hamilton JA, Kirkland JL, Corkey BE, Guo W. Medium-chain oil reduces fat mass and down-regulates expression of adipogenic genes in rats. Obes Res 2003;11:734–44. [14] Han JR, Deng B, Sun J, Chen CG, Corkey BE, Kirkland JL, et al. Effects of dietary medium-chain triglyceride on weight loss and insulin sensitivity in a group of moderately overweight free-living type 2 diabetic Chinese subjects. Metabolism 2007;56:985–91. [15] Eckel RH, Hanson AS, Chen AY, Berman JN, Yost TJ, Brass EP. Dietary substitution of medium-chain triglycerides improves insulin-mediated glucose metabolism in NIDDM subjects. Diabetes 1992;41:641–7. [16] Turner N, Hariharan K, TidAng J, Frangioudakis G, Beale SM, Wright LE, et al. Enhancement of muscle mitochondrial oxidative capacity and alterations in insulin action are lipid species dependent: potent tissue-specific effects of medium-chain fatty acids. Diabetes 2009;58:2547–54. [17] Takeuchi H, Noguchi O, Sekine S, Kobayashi A, Aoyama T. Lower weight gain and higher expression and blood levels of adiponectin in rats fed mediumchain TAG compared with long-chain TAG. Lipids 2006;41:207–12. [18] Negishi N, Shirasawa S, Arai Y, Suzuki J, Mukataka S. Activation of powdered lipase by cluster water and the use of lipase powders for commercial esterification of food oils. Enzyme Microb Technol 2003;32:66–70. [19] Aoyama T, Nosaka N, Kasai M. Research on the nutritional characteristics of medium-chain fatty acids. J Med Invest 2007;54:385–8.
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[20] Ogawa A, Nosaka N, Kasai M, Aoyama T, Okazaki M, Igarashi O, Kondo K. Dietary medium- and long-chain triacylglycerols accelerate diet-induced thermogenesis in humans. J Oleo Sci 2007;56:283–7. [21] Shinohara H, Ogawa A, Kasai M, Aoyama T. Effect of randomly interesterified triacylglycerols containing medium- and long-chain fatty acids on energy expenditure and hepatic fatty acid metabolism in rats. Biosci Biotechnol Biochem 2005;69:1811–8. [22] Matsuo T, Matsuo M, Kasai M, Takeuchi H. Effects of a liquid diet supplement containing structured medium- and long-chain triacylglycerols on body fat accumulation in healthy young subjects. Asia Pac J Clin Nutr 2001;10:46–50. [23] Noguchi O, Shimada H, Kubota F, Tsuji H, Aoyama T. Nutritional effects of randomly interesterified and physically mixed oil containing mediumchain fatty acids on rats. J Oleo Sci 2002;51:699–703. [24] Shinohara H, Wu J, Kasai M, Aoyama T. Randomly interesterified triacylglycerol containing medium- and long-chain fatty acids stimulates fatty acid metabolism in white adipose tissue of rats. Biosci Biotechnol Biochem 2006;70:2919–26. [25] Takeuchi H, Kubota F, Itakura M, Taguchi N. Effect of triacylglycerols containing medium- and long-chain fatty acids on body fat accumulation in rats. J Nutr Sci Vitaminol 2001;47:267–9. [26] Kasai M, Nosaka N, Maki H, Negishi S, Aoyama T, Nakamura M, et al. Effect of dietary medium- and long-chain triacylglycerols (MLCT) on accumulation of body fat in healthy humans. Asia Pac J Clin Nutr 2003;12:151–60. [27] Han DH, Hansen PA, Host HH, Holloszy JO. Insulin resistance of muscle glucose transport in rats fed a high-fat diet: a reevaluation. Diabetes 1997;46:1761–7. [28] Hancock CR, Han DH, Chen M, Terada S, Yasuda T, Wright DC, Holloszy JO. High-fat diets cause insulin resistance despite an increase in muscle mitochondria. Proc Natl Acad Sci U S A 2008;105:7815–20. [29] Kim JY, Nolte LA, Hansen PA, Han DH, Ferguson K, Thompson PA, Holloszy JO. High-fat diet-induced muscle insulin resistance: relationship to visceral fat mass. Am J Physiol Regul Integr Comp Physiol 2000;279:R2057–65. [30] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [31] Katz A, Nambi SS, Mather K, Baron AD, Follmann DA, Sullivan G, Quon MJ. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab 2000;85:2402–10. [32] Folch J, Sloane M, Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497–509. [33] Frayn KN, Maycock PF. Skeletal muscle triacylglycerol in the rat: methods for sampling and measurement, and studies of biological variability. J Lipid Res 1980;21:139–44. [34] Denton RM, Randle PJ. Concentrations of glycerides and phospholipids in rat heart and gastrocnemius muscle: effects of alloxan-diabetes and perfusion. Biochem J 1967;104:416–22. [35] Catalano KJ, Stefanovski D, Bergman RN. Critical role of the mesenteric depot versus other intra-abdominal adipose depots in the development of insulin resistance in young rats. Diabetes 2010;59:1416–23. [36] Rabe K, Lehrke M, Parhofer KG, Broedl UC. Adipokines and insulin resistance. Mol Med 2008;14:741–51. [37] Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin Invest 2006;116:1784–92. [38] Pravenec M, Kazdov a L, Landa V, Zidek V, Mlejnek P, Jansa P, et al. Transgenic and recombinant resistin impair skeletal muscle glucose metabolism in the spontaneously hypertensive rat. J Biol Chem 2003;278:45209–15. [39] Sell H, Dietze-Schroeder D, Kaiser U, Eckel J. Monocyte chemotactic protein-1 is a potential player in the negative cross-talk between adipose tissue and skeletal muscle. Endocrinology 2006;147:2458–67. [40] Hegarty BD, Furler SM, Ye J, Cooney GJ, Kraegen EW. The role of intramuscular lipid in insulin resistance. Acta Physiol Scand 2003;178:373–83. [41] DeFronzo RA, Ferrannini E, Sato Y, Felig P, Wahren J. Synergistic interaction between exercise and insulin on peripheral glucose uptake. J Clin Invest 1981;68:1468–74. [42] Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 2002;8:1288–95. [43] Lin E, Phillips LS, Ziegler TR, Schmotzer B, Wu K, Gu LH, et al. Increases in adiponectin predict improved liver, but not peripheral, insulin sensitivity in severely obese women during weight loss. Diabetes 2007;56:735–42.
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Basic nutritional investigation
Dietary intake of medium- and long-chain triacylglycerols ameliorates insulin resistance in rats fed a high-fat diet Shin Terada Ph.D. *, Sayuri Yamamoto M.Sc., Seiji Sekine Ph.D., Toshiaki Aoyama Ph.D., R.D. Central Research Laboratory, The Nisshin OilliO Group, Ltd., Yokosuka City, Kanagawa Prefecture, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2011 Accepted 30 April 2011
Objective: Excessive accumulation of visceral fat is strongly associated with insulin resistance. The present investigation examined the effects of dietary intake of medium- and long-chain triacylglycerols (MLCTs), which have been shown to induce significantly lower visceral fat accumulation in rats and humans, on high-fat diet–induced obesity and insulin resistance in rats. These effects were then compared with those observed in long-chain triacylglycerol (LCT)-fed rats. Methods: After an 8-wk feeding of a high-fat diet, which induced severe whole-body insulin resistance, male Sprague-Dawley rats were fed a standard diet containing LCTs or MLCTs for 6 wk. After the dietary treatment, an oral glucose tolerance test was performed. Results: Although body weight and total intra-abdominal fat mass did not differ between the two groups, mesenteric fat weight in the MLCT-fed group was significantly lower than that in the LCT group (P < 0.05). The increase in plasma insulin concentrations, but not in glucose, after glucose administration (area under the curve) was significantly smaller in the MLCT group than in the LCT group (P < 0.01) and was significantly associated with mesenteric fat weight (P < 0.05). MLCT-fed rats had significantly higher plasma adiponectin concentrations compared with LCT rats (P < 0.05). Adiponectin concentrations were negatively correlated with the area under the curve for plasma insulin (P < 0.05) and tended to be inversely related to mesenteric fat weight (P ¼ 0.08). Conclusion: These results suggest that dietary intake of MLCTs may improve insulin resistance in rats fed a high-fat diet, at least in part through increased adiponectin concentrations caused by a lower mesenteric fat mass. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Medium-chain fatty acids Insulin resistance High-fat diet Adiponectin Rat
Introduction Over the past three decades, there has been an explosive increase in the prevalence of type 2 diabetes [1]. Insulin resistance is an early and key defect associated with type 2 diabetes [2] and is postulated to be caused by an excessive accumulation of visceral fat [3,4]. The typical Western diet, which is very high in fat and sucrose, is considered a major factor in the visceral fat accumulation and the development of insulin resistance. Foods that are designed to decrease visceral fat mass may help to prevent the development of diet-induced insulin resistance and diabetes. Compared with long-chain fatty acids (LCFAs), mediumchain fatty acids (MCFAs; composed of chains of 8–12 carbon chains) have several unique nutritional and physiologic * Corresponding author. Tel.: þ81-46-837-2438; fax: þ81-46-837-2471. E-mail address: [email protected] (S. Terada). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.04.008
properties. Although LCFAs are absorbed through the intestinal lymphatic ducts and are transported as chylomicrons to the systemic circulation, it is well documented that MCFAs are absorbed through the portal system and are easily oxidized in the liver because the intramitochondrial transport of MCFAs does not require carnitine palmitoyltransferase, a rate-limiting enzyme of mitochondrial b-oxidation [5–8]. These characteristics make medium-chain triacylglycerols (MCTs), composed exclusively of MCFAs, a useful dietary treatment for preventing and reversing obesity and lifestyle-related disease [5]. Many investigators have reported the body weight- and body fat–decreasing effects of MCTs in animal studies [9–11], and we previously demonstrated that sufficiently calorie-controlled diets containing 10 g of MCT per day can induce significant decreases in body weight and fat mass in human subjects [12]. In addition, there has been accumulating evidence from animal and human studies that MCTs improve insulin sensitivity and glucose tolerance in diabetic and non-diabetic states [13–16].
S. Terada et al. / Nutrition 28 (2012) 92–97
Our previous study also demonstrated that rats fed a diet containing 20% MCTs for 8 wk had lower visceral fat weight accompanied by better glucose tolerance compared with rats fed log-chain triacylglycerols (LCTs) [17]. Although MCTs may be a valuable tool for preventing or alleviating insulin resistance and diabetes, it is not feasible to substitute MCTs for LCTs in the diet for long-term therapy because their lower smoking point and greater tendency to bubble limit the daily use of MCTs as cooking oil. A cooking oil containing medium- and long-chain fatty acids in the same glycerol molecule (MLCT) prepared by random interesterification is commercially available and widely used in Japan [18,19]. MLCT has overcome the disadvantages encountered with MCTs and can easily replace LCTs in the daily diet. Despite the lower MCFA content (w13%), this edible oil has also been reported to increase energy expenditure [20,21] and to cause a significantly lower visceral fat accumulation in humans and rats compared with LCT oil [21–26]. These results have led us to hypothesize that MLCT, instead of MCT, could be a useful dietary treatment to ameliorate insulin resistance, which is associated with visceral fat accumulation. In this context, the present study evaluated the effects of a dietary intake of MLCT on high-fat diet–induced insulin resistance in rats. Materials and methods Materials The MLCT and LCT (rapeseed oil) oils were obtained from The Nisshin OilliO Group (Tokyo, Japan). Their fatty acid compositions, as measured by gas chromatography, are listed in Table 1. Treatment of animals Four-week-old male Sprague-Dawley rats (70–90 g of body weight) were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan) and fed a high-fat (28% wt/wt) diet (n ¼ 31) for 8 wk. The high-fat diet contained the following ingredients (grams per kilogram): sucrose, 347.286; casein, 293.4; lard, 180; rapeseed oil, 100; methionine, 5; AIN-93 vitamin mix, 22; AIN-93G mineral mix, 51; choline bitartrate, 1.3; and tert-butyl hydroquinone, 0.014 [27–29]. The rats were provided with the diets and water ad libitum. Feeding of the high-fat diet for 4 to 8 wk has been shown to cause severe whole-body insulin resistance in rats [27–29]. The rats were separated into two groups matched for body weight, plasma glucose, and insulin concentrations after 8 wk on the high-fat diet. Each group of rats was allowed free access to the experimental diet containing LCTs (n ¼ 15) or MLCTs (n ¼ 16) for 6 wk. The composition of the diet is presented in Table 2. Both diets were based on the AIN-93 formula with the modification that sucrose was decreased from 100 to 40 g/kg of diet [24]. The energy content of the high-fat diet was 5.2 kcal/g, whereas that of the experimental diet was 4.0 kcal/g. Food intake and body weight were recorded every second day. All rats were treated in accordance with the guidelines established by the Japanese Society of Nutrition and Food Science (Law No. 105 and Notification No. 6 of the Japanese Government).
Table 1 Fatty acid compositions of test lipids Fatty acid*
LCT
MLCT
8:0 10:0 16:0 18:0 18:1 18:2 18:3 Others
ND ND 5.6 2.4 62.9 18.3 6.6 4.2
8.3 2.7 2.8 1.2 57.5 18.1 7.2 2.2
LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols; ND, not detected * Numbers are carbon atoms:double bonds.
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Table 2 Composition of experimental diets Ingredients
Dietary group (g/kg diet) LCT
MLCT
Cornstarch Casein Alpha-cornstarch Sucrose LCT oil MLCT oil Cellulose powder Mineral mix (AIN-93M) Vitamin mix (AIN-93) L-Cystine Choline bitartrate tert-Butyl hydroquinone
457.5 200 132 40 70 – 50 35 10 3 2.5 0.014
457.5 200 132 40 – 70 50 35 10 3 2.5 0.014
LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols
Oral glucose tolerance test After 6 wk of dietary treatment, an oral glucose tolerance test (OGTT) was performed. After overnight (16-h) fasting, glucose (1.5 g/kg of body weight) was orally administered using a stainless-steel gavage needle with a ball diameter. Blood samples were collected into capillary tubes from the tail vein immediately before and 30, 60, and 120 min after glucose administration. The capillary tubes were then centrifuged, and plasma samples were stored at 80 C until analysis. The areas under the curve (AUCs) for plasma glucose and insulin were calculated with the trapezoidal rule.
Tissue collection At the end of the feeding period, rats were sacrificed under anesthesia with diethyl ether without fasting. Blood samples were collected by cardiac puncture, and the triceps muscle and liver were then removed and weighed. After the muscle and liver dissections were completed, epididymal, mesenteric, and retroperitoneal fat pads were removed and weighed. Liver and muscle samples were frozen in liquid N2 and stored at 80 C until analysis. Analytical procedure Plasma glucose, free fatty acid, and triacylglycerol concentrations were determined using kits (Glucose C2 Test Wako, NEFA-C Test Wako, and Triglyceride E Test Wako, respectively) obtained from Wako Pure Chemical (Osaka, Japan). Plasma concentrations of insulin, adiponectin, resistin, and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (insulin, Mercodia AB, Uppsala, Sweden; adiponectin, Otsuka Pharmaceutical, Tokyo, Japan; resistin, BioVendor, Karasek, Czech Republic; MCP-1: Bender MedSystems, San Diego, CA, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as the product of the fasting serum glucose and insulin levels divided by a constant, assuming that rats have an average HOMA-IR of 1, analogous to the assumptions applied in the development of HOMA-IR in humans [30]. The equation was as follows: HOMA-IR ¼ (fasting glucose level [mg/dL] fasting insulin level [mU/mL]/2430 [31]. Muscle and liver triacylglycerol concentrations were determined by extracting total lipids from frozen triceps muscle and liver with chloroformmethanol (2:1 vol/vol), as described by Folch et al. [32], separating the chloroform and methanol water phases, removing phospholipids, and further processing the sample using Frayn and Maycock’s [33] modification of the method of Denton and Randle [34]. Triacylglycerols were then quantified spectrophotometrically as glycerol using an assay kit (Triglyceride E TEST Wako).
Statistical analysis The data are presented as mean standard error of the mean. In the experiment that involved OGTT, two-way analysis of variance (Jandel Sigma Stat, San Jose, CA, USA) was performed to examine the effects of time and dietary conditions. For the other experiments, statistical analysis was performed using the Student t test (Jandel Sigma Stat). Statistical significance was defined as P < 0.05.
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Results Body weight, food intake, intra-abdominal fat, and liver weights
Table 3 Body weight, food intake, food efficacy, and liver and intra-abdominal fat weights in LCT and MLCT rat groups LCT
Figure 1 shows the body weight change in rats. During the 8wk feeding of a high-fat diet, body weight progressively increased. Although body weights slightly increased in response to the LCT and MLCT diets, no significant differences in the final body weight and body weight gain were observed between the two groups (Table 3). Food intake and food efficiency were not significantly different between the LCT and MLCT groups (Table 3). Although epididymal, retroperitoneal, and total intra-abdominal fat weights did not significantly differ between the two groups, the mesenteric fat weight, expressed relative to body weight, in the MLCT group was significantly lower than that observed in the LCT group (P < 0.05; Table 3). There was no significant difference in liver weight between the two groups (Table 3). Oral glucose tolerance test The plasma glucose and insulin concentrations before glucose administration (after 16-h fasting) were not significantly different between the LCT and MLCT groups (Table 4, Fig. 2), resulting in similar values for HOMA-IR in the two groups (Table 4). Plasma glucose levels during the OGTT and the AUC for plasma glucose were not significantly different between the two groups (Fig. 2A, B). Two-way analysis of variance revealed that dietary treatment had a significant effect on plasma insulin levels during the OGTT, resulting in significantly lower plasma insulin concentrations in the MLCT group (P < 0.001; Fig. 2C). The increase in plasma insulin concentrations (plasma insulin AUC) in the MLCT group was also significantly smaller than that observed in the LCT group (Fig. 2D). As shown in Figure 3, there was a significant correlation between plasma insulin AUC and mesenteric fat weight (r ¼ 0.420, P < 0.05). No such correlation was observed with the other adipose tissues examined (data not shown). Blood lipid and adipocytokine concentrations and their relation to the OGTT No significant differences in plasma free fatty acid and triacylglycerol concentrations were observed between the LCT and
Initial body weight (g) Final body weight (g) Body weight gain (g) Food intake (g/d) Food efficiency (%)* Liver weight (g/100 g BW) Epididymal fat (g/100 g BW) Retroperitoneal fat (g/100 g BW) Mesenteric fat (g/100 g BW) Total intra-abdominal fat (g/100 g BW)
459 519 60 19.0 7.8 3.78 2.81 3.85 2.22 8.87
MLCT
10 10 7 0.4 0.8 0.08 0.13 0.14 0.10 0.34
459 516 58 18.4 7.8 3.71 2.81 3.93 1.86 8.60
9 10 5 0.5 0.6 0.06 0.11 0.14 0.10y 0.32
BW, body weight; LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols Values are presented as mean SEM * Equal to weight gain (grams)/food intake (grams) 100. y Significant difference from the LCT group at P < 0.05. The initial body weight was measured at the beginning of feeding a diet containing LCT or MLCT.
MLCT groups (Table 4). There were also no significant differences in plasma resistin and MCP-1 concentrations between the two groups (Table 4). In contrast, plasma adiponectin concentrations were significantly higher in the MLCT than the LCT group (P < 0.05). As shown in Figure 4A, plasma adiponectin concentrations tended to be inversely correlated with mesenteric fat mass, although the relation did not reach statistical significance (P ¼ 0.08). There was a significant negative correlation between adiponectin concentrations and plasma insulin AUC (r ¼ 0.420, P < 0.05; Fig. 4B). Muscle and liver triacylglycerol concentrations There were no significant differences in muscle and liver triacylglycerol concentrations between the LCT and MLCT groups (Table 5). No significant correlation was observed between plasma insulin AUC and liver/muscle triacylglycerol content (data not shown). Discussion To examine the effects of dietary MLCT consumption on glucose tolerance and insulin sensitivity, we performed the OGTT after 6-wk feeding of meals containing MLCTs or LCTs in rats previously fed a high-fat diet, which is an animal model frequently used to induce obesity and severe insulin resistance [27–29]. In consequence, we found that rats fed MLCTs had
Table 4 Plasma glucose, FFA, triacylglycerol, insulin, and adipocytokine concentrations in the fasting state LCT Glucose (mg/dL) FFA (mEq/L) Triacylglycerol (mg/dL) Insulin (ng/mL) MCP-1 (ng/mL) Resistin (ng/mL) Adiponectin (mg/mL) HOMA-IR Fig. 1. Body weight of rats during feedings of high-fat and experimental diets. The final data points represent final values the day before sacrifice. Values are presented as mean SEM. LCT, long-chain triacylglycerols; MLCT, medium- and long-chain triacylglycerols.
107 1.53 265 1.61 7.7 23.0 1.87 2.0
MLCT
4 0.07 22 0.23 0.4 0.6 0.09 0.3
107 1.52 258 1.70 7.8 23.4 2.12 2.2
3 0.06 17 0.27 0.5 0.9 0.07* 0.4
FFA, free fatty acid; HOMA-IR, homeostasis model assessment of insulin resistance; LCT, long-chain triacylglycerols (rapeseed oil); MCP-1, monocyte chemoattractant protein-1; MLCT, medium- and long-chain triacylglycerols Values are presented as mean SEM * Significant difference from the LCT group at P < 0.05.
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Fig. 2. Plasma glucose (A) and insulin (C) responses after oral glucose administration (1.5 g/kg of body weight) in rats fed an MLCT- or LCT-containing diet for 6 wk. The AUCs for plasma glucose (B) and insulin (D) during the 120-min period after oral glucose administration was calculated in accordance with the trapezoidal rule. Values are presented as mean SEM. **P < 0.01 and ***P < 0.001, significant differences from LCT. AUC, area under the curve; LCT, long-chain triacylglycerols; MLCT, medium- and longchain triacylglycerols.
a markedly blunted insulin response to a glucose load compared with the LCT-fed group (Fig. 2C, D). These results suggest that MLCTs compared with LCTs could be a valuable dietary intervention to alleviate excess calorie-induced insulin resistance.
Fig. 3. Correlation between the AUC of plasma insulin during the oral glucose tolerance test and mesenteric fat mass in rats fed an LCT- or MLCT-containing diet for 6 wk. AUC, area under the curve; BW, body weight; LCT, long-chain triacylglycerols; MLCT, medium- and long-chain triacylglycerols.
Previous studies have shown that MCT exerts favorable effects on insulin resistance and glucose intolerance in rodents [13,16, 17]. In those studies [13,16,17], a relatively larger amount of MCT (w200 g/kg of diet in animals), which consisted exclusively of MCFAs, was administered. In the present investigation, the MLCT-containing diet, in which the total MCFA content was only w0.8% (wt/wt), ameliorated insulin resistance in rats fed a high-fat diet (Fig. 2B, D), suggesting that even a small amount of MCFA is enough to prevent or alleviate insulin resistance in rats. However, because MLCTs consist of MCFAs and LCFAs in the same glycerol molecule by random interesterification [18,19], we could not rule out the possibility that the unique structure, not MCFA itself, might be responsible for its beneficial effects. Further studies will be required to elucidate the mechanism by which MLCT improves excess calorie-induced insulin resistance. Our results showed that rats fed an MLCT diet had significantly lower mesenteric fat mass than the LCT rats (Table 3). This result confirmed our previous findings observed in non-obese rats [21,23–25] and might provide further evidence that MLCT intake has a small but significant effect on visceral fat accumulation even in excess calorie-induced obese rats. It is well documented that an increase in visceral fat is associated with the development of insulin resistance [3,4]. Kim et al. [29] reported that calorie restriction, leptin injection, and fish oil feeding, which are well known to decrease visceral fat mass, completely prevented high-fat diet–induced insulin resistance in rats,
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Fig. 4. Correlation between plasma adiponectin concentrations and the AUC of plasma insulin during the oral glucose tolerance test (A) or mesenteric fat mass (B) in rats fed an LCT- or MLCT-containing diet for 6 wk. AUC, area under the curve; LCT, long-chain triacylglycerols; MLCT, medium- and long-chain triacylglycerols.
suggesting that an intervention that prevents or reverses increased visceral fat accumulation should prevent or reverse insulin resistance [29]. In the present investigation, lower mesenteric fat mass in the MLCT-fed group (Table 3) was accompanied by an improvement of insulin resistance (Fig 2B, D). Furthermore, a significant correlation was observed between plasma insulin AUC during the OGTT and mesenteric fat mass (Fig. 3). It is therefore plausible that the decreased mesenteric fat mass is associated with the MLCT-induced improvement of insulin resistance in rats fed a high-fat diet. In support of this possibility is the recent observation by Catalano et al. [35] that mesenteric fat is a better predictor of insulin sensitivity than other abdominal depots in rats. The concept that adipocytokines, bioactive proteins secreted from adipocytes especially in the visceral compartment, regulate energy metabolism and insulin sensitivity in peripheral tissues is now widely accepted [4,36]. Of these adipocytokines, adiponectin has recently attracted much attention because of its antidiabetic and antiatherogenic effects and is expected to be a novel therapeutic tool for diabetes and the metabolic syndrome [37]. Indeed, a decrease in the circulating levels of adiponectin by genetic and environmental factors causing obesity has been shown to contribute to the development of diabetes and metabolic syndromes. In contrast, Takeuchi et al. [17] reported that MCT intake suppresses adipocyte hypertrophy, leading to higher serum adiponectin concentrations in rats. In our study, plasma adiponectin concentrations were significantly higher in the MLCT than in the LCT group (Table 4), although resistin and MCP-1, which have been suggested to be involved in abdominal obesity-induced insulin resistance [38,39], did not significantly differ between the two groups (Table 4). In addition, plasma adiponectin concentrations tended to be negatively associated with mesenteric fat mass (Fig. 4A). Taken together, the beneficial
Table 5 Triacylglycerol concentrations in liver and skeletal muscle
Liver triacylglycerol (mg/g) Muscle triacylglycerol (mg/g)
LCT
MLCT
47.9 3.2 4.1 0.5
44.8 3.0 4.6 0.7
LCT, long-chain triacylglycerols (rapeseed oil); MLCT, medium- and long-chain triacylglycerols Values are presented as mean SEM
effects of MLCT on plasma adiponectin levels could be attributed to a decrease in the adipocyte size in mesenteric fat tissue. Furthermore, a significant inverse correlation was observed between plasma adiponectin concentrations and insulin AUC (Fig. 4B). These results may provide evidence that dietary intake of MLCT improves high-fat diet–induced insulin resistance, at least in part through increased adiponectin concentrations owing to a lower mesenteric fat mass. There has been interest in the possibility that increased triacylglycerol content is involved in insulin resistance in skeletal muscle [40], which accounts for w80% of insulin-stimulated glucose disposal in human [41]. It has been shown that longterm treatment with adiponectin increases fatty acid oxidation by the activation of adenosine monophosphate–activated protein kinase (AMPK) and decreases triacylglycerol concentrations in skeletal muscle, resulting in increased insulin sensitivity [41]. However, studies from the laboratory of Holloszy have indicated that increased muscle triacylglycerol concentrations do not always account for insulin resistance in skeletal muscle [29]. Our results also showed that no significant difference in muscle triacylglycerol content was observed between the LCT and MLCT groups (Table 5). It is therefore unlikely that higher plasma adiponectin levels in the MLCT group improved insulin resistance through a decrease in muscle triacylglycerol concentrations. Even acute activation of AMPK by adiponectin has been shown to potentiate insulin-stimulated glucose uptake in cultured myotubes in vitro [42]. Although we did not evaluate AMPK- and insulin-signaling pathways and glucose transport in skeletal muscle, the activation of AMPK by increased plasma adiponectin concentrations in MLCT-fed rats may have increased the sensitivity of muscle glucose transport to insulin independently of triacylglycerol concentrations, resulting in a blunted plasma insulin response during the OGTT (Fig. 2C, D). An intake of MCTs at a relatively high level has been shown to increase liver triacylglycerol concentrations in rats because of an increased lipogenic gene expression [21]. Turner et al. [16] reported that MCT intake induces steatosis and insulin resistance in the liver, despite decreasing adiposity and preservation of insulin action in muscle and adipose tissue. In the present study, liver triacylglycerol concentrations in the MLCT group were comparable to those observed in the LCT group (Table 5). Furthermore, HOMA-IR, which is thought to largely reflect insulin resistance in the liver [43], was not significantly different
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between the two groups (Table 4). It is therefore unlikely that a dietary intake of MLCT worsens hepatic insulin resistance induced by a high-fat diet. Conclusion A dietary intake of MLCT was found to have favorable effects on insulin resistance in rats induced by long-term feeding of a high-fat diet. The mechanism responsible for this phenomenon appears to be, at least in part, higher plasma adiponectin concentrations cause by a decreased mesenteric fat mass. Acknowledgments The authors thank Yumiko Ishikawa and Shihoko Kudo for their conscientious assistance with the animal care. References [1] Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27:1047–53. [2] Martin BC, Warram JH, Krolewski AS, Bergman RN, Soeldner JS, Kahn CR. Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet 1992;340:925–9. [3] Kissebah AH. Insulin resistance in visceral obesity. Int J Obes 1991;15: 109–15. [4] Matsuzawa Y. The role of fat topology in the risk of disease. Int J Obes 2008;32:S83–92. [5] Bach AC, Babayan VK. Medium-chain triglycerides: an update. Am J Clin Nutr 1982;36:950–62. [6] Chow BP, Shaffer EA, Parsons HG. Absorption of triglycerides in the absence of lipase. Can J Physiol Pharmacol 1990;68:519–23. [7] Greenberger NJ, Skillman TG. Medium-chain triglycerides. N Engl J Med 1969;280:1045–58. [8] Zurier RB, Campbell RG, Hashim SA, Van Itallie TB. Enrichment of depot fat with odd and even numbered medium chain fatty acids. Am J Physiol 1967;212:291–4. [9] Lavau MM, Hashim SA. Effect of medium chain triglyceride on lipogenesis and body fat in the rat. J Nutr 1978;108:613–20. [10] Bray GA, Lee M, Bray TL. Weight gain of rats fed medium-chain triglycerides is less than rats fed long-chain triglycerides. Int J Obes 1980;4:27–32. [11] Crozier G, Bois-Joyeux B, Chanez M, Girard J, Peret J. Metabolic effects induced by long-term feeding of medium-chain triglycerides in the rat. Metabolism 1987;36:807–14. [12] Tsuji H, Kasai M, Takeuchi H, Nakamura M, Okazaki M, Kondo K. Dietary medium-chain triacylglycerols suppress accumulation of body fat in a double-blind, controlled trial in healthy men and women. J Nutr 2001;131:2853–9. [13] Han J, Hamilton JA, Kirkland JL, Corkey BE, Guo W. Medium-chain oil reduces fat mass and down-regulates expression of adipogenic genes in rats. Obes Res 2003;11:734–44. [14] Han JR, Deng B, Sun J, Chen CG, Corkey BE, Kirkland JL, et al. Effects of dietary medium-chain triglyceride on weight loss and insulin sensitivity in a group of moderately overweight free-living type 2 diabetic Chinese subjects. Metabolism 2007;56:985–91. [15] Eckel RH, Hanson AS, Chen AY, Berman JN, Yost TJ, Brass EP. Dietary substitution of medium-chain triglycerides improves insulin-mediated glucose metabolism in NIDDM subjects. Diabetes 1992;41:641–7. [16] Turner N, Hariharan K, TidAng J, Frangioudakis G, Beale SM, Wright LE, et al. Enhancement of muscle mitochondrial oxidative capacity and alterations in insulin action are lipid species dependent: potent tissue-specific effects of medium-chain fatty acids. Diabetes 2009;58:2547–54. [17] Takeuchi H, Noguchi O, Sekine S, Kobayashi A, Aoyama T. Lower weight gain and higher expression and blood levels of adiponectin in rats fed mediumchain TAG compared with long-chain TAG. Lipids 2006;41:207–12. [18] Negishi N, Shirasawa S, Arai Y, Suzuki J, Mukataka S. Activation of powdered lipase by cluster water and the use of lipase powders for commercial esterification of food oils. Enzyme Microb Technol 2003;32:66–70. [19] Aoyama T, Nosaka N, Kasai M. Research on the nutritional characteristics of medium-chain fatty acids. J Med Invest 2007;54:385–8.
97
[20] Ogawa A, Nosaka N, Kasai M, Aoyama T, Okazaki M, Igarashi O, Kondo K. Dietary medium- and long-chain triacylglycerols accelerate diet-induced thermogenesis in humans. J Oleo Sci 2007;56:283–7. [21] Shinohara H, Ogawa A, Kasai M, Aoyama T. Effect of randomly interesterified triacylglycerols containing medium- and long-chain fatty acids on energy expenditure and hepatic fatty acid metabolism in rats. Biosci Biotechnol Biochem 2005;69:1811–8. [22] Matsuo T, Matsuo M, Kasai M, Takeuchi H. Effects of a liquid diet supplement containing structured medium- and long-chain triacylglycerols on body fat accumulation in healthy young subjects. Asia Pac J Clin Nutr 2001;10:46–50. [23] Noguchi O, Shimada H, Kubota F, Tsuji H, Aoyama T. Nutritional effects of randomly interesterified and physically mixed oil containing mediumchain fatty acids on rats. J Oleo Sci 2002;51:699–703. [24] Shinohara H, Wu J, Kasai M, Aoyama T. Randomly interesterified triacylglycerol containing medium- and long-chain fatty acids stimulates fatty acid metabolism in white adipose tissue of rats. Biosci Biotechnol Biochem 2006;70:2919–26. [25] Takeuchi H, Kubota F, Itakura M, Taguchi N. Effect of triacylglycerols containing medium- and long-chain fatty acids on body fat accumulation in rats. J Nutr Sci Vitaminol 2001;47:267–9. [26] Kasai M, Nosaka N, Maki H, Negishi S, Aoyama T, Nakamura M, et al. Effect of dietary medium- and long-chain triacylglycerols (MLCT) on accumulation of body fat in healthy humans. Asia Pac J Clin Nutr 2003;12:151–60. [27] Han DH, Hansen PA, Host HH, Holloszy JO. Insulin resistance of muscle glucose transport in rats fed a high-fat diet: a reevaluation. Diabetes 1997;46:1761–7. [28] Hancock CR, Han DH, Chen M, Terada S, Yasuda T, Wright DC, Holloszy JO. High-fat diets cause insulin resistance despite an increase in muscle mitochondria. Proc Natl Acad Sci U S A 2008;105:7815–20. [29] Kim JY, Nolte LA, Hansen PA, Han DH, Ferguson K, Thompson PA, Holloszy JO. High-fat diet-induced muscle insulin resistance: relationship to visceral fat mass. Am J Physiol Regul Integr Comp Physiol 2000;279:R2057–65. [30] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [31] Katz A, Nambi SS, Mather K, Baron AD, Follmann DA, Sullivan G, Quon MJ. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab 2000;85:2402–10. [32] Folch J, Sloane M, Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497–509. [33] Frayn KN, Maycock PF. Skeletal muscle triacylglycerol in the rat: methods for sampling and measurement, and studies of biological variability. J Lipid Res 1980;21:139–44. [34] Denton RM, Randle PJ. Concentrations of glycerides and phospholipids in rat heart and gastrocnemius muscle: effects of alloxan-diabetes and perfusion. Biochem J 1967;104:416–22. [35] Catalano KJ, Stefanovski D, Bergman RN. Critical role of the mesenteric depot versus other intra-abdominal adipose depots in the development of insulin resistance in young rats. Diabetes 2010;59:1416–23. [36] Rabe K, Lehrke M, Parhofer KG, Broedl UC. Adipokines and insulin resistance. Mol Med 2008;14:741–51. [37] Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin Invest 2006;116:1784–92. [38] Pravenec M, Kazdov a L, Landa V, Zidek V, Mlejnek P, Jansa P, et al. Transgenic and recombinant resistin impair skeletal muscle glucose metabolism in the spontaneously hypertensive rat. J Biol Chem 2003;278:45209–15. [39] Sell H, Dietze-Schroeder D, Kaiser U, Eckel J. Monocyte chemotactic protein-1 is a potential player in the negative cross-talk between adipose tissue and skeletal muscle. Endocrinology 2006;147:2458–67. [40] Hegarty BD, Furler SM, Ye J, Cooney GJ, Kraegen EW. The role of intramuscular lipid in insulin resistance. Acta Physiol Scand 2003;178:373–83. [41] DeFronzo RA, Ferrannini E, Sato Y, Felig P, Wahren J. Synergistic interaction between exercise and insulin on peripheral glucose uptake. J Clin Invest 1981;68:1468–74. [42] Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 2002;8:1288–95. [43] Lin E, Phillips LS, Ziegler TR, Schmotzer B, Wu K, Gu LH, et al. Increases in adiponectin predict improved liver, but not peripheral, insulin sensitivity in severely obese women during weight loss. Diabetes 2007;56:735–42.