2018 Volume 24 Issue 1 Pages 119-127
Previously, we reported that 4-week enzymatically synthesized glycogen (ESG) supplementation reduced lipid accumulation in diet-induced obese rats. The aim of this study was to investigate the effects of long-term dietary ESG supplementation on lipid metabolism of diet-induced obese mice. Male C57BL/6NCr mice were fed a control or a high-fat diet containing 0%, 10%, or 20% ESG for 15 weeks. In the high-fat diet groups, ESG showed significant suppressive effect on adipose tissue weight. Supplementation of ESG decreased plasma cholesterol and liver lipid levels. Although, ESG substantially increased fecal lipid, ESG did not affect lipid metabolism related gene expression. In control groups ESG increased body temperature and plasma NO, although the effects were not observed in high-fat groups. In the test of single administration, ESG significantly suppressed lipid absorption. In this study, we confirmed the anti-obese effect of ESG. It is mainly due to inhibition of lipid absorption by ESG.
According to the World Health Organization (WHO) fact sheet, global obesity has more than doubled since 1980, and more than 1.9 billion adults were overweight (body mass index (BMI) ≥ 25) during 2014. Of these overweight adults, over 600 million were obese (BMI ≥ 30). The accumulation of excess fat increases the risk of many diseases, including type II diabetes mellitus, hyperlipidemia, ischemia heart diseases, Alzheimer disease, hypertension, and certain cancers (Seidell 2000, Garrison et al., 1996, Alexander 2001, Arnoldussen et al., 2014, Hall et al., 2015, Wolin et al., 2010, Iyengar et al., 2015). Recently, increasing evidence has found that many foods and food components have beneficial effects in the suppression of obesity such as green tea, catechins, black tea, almonds, and wasabi leaf (Ashida et al., 2004, Huang et al., 2014, Kao et al., 2000, Imada et al., 2011, Foster et al. 2012, Yamada-Kato 2016).
Glycogen is a highly branched (1→4) and (1→6) linked α-D-glucan with a large molecular weight, and exists widely in animals as a storage form of glucose. In the human body, although glycogen is mainly contained in the liver and muscle at levels of 5% and 1% of wet weight, respectively, many other organs such as the skin, brain, fat pads, kidney, and erythrocytes contain glycogen (Adeva-Andany et al., 2016, Vukas et al., 1978, Farquharson et al., 1990). We recently developed a new method for enzymatically synthesizing glycogen (ESG) from starch using enzyme technology (Kajiura et al., 2008). The results of physicochemical analyses clarified that ESGs are equivalent to natural source glycogen (Kajiura et al., 2010). In the field of physiological study, we reported that ESG has immunomodulatory activity in macrophage cells and mice (Kakutani et al., 2007, Kakutani et al., 2012a, Kakutani et al., 2012b). ESG has slight resistant property against the digestive enzyme alpha-amylase, and a single oral administration test in rats determined that the glycemic index of ESG is approximately 80 (Takata et al., 2009, Furuyashiki et al., 2011). Our recent study suggested that the resistant part of ESG reaches the cecum in rats and is converted to short chain fatty acids (SCFAs), and increases Lactobacillus and Bifidobacterium (Furuyashiki et al., 2011). Thus, we considered that the digestion-resistant part of ESG possesses a dietary fiber like effect. As mentioned in many reports, dietary fiber provides beneficial effects to lipid metabolism (Otles 2014, Goff et al., 2013). ESG supplementation inhibited an accumulation of body fat mass (Furuyashiki et al., 2013). However, these results revealed only the restrictive effects of ESG on lipid metabolism.
In the present study, the effects of long-term ESG ingestion on lipid metabolism were investigated in a diet-induced obese mice model. After 15 week ingestion of ESG with or without a high-fat diet, the body lipid accumulation, liver lipids, blood lipid profiles, fecal lipid excretion, body temperature, lipid metabolism and thermogenesis related factors in the liver and adipose tissue, and nitric oxide (NO) production were measured to assess the change in overall lipid metabolism.
Materials ESG was synthesized in our laboratory using a previously described method (Kajiura et al., 2008). Briefly, (1→6) linkage of waky cornstarch was hydrolyzed by isoamylase (EC 22.214.171.124), then reconstructed to glycogen by the action of branching enzyme (EC 126.96.36.199) and amylomaltase (EC 188.8.131.52). Mice were obtained from Japan SLC (Shizuoka, Japan). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise specified.
Animal experiments All experiments with animals were approved by the Institutional Animal Care and Use Committee (Permission number: 21-07-02), and were conducted according to the Kobe University Animal Experimentation Regulations. Male C57BL/6NCr mice (4 weeks old) were housed in a temperature controlled (25 ± 3°C) room at 60 ± 5% humidity under a 12 h:12 h light–dark cycle. After 1 week quarantine, the mice were randomly divided into six groups (n = 5), and fed with normal diet (No. D12450B: Research Diets, Inc., New Brunswick, NJ) or a high fat diet (No. D12492) containing 60% kcal lard for 15 weeks. ESG was added to each diet at the levels of 0%, 10%, or 20% (w/w), and assigned as C-0%, C-10%, C-20%, respectively, as the controls, and HF-0%, HF-10%, and HF-20%, respectively, as the high-fat groups. The compositions of the diets are described in Table 1. The dietary fiber content of glycogen was estimated approximately 17–22% from analyses data (Takata et al 2009), thus glycogen was replaced with cornstarch and cellulose in this range to equate the calorie of the diet in each diet groups. During the feeding period, food intake and body weight were measured once a week. At the end of the feeding period, the mice were fasted for 14 h, and then sacrificed by collecting blood via cardiac puncture using a heparinized syringe under anesthesia with sodium pentobarbital. Mesenteric, epididymal, retroperitoneal, and subcutaneous white adipose tissue (WAT), interscapular brown adipose tissue (BAT), muscle, liver, and cecum samples were collected and weighed. Each tissue was washed with 1.15% (w/v) KCl and stored at −80°C until use.
HF, high fat; ESG, enzymatically synthesized glycogen
Body temperature Measurement of the body temperature was conducted the day before dissection. The mice were sedated and restrained for 30 s during the measurement. A digital thermometer TD-300 (Shibaura Electronics Co., Ltd., Saitama, Japan) was used with the probe placed in the rectum at 2.5 cm depth. The temperature of the measurement room was maintained at 25 ± 3°C.
Body composition After 14 weeks, the mice were anesthetized with isoflurane and scanned along the body axis using an experimental animal X-ray computed tomographic (X-ray CT) system (Latheta LCT-100, Hitachi Aloka Medical, LTD., Tokyo, Japan). Contiguous 1 mm slice images of the trunk and lower extremities were examined for quantitative assessment using Latheta software (version 2.10, ALOKA). Total fat mass, which consists of visceral fat mass, subcutaneous fat mass, and lean mass were measured, and the ratio of fat mass was calculated.
Measurement of the blood lipids and NO The blood samples were centrifuged (9,700 × g, 10 min), and the collected plasma samples were stored at −80°C until use. Plasma triacylglycerol, total cholesterol, non-esterified fatty acids (NEFA), and glucose were measured using the appropriate commercial assay kit for each target (Triglyceride-E test, LabAssay™ cholesterol, NEFA-C test, and LabAssay™ glucose). After deproteinization of each plasma sample with acetonitrile, plasma NO was measured using the NO2/NO3 Assay Kit-FX (Fluorometric) DOJINDO LABORATORIES (Dojin Chemical, Tokyo, Japan).
Measurement of the hepatic and fecal lipid levels Feces were collected on the day before the end of the feeding period. The collected feces were freeze-dried, weighed, and ground to powder. Approximately 100 mg of liver or feces powder was homogenized with 0.35 mL of distilled water, and the homogenate was extracted three times with 0.7 mL of chloroform–methanol (2:1, v/v) solution. The chloroform layer was collected by centrifugation at 1,800 × g for 10 min, and then washed with a 1/4 volume of 0.88% (w/v) KCl. The obtained chloroform layer was evaporated, and the weight of the residue was measured as total lipids. The residue of hepatic lipids was dissolved in isopropanol containing 10% (v/v) Triton-X, and the triglyceride and cholesterol levels were measured using the corresponding commercial kit as described above.
Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) To assess the change in the expression of genes involved in energy metabolism and lipid metabolism, total RNAs were prepared using Sepazol-RNA I (Nacalai Tesque, Inc., Kyoto, Japan) from the frozen tissues (maintained at −80°C); i.e., 50 mg of muscle and liver, respectively, and 100 mg of BAT and retroperitoneal WAT, respectively. First-strand complementary DNA (cDNA) was synthesized from 0.15 µg of total RNA treated with DNase I (Ambion Inc., Austin, Texas, USA) using a ReverTra Ace® quantitative PCR (qPCR) RT Kit (Toyobo Co. LTD., Osaka, Japan) with random primers. The forward and reverse primers used are listed in Table 2. The levels of mRNA expression were quantified using THUNDERBIRD™ SYBR® qPCR Mix (Toyobo Co. Ltd., Osaka, Japan) in duplicate by the Applied Biosystems 7300 Real-Time PCR system according to the supplier's recommendations. The specificity of amplifications in each sample was confirmed by dissociation analysis showing that each sample provided a single melting peak. Relative gene expression was calculated by comparing the number of thermal cycles necessary to generate threshold amounts of product (Ct). Ct was calculated for the target gene and for the ribosomal protein S 17 (RPS17). For each cDNA sample, the Ct for RPS17 was subtracted from the CT for the target gene to provide the parameter ΔCt, thereby normalizing the initial amount of RNA used. The amount of the target gene mRNA was calculated as 2–ΔΔCt, where ΔΔCt is the difference between the ΔCt of the two cDNA samples to be compared.
ACCα, acyl-CoA carboxylase; FAS, fatty acid synthase; CPT1a, carnitine palmitoyltransferase I; ACO, acyl-CoA oxidase; LCAD, long-chain acyl-CoA dehydrogenase; UCP, uncoupling protein; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; CYP7A1; cholesterol 7 alpha-hydroxylase; LDLR, low density lipoprotein receptor
The relative gene expression comparisons were conducted using an endogenous control (ribosomal protein S 17 [RPS17]).
The ΔΔCT method was used for relative quantification.
Single oral lipid administration test To assess the effect of ESG on lipid absorption, 36 mice were divided to 2 groups, ESG and control. Mice were starved for 16 hours before the test. Corn oil emulsion (corn oil 3 mL, water 3 mL, cholic acid 50 mg, and lecithin 50 mg) was made by sonication. Mice were orally administered with 5 mL/kg B.W. of corn oil emulsion simultaneously with 5% ESG solution or water as control at dosage of 5 mL/kg B.W.. Before, 1, 2, 3, 4 and 6 hours after administration, blood samples were collected from tail vein and plasma TG levels were measured by commercial kit (Wako Pure Chemical Industries (Osaka, Japan)).
Statistical analysis All data are presented as the means ± standard error (SE). Statistical analysis of long-term supplementation experiment was conducted by one-way analysis of variance (ANOVA) and subsequently by a post hoc Tukey-Kramer test. Differences were considered significant when P values were <0.05. Statistical analysis of single oral administration test was conducted by student's t-test.
Effect of ESG on body composition, body weight, and adipose tissue weight In the present study, mice were fed a control diet (C) or high-fat diet (HF) containing 0%, 10%, or 20% ESG for 15 weeks. Results of CT analysis at 14 weeks demonstrated that total fat mass was significantly higher in the HF-0% group than in the C-0% group. Compared with the HF-0% group, the total fat masses including visceral fat mass and subcutaneous fat mass in the HF-10% and HF-20% groups were lowered by 7.5% and 31.9%, respectively (Fig. 1). Total fat mass in HF-20% group was significantly lower than that in HF-0% group. Food intake in control diet groups and HF diet groups was not affected by ESG during the experimental period. The average food intake of C-0%, C-10%, C20%, HF-0%, HF-10% and HF20% group were 2.49, 2.57, 2.45, 2.32, 2.44 and 2.43 g/day/mouse, respectively. At the end of the feeding period, the body weight of the HF-0% group was significantly higher than that of C-0% group (Table 3). The significant increase was observed from week 7. In HF-20% group body weight gain was significantly suppressed as compared with HF-0% group, whereas supplementation of ESG had little effect on body weight in the control groups. The relative weights of mesenteric, epididymal, retroperitoneal, and subcutaneous WAT were increased by HF diet. In the HF groups, 20% ESG significantly decreased the relative weights of mesenteric, subcutaneous, and total WAT (Table 3). The relative weight of BAT did not change in any experimental group.
Computed tomographic (CT) analysis for the effect of enzymatically synthesized glycogen (ESG) on body fat percentage
At 14 weeks, body fat percentages of mice were analyzed by X-ray CT. Values without a common letter in a row differ significantly among the groups (P < 0.05, Tukey-Kramer multiple comparison test).
HF, high fat
Mice were fed the control or high-fat diet containing 0%, 10%, and 20% ESG for 15 weeks. At the end of experiment, body weight and tissue weights were measured after 14 h fasting.
Values are the mean ± standard error (SE) (n = 4, 5). Values without a common letter in a row differ significantly (P < 0.05) by the Tukey-Kramer multiple comparison test.
Effect of ESG on blood lipids and NO level The plasma glucose level was significantly higher in the HF-0% group compared with the C-0% group (Table 4). ESG supplementation tended to reduce the plasma glucose level, although the reduction was not significant. The total cholesterol level was significantly increased by HF diet, and the increase was significantly inhibited by 20% ESG supplementation. The plasma levels of triglyceride and NEFA remained unchanged. It has been reported that NO increases thermogenesis in brown adipose tissue (Saha et al, 1996). Thus, we measured plasma NO level as a parameter of thermogenesis. The plasma NO level of the C-20% group was significantly higher than that of C-0% group. A tendency to increase NO level with supplementation of ESG was observed in the HF groups (Table 4).
HF, high fat; NEFA, non-esterified fatty acid; NO, nitric oxide
Mice were fed the control or high-fat diet containing 0%, 10%, and 20% ESG for 15 weeks. At the end of experiment, blood parameters were measured after 14 h fasting.
Values are the mean ± standard error (SE) (n = 4, 5). Values without a common letter in a row differ significantly (P < 0.05) by the Tukey-Kramer multiple comparison test.
Body temperature The body temperature measured in the rectum was 37.4 ± 0.2, 37.8 ± 0.1, 38.3 ± 0.2, 37.6 ± 0.1, 37.5 ± 0.1, and 37.4 ± 0.2°C in the C-0%, C-10%, C-20%, HF-0%, HF-10%, and HF-20% groups, respectively. Although no difference in the body temperatures was found between the C-0% and the HF-0% groups, that of the C-20% group was markedly higher than the C-0% group. This effect of ESG on the body temperature was not observed in the high-fat diet groups.
Hepatic lipids Because intake of a high-fat diet induces fatty liver and hepatic lipid accumulation, we investigated the liver lipid levels. The relative and net weight of the liver did not change in any experimental groups (Table 3 and 5). The hepatic total lipid level was significantly higher in the HF-0% group than in the C-0% group. ESG suppressed the high-fat diet-induced hepatic lipid accumulation in a dose-dependent manner, and the hepatic total lipid level was significantly lower in the HF-20% group than in the HF-0% group. The hepatic phospholipid, triglyceride, and cholesterol levels in the HF-0% group were also higher than those in the C-0% group, and these increases were significantly lowered by supplementation with 20% ESG.
HF, high fat
Mice were fed the control or high-fat diet containing 0%, 10%, and 20% ESG for 15 weeks.
Values are the mean ± standard error (SE) (n = 4, 5). Values without a common letter in a row differ significantly (P < 0.05) by the Tukey-Kramer multiple comparison test
Fecal lipid and cecum weight The cecum weights of C-20% and HF-20% groups were markedly higher than that of the respective C-0% and HF-0% groups (Fig. 2A). Moreover, ESG supplementation increased cecal content (Fig. 2B). The fecal lipids increased by 49% and 56% in the HF-10% and HF-20% groups, respectively, compared with the HF-0% group (Fig. 2C). On the one hand, there was no significant difference in the fecal lipids among the control groups. Dietary supplementation of ESG in particular increases the fecal lipid in high-fat diet-fed mice.
Effects of enzymatically synthesized glycogen (ESG) on cecum weight, cecal content weights, and fecal lipids levels. At the end of experiment, the weights of cecum (A) and cecal contents (B) were measured after 14 h fasting. Three days before the end of experiment, the feces were collected and the fecal lipids were measured (C).
Values without a common letter in a row differ significantly among the groups (P < 0.05, Tukey-Kramer multiple comparison test).
The expression of genes related to thermogenesis and lipid metabolism Supplementation of 20% ESG significantly prevented the accumulation of liver fat as shown above (Table 5). We focused on hepatic lipid metabolism-related gene expression and compared the expression level of control, control 20%, HF and HF20% groups. We investigated the mRNA levels of fatty acid oxidation-related genes, carnitine palmitoyltransferase I (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), and acyl-CoA oxidase (ACO), and fatty acid synthesis-related genes acyl-CoA carboxylase (ACCa) and fatty acid synthase (FAS) in the liver. The gene expression levels of CPT1a, LCAD, ACO, ACCa, and FAS in the HF-20% group tended to be lower than those in the HF-0% group; however, there were no significant differences among the groups (Fig. 3A). The mRNA levels of cholesterol metabolism-related genes, HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), cholesterol 7 alpha-hydroxylase (CYP7A1), and low density lipoprotein receptor (LDLR), were not altered by ESG (Fig. 3B).
Expression of genes related to lipid metabolism and thermogenesis in mice fed control and high-fat diet containing 0% and 20% enzymatically synthesized glycogen (ESG).
Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on RNA extracted from liver (A, B), brown adipose tissue (BAT), retroperitoneal white adipose tissue (RWAT) and muscle (C) for analysis of the indicated mRNA levels. Values are the means ± standard error (SE) (n = 4, 5). Values without a common letter in a row show significant differences (P < 0.05, Tukey-Kramer multiple comparison test)
The uncoupling proteins (UCPs) are a proton transporter family located in the mitochondrial inner membrane and involved in the regulation of energy metabolism and thermogenesis. UCP-1, a member of this family, plays an important role in energy expenditure by fat oxidation and subsequent heat generation in BAT. UCP-2 and UCP-3 are also involved in energy metabolism and obesity (Rousset S., Alves-Guerra M.C., et al., 2004). The mRNA expression levels of UCP-1 in BAT, UCP-2 in WAT, and UCP-3 in skeletal muscle of 0% and 20% ESG groups were investigated, and no changes were detected (Fig. 3C).
Single oral lipid administration test To evaluate inhibitory effect of ESG on lipid absorption, the concentrations of plasma samples of TG were measured. As shown in Fig. 4, ESG significantly suppressed the plasma TG concentration after 2 hours of administration.
Effects of enzymatically synthesized glycogen (ESG) on lipid absorption in mice. Mice were fasted 16 hours and then orally administered with corn oil emulsion simultaneously with ESG or water as control. Before, 1, 2, 3, 4 and 6 hours after administration, blood samples were collected from tail vein and plasma TG levels were measured by commercial kit.
Data are means ± S.E. (n= 18). Asterisk shows significant difference from control group at same time point (P < 0.05, student's t-test).
In the present study, the effects of long-term ingestion of ESG on the lipid accumulation and metabolism were evaluated in diet-induced obese mice. It was found that the excretion of lipids into the feces was increased by ESG in high-fat diet-ingested groups, but not in normal groups. These data indicated that ESG suppresses lipid absorption in the intestine after intake of a high-fat diet. In fact, ESG reduced the rise of plasma triacylglycerol after oral administration together with corn oil emulsion (Fig. 4). Thus we considered the main mechanism of the anti-obese effect of ESG is due to the inhibition of lipid absorption.
There are many food ingredients that are reported to inhibit the increase of postprandial plasma triacylglycerol. For example, green tea and oolong tea catechins and flavonoids are reported to inhibit lipase activity (Wang et al., 2014, Nakai et al., 2005). Kishimoto et al. reported that resistant dextrin, a water soluble dietary fiber, inhibits an increase of postprandial triacylglycerol in human and animal studies. The mechanism behind this inhibition is considered to be a delay of the release of fatty acids from micelle due to resistant maltodextrin by inhibition of the decomposition of micelle and stabilization of micellar structure (Kishimoto et al., 2007). ESG affected neither micellar stability nor lipase activity, although ESG possesses water soluble dietary fiber portion (data not shown). We should do farther study to examine the detail of mechanism in future.
On the one hand, in the current study, subcutaneous adipose tissue, triacylglycerol, and cholesterol accumulation in the liver, as well as plasma total cholesterol were significantly lower in the HF-20% group than those in the HF-10% group, although there was no difference in the amount of lipid excreted to the feces and the amount of cecum content between the HF-10% and HF-20% groups. This suggested that the effect of ESG on reduction of lipid accumulation is related not only in increase of lipid excretion but other mechanisms.
Dietary lipids digested and absorbed in the small intestine are carried in the form of chylomicron to the liver and recomposed to circulate through the entire body in the form of lipoprotein. We measured the lipid metabolism related enzymes (Fig. 3); however, no significant change was observed in the ESG treated groups. It is considered that thermogenesis is also closely related to energy expenditure. The body temperature was increased significantly in the C-20% group, and plasma concentration of NO, which is considered to thermogenesis related factor, was also significantly increased. We have reported that ESG increases NO production of macrophages (Kakutani et. al. 2007). Thus, we considered that ESG increased plasma NO level through enhancement of NO production of macrophages in small intestine of mice. However, in HF groups ESG did not affect body temperature or NO level at all. Thus, we could not conclude the effect of ESG on thermogenesis.
We should discuss the other possibility, which is the relationship of gut bacteria. Although glycogens are (1→4/1→6) α-D-glucan, some kinds of glycogen, particularly ESG, have a property which is resistant to the digestion of α-amylase (Takata et al., 2009). Our recent study showed that the resistant part of ESG reaches the cecum and works as prebiotics in rats, i.e. ESG is converted to SCFAs by microbiota and increases the counts of Lactobacillus and Bifidobacterium. (Furuyashiki et al., 2011). In the present study, the increase of the cecal content convinced us of the increment of the prebiotic effect of ESG. Recently many researches have suggested that prebiotics prevent obesity through the changes of intestinal microbiota (Barczynska et al., 2015). Thus, the prebiotics effect of ESG, at least in part, resulted in the reduction of the adipose tissue weight, plasma cholesterol, and the liver lipids observed in the present study. Indeed, although the change of cecal content was not significantly changed in HF groups, the amount of cecal content was closely related with the reduction in body fat mass, liver lipid and plasma cholesterol.
From the results of the present study, ESG inhibited adipose tissue hypertrophy and lipid accumulation in the liver, and decreased the plasma cholesterol level in diet-induced obese model mice. However, antiobesity effects of ESG on humans have not been studied, and the mechanism behind the antiobesity effects of ESG remains unclear; thus, future human clinical studies and experiments to further elucidate the mechanism are necessary to establish the evidence for ESG contributing to human health.
Acknowledgment This work was supported in part by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.