Hydrophilic β-conglycinin Peptide Reduces Hepatic Triglyceride Accumulation in Obese Model OLETF Rats

Abstract

We have previously demonstrated that soy β-conglycinin (βCG) peptide improves obesity and reduces lipid abnormalities in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we fractionated βCG peptides by using hydrophobic synthetic absorbent materials and screened for peptide fractions with anti-obesity or hypolipidemic bioactivities. Wild-type Long-Evans Tokushima Otsuka (LETO group) and OLETF rats (control group) were provided a normal diet containing 20% casein. We prepared hydrophobic and hydrophilic peptides from βCG and fed OLETF rats diets in which 5% of the casein was replaced by either hydrophobic (the hydrophobic peptide group) or hydrophilic (the hydrophilic peptide group) peptides. After four weeks, hepatic triglyceride levels were lower in OLETF rats of the hydrophilic peptide group than in those of the control group due to decreased activity of hepatic fatty acid synthase, caused by the consumption of hydrophilic peptides. Although anti-obesity activity was not found in either βCG peptide fraction, the hydrophilic fraction of the βCG peptide could be a candidate for the identification of new peptide sequences that suppress hepatic lipid synthesis.

Introduction

Lifestyle-related diseases are important risk factors for the increases in cardiovascular morbidity and mortality (Formiguera and Canton, 2004; Nagao and Yanagita, 2008). Many studies have suggested that dietary proteins and peptides are useful as modulators of the risks associated with these diseases (Sirtori et al., 2009; Yoshikawa et al., 2000; Erdmann et al., 2008). It has been reported that dietary soy protein reduces lipid levels in animals and humans (Ramdath et al., 2017). In particular, soy β-conglycinin (βCG) has been shown to have physiological effects such as lipid lowering effects and anti-obesity effects (Tachibana et al., 2010; Wanezaki et al., 2015; Kawabeta et al., 2019; Kohno et al., 2006). It has been suggested that chemical or enzymatic hydrolysis of proteins can modify and improve their physiological functions (Singh et al., 2014; Nagaoka et al., 2019; Chatterjee et al., 2018). Many soybean-derived hypocholesterolemic oligopeptides have been identified in both in vitro and in vivo systems (Cho et al., 2008; Lammi et al., 2015a; Lammi et al., 2015b; Zhang et al., 2013; Nagaoka et al., 2010). Additionally, an antihypertensive dipeptide, immunostimulating tetrapeptide, antidiabetic pentapeptide, and the appetite suppressive oligopeptide have also been identified from in vivo studies on soy glycinin and βCG (Nakahara et al., 2011; Koyama et al., 2019; Tsuruki et al., 2005; Yamada et al., 2012; Nishi et al., 2003).

Otsuka Long-Evans Tokushima fatty (OLETF) rats develop metabolic disorders such as obesity, dyslipidemia and fatty liver (Yagi et al., 1997). We previously reported that the replacement of 10% casein with βCG peptide in the diet reduced obesity, hypertriglyceridemia and hepatic lipid accumulation through the enhancement of lipolysis and the suppression of lipogenesis in obese model OLETF rats (Wanezaki et al., 2020). In the present study, we sought to identify novel βCG peptide sequences with anti-obese activity, lipolysis-enhancing, and lipogenesis-suppressing activities using column adsorption chromatography, as well as to investigate the effects of fractionated hydrophobic and hydrophilic peptides on obesity and obesity-induced lipid abnormalities in OLETF rats.

Materials and Methods

Animals and diets    As shown in Figure 1, βCG peptide was prepared by the hydrolyzation of soy βCG (Lipoff, Fuji oil Co., Osaka, Japan) with three-kinds of protease (pepsin, trypsin, and chymotrypsin) via heat inactivation and centrifugation (with the yield of approximately 98%). Next, βCG peptide was separated into hydrophobic and hydrophilic fractions using a hydrophobic synthetic absorbent (DIAION HP20; Mitsubishi Chemical Corporation, Tokyo, Japan). The amino acid composition of each fraction was analyzed using an automatic amino acid analyzer (Table 1), and the molecular weight distribution of both fractions, estimated by gel filtration, ranged from 500–15 000 Da. Animal experiments were conducted according to the guidelines provided by the Ethics Committee for Experimental Animal Care of Saga University (No. 22-042-1, 2013-2015). Six-week-old male OLETF and Long-Evans Tokushima Otsuka (LETO) wild-type rats were purchased from Hoshino Laboratory Animals, Inc. (Ibaraki, Japan), and housed individually in metal cages in a temperature-controlled room at 24 °C with 12 h light/dark cycle. After an adaptation period of one week on a powder chow diet (CE-2; Clea Japan, Tokyo, Japan), the rats were fed one of three diets (Table 2). As the nitrogen content of casein, hydrophobic βCG peptide, and hydrophilic βCG peptide was 13.95%, 14.26%, and 11.47%, respectively, we prepared test diets with adjusted total nitrogen content. LETO and control OLETF rats (n = 5 of each) were maintained on the same casein-based diet, while a second group of OLETF rats (n = 5) were pair-fed to the control OLETF group with a similar diet containing 4.89% (w/w) hydrophobic βCG peptide at the expense of casein (5%) and sucrose. A third group of OLETF rats (n = 5) were pair-fed to the control OLETF group with a similar diet containing 6.08% (w/w) hydrophilic βCG peptide at the expense of casein (5%) and sucrose.

Table 1. Composition of fractionated βCG peptides

	Hydrophobic fraction	Hydrophilicfraction
Amino acid	(%)
Arg	8.80	8.08
Lys	6.45	5.90
His	2.55	2.12
Phe	9.07	3.67
Tyr	4.45	2.22
Leu	10.28	6.86
Ile	6.01	3.43
Met	0.55	0.48
Val	3.96	3.86
Ala	3.80	2.93
Gly	2.96	3.22
Pro	6.33	3.83
Glx	14.34	31.72
Ser	4.88	5.40
Thr	3.05	1.95
Asx	11.51	13.38
Trp	0.45	0.60
Cys	0.55	0.34
Total	100	100

Table 2. Composition of experimental diets

		Control	Hydrophobic	Hydrophilic
	LETO		OLETF
Casein	20.0	20.0	15.0	15.0
Hydrophobic βCG peptide	-	-	4.89*	-
Hydrophilic βCG peptide	-	-	-	6.08*
Cornstarch	15.0	15.0	15.0	15.0
Cellulose	5.0	5.0	5.0	5.0
Mineral mixture (AIN-76)	3.5	3.5	3.5	3.5
Vitamin mixture (AIN-76)	1.0	1.0	1.0	1.0
dl-Methionine	0.3	0.3	0.3	0.3
Choline bitartrate	0.2	0.2	0.2	0.2
Corn oil	7.0	7.0	7.0	7.0
Sucrose	48.0	48.0	48.1	46.9

*  Nitrogen content was adjusted to that in 5% casein.

Fig. 1.

Preparation of fractionated βCG peptides.

After four weeks of feeding followed by 9 h starvation, blood was collected from the abdominal aorta under isoflurane anesthesia. Serum was obtained by centrifugation of the blood and excised abdominal (epididymal, perirenal, and omental) white adipose tissues (WAT) and liver were stored at −80 °C until further analysis.

Measurement of serum parameters    Serum triglyceride, cholesterol, phospholipid, and glucose levels were measured using commercial enzyme assay kits (Wako Pure Chemicals).

Measurement of lipid levels in the liver    Hepatic lipids were extracted from the liver by the method described by Folch et al. (1957) Hepatic concentrations of triglyceride and phospholipid were measured according to the methods described by Fletcher et al. (1968) and Rouser et al. (1966), respectively. Hepatic cholesterol concentration was analyzed using the Cholesterol E-Test Wako (Wako Pure Chemical Industries, Ltd.).

Assays for hepatic enzyme activity    A piece of liver from each rat was homogenized in six volumes of a 0.25 M sucrose solution containing 1 mM ethylenediaminetetraacetic acid in a 10 mM Tris-HCl buffer (pH 7.4). After the nuclear fractions were precipitated, the supernatants were centrifuged at 10 000 × g for 10 min at 4 °C to obtain mitochondrial fractions. The resulting supernatants were recentrifuged at 125 000 × g for 60 min to precipitate microsomes, and the remaining supernatant constituted the cytosol fraction. The enzyme activities of fatty acid synthase (FAS), malic enzyme, glucose 6-phosphate dehydrogenase (G6PDH), and carnitine palmitoyltransferase (CPT) were determined according to the methods described by Kelly et al. (1986), Ochoa (1955), Kelly and Kletzien (1984) and Markwell et al. (1973), respectively.

Statistical analysis    The data are presented as the mean ± standard error (SE). To assess differences among four groups, data were analyzed by one-way ANOVA, and all differences were analyzed by the Fisher's Least Significant Difference post-hoc test using the KaleidaGraph software version 4.5 (Synergy Software, Reading, PA). P < 0.05 was considered statistically significant.

Results and Discussion

The amino acid composition of dietary proteins and peptides is known to influence their bioactivity; for example, hydrophobic peptides can exert antioxidant properties and hypocholesterolemic effects (Chen et al., 1998; Zhang et al., 2012). In this study, we evaluated whether the consumption of fractionated βCG peptides could affect obesity and lipid abnormality in obese model OLETF rats. Although the properties of several amino acids were not observed as they were present as peptides, the hydrophobic fraction was found to contain higher amounts of phenylalanine, tyrosine, leucine, isoleucine, and proline whereas the hydrophilic fraction contained a higher amount of glutamine (Table 1).

Food efficiencies were not significantly different among groups (Table 3). With higher food intake, the weight of WATs increased in control diet-fed OLETF rats compared to LETO rats (Table 3). Although there was no difference in the weights of the livers and perirenal or omental WATs, epididymal WATs in the hydrophobic group were significantly heavier than those in the control group of OLETF rats (Table 3). Because our previous study demonstrated that βCG peptide administration reduces WAT weights in OLETF rats (Wanezaki et al., 2020), we expected that either fraction of βCG peptides would have anti-obesity effects in OLETF rats. However, these results suggest that neither of the two fractions of βCG peptides is suitable for screening body fat-reducing peptides. Previous studies found that peptides from the β-subunit of βCG, such as soymorphins (Yamada et al., 2012) and VRIRLLQRFNKRS (Nishi et al., 2003), suppress appetite in vivo; so we attempted to find peptides that had anti-obesity effects without affecting food intake. However, we were unable to do so using our approach.

Table 3. Effect of experiment diet on growth parameters in LETO and OLETF rats

		Control	Hydrophobic	Hydrophilic
	LETO		OLETF
Initial B.W. (g)	113±2 a	151±3 b	151±3 b	151±3 b
Final B.W. (g)	240±4 a	336±4 b	330±3 b	327±4 b
Food intake (g)	399±3 a	582 ± 1 b	580 ± 7 b	583 ± 2 b
Food efficiency (g gain/g intake)
	0.320±0.003	0.319±0.003	0.310±0.009	0.304±0.004
Liver weight (g/100 g B.W. )	3.70±0.04	3.62±0.07	3.56±0.09	3.70±0.05
White adipose tissue weight (g/100 g B.W. )
Total	2.74±0.13 a	5.57±0.29 b	5.95±0.37 b	5.65±0.20 b
Epididymal	0.965 ± 0.068 a	1.54±0.09 b	1.75±0.07 c	1.56±0.03 bc
Perirenal	1.15 ± 0.08 a	2.73±0.14 b	2.84 ± 0.17 b	2.79±0.10 b
Omental	0.631±0.065 a	1.30 ± 0.08 b	1.36±0.13 b	1.29±0.09 b

Each value represents mean ± SE.

^abc  Different letters show significant difference at P < 0.05.

The responsive molecules involved in the anti-obesity effect of the investigated βCG peptide are still unknown, and the results of the present study indicate that the anti-obesity effect of this βCG peptide was not related to a single peptide but to a combination of several peptides. Therefore, the identification of novel anti-obesity peptides from βCG will require the use of other strategies for fractionation and screening.

Although the serum glucose levels did not differ between the LETO and OLETF rats, hyperlipidemia and hepatic lipid accumulation were observed in the OLETF rats fed the control diet (Table 4 and Figure 2). There were no significant differences in the levels of serum components, hepatic cholesterol, or hepatic phospholipids among the groups in OLETF rats (Table 4 and Figure 2). Hepatic triglyceride levels, however, were significantly lower in hydrophilic peptide-fed OLETF rats than in the control rats. Previous studies have shown that the administration of βCG peptides reduces hepatic triglyceride levels in OLETF rats (Wanezaki et al., 2020), and the present results suggest that the triglyceride-lowering peptides were restricted to the hydrophilic fraction of the βCG peptide.

Table 4. Effect of experiment diet on serum parameters in LETO and OLETF rats

		Control	Hydrophobic	Hydrophilic
	LETO		OLETF
Triglyceride(mg/dL)	36.6±5.0 a	104±8 b	87.9±7.7 b	110±16 b
Cholesterol (mg/dL)	94.8±1.9 a	151±12 b	147±10 b	156±15 b
Phospholipid (mg/dL)	142±7 a	223±15 b	211±10 b	216±13 b
Glucose (mg/dL)	158±5	179±12	174±5	174±7

Each value represents mean ± SE.

^ab  Different letters show significant difference at P < 0.05.

Fig. 2.

Effect of fractionated βCG peptides on hepatic lipid levels.

Rats were fed the control diet, a hydrophobic βCG peptide diet, or a hydrophilic βCG peptide diet for four weeks. Values are expressed as the mean ± standard error for five rats. See Table 2 for composition of diets. ^abc Different letters show significant difference among groups (P < 0.05).

We then investigated the effects of fractionated βCG peptides on hepatic enzymes related to triglyceride metabolism were analyzed.

It has been reported that a βCG-derived pentapeptide (soymorphin-5: YPFVV) activates β-oxidation and energy expenditure in obese mice (Yamada et al., 2012). We therefore expected that feeding either fraction of βCG peptides would reveal lipolysis-enhancing effect in OLETF rats. Although the lipolytic enzyme CPT activity was markedly enhanced by βCG peptide feeding in our previous study using OLETF rats (Wanezaki et al., 2020), there was no significant difference among groups in the present study. These results suggest that neither fractions of the βCG peptide is a suitable candidates for screening lipolysis-enhancing peptides.

We evaluated the activities of FAS, the cytosolic enzyme complex for de novo fatty acid synthase, and G6PDH and malic enzyme, two cytosolic NADPH-generating enzymes. The levels of these enzymes were significantly higher in the livers of OLETF rats than in LETO rats (Figure 3). As shown in Figure 3, although the activities of malic enzyme and G6PDH were not different among the groups, the activity of FAS was significantly lower in rats fed the hydrophilic peptide diet compared to the control OLETF rats. These data suggest that FAS-inhibiting peptides were distributed in the hydrophilic fraction of the βCG peptide, and their administration contributed to the reduced hepatic triglyceride levels in obese OLETF rats. We previously found that soybean protein derived dipeptides (KA, VK, SY) inhibit triglyceride synthesis in the liver model HepG2 cells (Inoue et al., 2011). However, these peptides did not display hypotriglyceridemic action in the follow-up study using OLETF rats (0.2% dipeptides containing diets, unpublished data). Another study indicated that βCG derived oligopeptides (KNPQLR, EITPEKNPWLR, and RKQEEDEDEEQQRE) inhibit FAS activity in vitro (Martinez-Villaluenga et al., 2010). However, the ability of these peptides to inhibit FAS activity in vivo has not been tested. Additionally, the effect of the hydrophilic fraction of βCG peptide on the activity of acetyl-CoA carboxylase, another key enzyme in fatty acid synthesis, has not been evaluated in this study. More studies are required to further identify lipogenesis-suppressive sequences from the hydrophilic fraction and elucidate their action in vivo.

Fig. 3.

Effect of fractionated βCG peptides on hepatic enzyme activities.

Rats were fed the control diet, a hydrophobic βCG peptide diet, or a hydrophilic βCG peptide diet for four weeks. Values are expressed as the mean ± standard error for five rats. See Table 2 for composition of diets. ^abc Different letters show significant difference among groups (P < 0.05).

In conclusion, the present study showed that a dietary intake of the hydrophilic fraction of βCG peptides resulted in the alleviation of hepatic triglyceride accumulation through the suppression of hepatic FAS activity in OLETF rats. Although bioactive peptides that have anti-obese or lipolysis activation effects may not be contained in either fractions, new peptide sequences that suppress hepatic lipid synthesis are expected to be found in the hydrophilic fraction of the βCG peptides in future studies.

Acknowledgements    This work was supported by a research grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (JSPS KAKENHI Grant No. 23580173). We would like to thank Editage (www.editage. com) for English language editing.

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