Beneficial Effects of Pterocarpan-High Soybean Leaf Extract on Metabolic Syndrome in Overweight and Obese Korean Subjects: Randomized Controlled Trial

Pterocarpans are known to have antifungal and anti-inflammatory properties. However, little is known about the changes in transcriptional profiles in response to a pterocarpan-high soybean leaf extract (PT). Therefore, this study investigated the effects of PT on blood glucose and lipid levels, as well as on the inflammation-related gene expression based on a peripheral blood mononuclear cells (PBMCs) mRNA sequencing analysis in Korean overweight and obese subjects with mild metabolic syndrome. The participants were randomly assigned to two groups and were administered either placebo (starch, 3 g/day) or PT (2 g/day) for 12 weeks. The PT intervention did not change body weight, body fat percentage and body mass index (BMI). However, PT significantly decreased the glycosylated hemoglobin (HbA1c), plasma glucose, free fatty acid, total cholesterol, and non-HDL cholesterol levels after 12 weeks. Furthermore, PT supplementation significantly lowered the homeostatic index of insulin resistance, as well as the plasma levels of inflammatory markers. Finally, the mRNA sequencing analysis revealed that PT downregulated genes related to immune responses. PT supplementation is beneficial for the improvement of metabolic syndrome by altering the fasting blood and plasma glucose, HbA1c, plasma lipid levels and inflammation-related gene expression in PBMCs.


Introduction
Obesity increases the risk of metabolic abnormalities associated with insulin resistance, hyperglycemia, type 2 diabetes, and dyslipidemia. The increasing prevalence of obesity and obesity-related diseases increases healthcare costs and also downgrades the quality of life [1]. Obesity, as well as excess fat intake, leads to the accumulation of body fat mass. In particular, central obesity or abnormally high deposition of visceral adipose tissue is considered a risk factor for metabolic complications [2]. In obesity, excess triglycerides promote the lipolysis and release of free fatty acids (FFA), which results in the dysfunction of insulin action and in insulin resistance. Elevated levels of circulating FFA can disrupt the insulin signaling in peripheral tissues and decrease the

Study Design
This study was a randomized double-blinded, placebo-controlled, parallel trial to evaluate the glucose-, and lipid-lowering effects of PT in overweight and obese subjects with mild metabolic syndrome.
The random allocation sequence was created using computer random numbers. At randomization, all subjects were randomly assigned in 1:1 ratio, two nutritional intervention groups: placebo (n = 25) and PT (n = 25). The mechanism used for allocation concealment was sequentially numbered containers by an independent laboratory researcher, and participants were kept blinded to the sequence and randomization until the end of the study. The capsules containing PT and placebo were no different, including undistinguishable size and color. Each participant received the capsules in the prepacked white plastic containers. The primary outcomes were fasting blood glucose, hemoglobin A1c, plasma glucose, and secondary outcomes were the homeostatic model assessment of insulin resistance, plasma lipid levels, and plasma cytokines-related metabolic syndrome and inflammation.
The subjects in the placebo group consumed six capsules containing starch (3 g per day), and those in the PT group consumed PT (2 g per day) in the morning, afternoon, and evening daily for 12 weeks. All participants were instructed to maintain their routine food intake and physical activity during the study. During the study period, we monitored the compliance of the subjects with the nutritional intervention and capsule consumption every week by telephone.

Anthropometric and Biochemical Analyses
At baseline and after the 4-, 8-, and 12-week nutritional intervention, the subjects attended the Science Research Center laboratory at Kyungpook National University between the hours of 07:00 and 11:00 a.m. after a 12-h overnight fast for anthropometric and physiological measurements. The waist circumference, hip circumference, blood pressure, fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), and lipids were determined at baseline and after the 12-week nutritional intervention. The body mass index (BMI), height, weight, and body composition were measured using an X-Scan plus II body composition analyzer (Jawon Medical Company, Daejeon, Korea). The waist and hip circumferences were measured with an anthropometric tape. The waist circumference was measured at the minimum circumference between the iliac crest and rib cage, and the hip circumference was measured at the maximum width over the greater trochanters. The waist-to-hip ratio (WHR) was then calculated by dividing the waist measurement by the hip measurement. The FBG, HbA1c, and blood pressure were measured using a glucose analyzer (LifeScan Inc., Milpitas, CA, USA), an HbA1c analyzer (Micormat™ Hemoglobin A1c Test, Bio-Rad, Hercules, CA, USA), and an automatic blood pressure (BP) monitor (Omron, Kyoto, Japan), respectively. In addition, blood samples were drawn into heparin-coated tubes and then centrifuged at 1000× g for 15 min at 4 • C for plasma assays. Dietary intake was recorded using 24-h dietary recalls for each subject before and during the nutritional intervention trial. Nutritional analysis was performed using the CAN-Pro 3.0 software (The Korean Nutrition Society, Seoul, Korea), which provides a comprehensive database for the nutritional content of general foods and specialty Korean foods.

Plasma Lipid Analyses
Plasma lipid concentrations were determined using commercially available kits for total cholesterol, triglycerides, HDL cholesterol (Asan Pharm. Co., Seoul, Korea), and free fatty acids (FFA) (Wako Chemicals, Richmond, VA, USA). The LDL cholesterol level was calculated using the Friedewald formula [32]: [total cholesterol-HDL cholesterol-(triglycerides/5)]. The non-HDL cholesterol level was calculated as follows: HDL cholesterol-total cholesterol. The atherogenic index (AI) was calculated as follows: (total cholesterol-HDL cholesterol)/HDL cholesterol.

Isolation of Peripheral Blood Mononuclear Cells and Extraction of RNA
Peripheral blood mononuclear cells (PBMCs) were isolated from the heparin-treated blood samples by density gradient centrifugation with the Ficoll-Paque reagent (GE Healthcare, Piscataway, NJ, USA) and were used for total RNA extraction. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The purity and integrity of the isolated RNA were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

mRNA Sequencing Analysis
For mRNA sequencing analysis, PBMCs were collected from three subjects randomly selected from the PT groups at baseline and 12 weeks. The mRNA in the total RNA was converted into a library of template molecules suitable for subsequent cluster generation using the reagents provided in the Illumina ® TruSeq™ RNA Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA). The first step in the workflow involved the purification of poly-A-containing mRNA molecules using poly-T oligo-attached magnetic beads. Following the purification, the mRNA was fragmented into small pieces using divalent cations at an elevated temperature. The cleaved RNA fragments were reverse-transcribed into first-strand cDNA using a reverse transcriptase and random primers. This was followed by the second-strand cDNA synthesis using DNA polymerase I and RNase H. These cDNA fragments then underwent an end repair process, the addition of a single "A" base, and ligation of the adapters. The products were then purified and enriched by polymerase chain reaction (PCR) to create the final cDNA library.

Preprocessing of RNA-seq Data
Quality control of the reads was performed using FastQC v. 0.10.0 (Babraham Bioinformatics, Cambridge, UK). The remaining reads were mapped onto a reference genome using the aligner software, TopHat version 1.3.3 (Johns Hopkins University, Baltimore, MD, USA). Then, the transcripts were assembled in Cufflink v. 2.0.2 using the gene annotation database of the TopHat Aligner. After the assembly, expression levels were measured in fragments per kilobase of transcript per million mapped reads (FPKM). The RNA-seq data has been submitted to the publicly available NCBI's Gene Expression Omnibus Database (http://www.ncbi.nlm.nih.gov/geo/) [34], accession number GSE80714.

Differential Transcriptome and Pathway Analysis
Differentially expressed genes were identified based on both the p-value threshold of less than 0.05 and a 1.5-fold change, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (www.genome.jp/kegg) were used for analyzing gene functions.

Statistical Analysis
The sample size for this trial was determined between two independent sample means (two tailed t-test, p < 0.05), a minimum of 20 participants per group was required. All data are presented as the mean ± standard error of the mean (SE). Statistical analysis was performed using SPSS Statistics, version 21 (IBM, Chicago, IL, USA). Significant changes within the groups between the baseline and 12-week values were assessed using a paired Student's t-test. The differences between the groups were evaluated by the General Linear Model with two-way repeated-measures ANOVA and baseline value, age, gender and BMI as covariates. Statistically significant differences were accepted as p < 0.05.

Baseline Clinical Characteristics
Fifty subjects were enrolled in this study. Among the enrolled subjects, one of the subjects dropped out of this study for personal reasons. Therefore, 49 subjects completed the trial [placebo (n = 25) and PT (n = 24)] from March to June 2014. Five subjects were excluded because of poor compliance [consuming <80% of the instructed amount, placebo (n = 2) and PT (n = 3)]. Serious adverse effects were not reported by the subjects consuming the PT or placebo supplements. In both males and females, there were no significant differences in the age, body weight, BMI, waist-to-hip ratio (WHR), blood pressure, and fasting blood glucose (FBG) levels between the groups before the trial (Table 1). In addition, all subjects were overweight or obese (27 kg·m −2 ≥ BMI ≥ 23 kg·m −2 ) [30,36]. Values are the mean ± SE. Placebo, starch; PT, pterocarpan-high soy leaf extract (2 g/day); BMI, body mass index; BP, blood pressure; WHR, waist-hip ratio; FBG, fasting blood glucose.

Nutrient Intake
Analysis of the participants' 24-h dietary recalls indicated no significant differences in the energy and nutrient intake between the groups at baseline or after 12 weeks of the supplementation ( Table 2). Values are the mean ± SE. Placebo, starch; PT, pterocarpan-high soy leaf extract (2 g/day).

Anthropometric Parameters and Blood Pressure
After 12 weeks of supplementation with PT, the systolic blood pressure (p = 0.045) was significantly lowered relative to the baseline values. However, there were no significant interaction effects (time × group) in the body weight, BMI, body fat percentage, WHR, and blood pressure versus the baseline values between the groups (Table 3). Two-way repeated-measures ANOVA showed significant interaction effects on FBG (p = 0.017) and HbA1c (p = 0.023), but there was no significant difference in the FBG concentration in the PT group after the trial (Table 4). After the 12-week trial, the plasma glucose level (p = 0.000) and the homeostatic model assessment of insulin resistance (HOMA-IR) index (p = 0.000) were significantly decreased in the PT group (Table 4). Furthermore, there were significant differences in the plasma glucose (p = 0.001) and HOMA-IR (p = 0.024) levels between the groups. PT significantly reduced the plasma insulin concentration (p = 0.012) after 12 weeks, although there was no significant interaction effect (time × group) ( Table 4).

Aspartate Transaminase and Alanine Transaminase Activities in Plasma
The plasma aspartate transaminase (AST) and alanine transaminase (ALT) activities were not different in the placebo and PT group during the trial, nor between the groups (Table 5).

Plasma Lipid Levels
At baseline, the plasma triglyceride, total cholesterol, FFA, HDL cholesterol, non-HDL cholesterol, LDL cholesterol levels, and the atherogenic index (AI) were not significantly different between the two groups ( Table 6). After 12 weeks, FFA (p = 0.000), total cholesterol (p = 0.020), and non-HDL cholesterol (p = 0.014) levels were significantly decreased in the PT group (Table 6). In addition, two-way repeated-measures ANOVA showed significant effects on the plasma FFA (p = 0.000), and non-HDL cholesterol (p = 0.014) levels between the groups (Table 6). Meanwhile, there were no significant interaction effects (time × group) in the plasma triglyceride, HDL cholesterol, LDL cholesterol, and AI values at interaction between the groups (Table 6).

Gene Expression Profiles of PBMCs Based on mRNA Sequencing Analysis
The PBMCs mRNA sequencing analysis identified genes that were differentially expressed before and after the PT supplementation. This comparison showed that 37 genes were upregulated and 128 genes were downregulated by the PT supplement.
To elucidate the functional differences in the PBMCs transcriptome caused by the PT supplement, we used the KEGG pathways mapper tool for the genes that were differentially expressed after the 12-week intervention. In the PT group, the differentially expressed gene-enriched pathways included chemokine signaling pathways, cytokine-cytokine receptor interactions, nuclear factor kappa B signaling, fatty acid metabolism, peroxisome proliferator-activated receptor signaling, and insulin signaling pathways (Table 8). In addition, genes involved in these pathways were associated with immune responses. Selected results of the mRNA sequencing analyses were confirmed by real-time-quantitative polymerase chain reaction (real-time-qPCR) (Figure 1). Analyses were performed using KEGG pathway analysis (www.genome.jp/kegg) with genes that were differentially expressed pterocarpan-high extract, soy leaf extract supplemented subjects, compared with before pterocarpan-high soybean leaf extract supplementation. Genes up-, or downregulated in pterocarpan-high soybean leaf extract supplemented subjects compared with before pterocarpan-high soybean leaf extract supplementation; AMPK, AMP-activated protein kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; mTOR, mammalian target of rapamycin; TNF, tumor necrosis factor.  Analyses were performed using KEGG pathway analysis (www.genome.jp/kegg) with genes that were differentially expressed pterocarpan-high extract, soy leaf extract supplemented subjects, compared with before pterocarpan-high soybean leaf extract supplementation. Genes up-, or downregulated in pterocarpan-high soybean leaf extract supplemented subjects compared with before pterocarpan-high soybean leaf extract supplementation; AMPK, AMP-activated protein kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; mTOR, mammalian target of rapamycin; TNF, tumor necrosis factor.

Discussion
In a previous study, PT ameliorated the insulin sensitivity and β-cell dysfunction in type 2 diabetic mice [27]. However, no clinical trial has been conducted to examine the anti-metabolic disorder effects with the transcriptome analysis in PT-supplemented subjects. Therefore, in the current study, we evaluated the effects of PT on diabetes-associated phenotype markers, plasma lipid, and plasma inflammatory cytokine levels, as well as on PBMCs transcriptional responses, in subjects with mild metabolic syndrome and compared the results with those obtained for placebosupplemented subjects.
Previous studies have shown that supplementation with pterocarpan (coumesterol and phaseol) -rich soy leaf reduced body weight and lipid accumulation by regulating adipogenic transcription factors in obese mice-fed HFD [37]. Additionally, Choi et al. [28] have demonstrated that supplementation with 70% ethanol extracts of soybean leaf has body fat-lowering effect in prediabetic patients, while Kim et al. [29] reported that 95% ethanol extracts of soybean leaf had a minimal effect on % body fat in overweight subjects. However, in the present study, the body weight, BMI, and body

Discussion
In a previous study, PT ameliorated the insulin sensitivity and β-cell dysfunction in type 2 diabetic mice [27]. However, no clinical trial has been conducted to examine the anti-metabolic disorder effects with the transcriptome analysis in PT-supplemented subjects. Therefore, in the current study, we evaluated the effects of PT on diabetes-associated phenotype markers, plasma lipid, and plasma inflammatory cytokine levels, as well as on PBMCs transcriptional responses, in subjects with mild metabolic syndrome and compared the results with those obtained for placebo-supplemented subjects.
Previous studies have shown that supplementation with pterocarpan (coumesterol and phaseol) -rich soy leaf reduced body weight and lipid accumulation by regulating adipogenic transcription factors in obese mice-fed HFD [37]. Additionally, Choi et al. [28] have demonstrated that supplementation with 70% ethanol extracts of soybean leaf has body fat-lowering effect in prediabetic patients, while Kim et al. [29] reported that 95% ethanol extracts of soybean leaf had a minimal effect on % body fat in overweight subjects. However, in the present study, the body weight, BMI, and body fat content were not significantly different compared to baseline after 12 weeks. Thus, our previous [29] and present studies suggest that the body fat-lowering effect of soybean leaf extracts may be associated with solvent and experimental conditions. The HbA1c level is a useful parameter for monitoring diabetes and glucose tolerance since HbA1c reflects the degree of blood glucose regulation over two to three months [38]. In the current study, the supplementation with PT led to a significant decrease in the blood HbA1c and FBG levels after 12-week intervention. Thus, the present study indicates that the supplementation with PT can regulate long-term hyperglycemia by decreasing the blood HbA1c and FBG levels. Our previous study has demonstrated that the soybean leaf extract (2 g per day) supplementation for 12 weeks significantly decreased the blood HbA1c and FBG levels compared to the baseline [28]. Similarly, Soy leaf extract containing KG improved diabetes-associated phenotypes and glucose homeostasis in db/db mice [39]. In addition, the plasma insulin level was significantly lowered after the PT supplementation. Thus, we evaluated the HOMA-IR, a useful index for assessing insulin resistance, insulin sensitivity, and β-cell function. The PT supplement significantly decreased the HOMA-IR value after the trial, as well as resulting in significant differences in the HOMA-IR value between the groups. The HOMA-IR lowering effect of the soybean leaf extracts were consistent between our previous human [28,29] and current studies. Taken together, these results suggest that the PT supplementation can provide improvements in hyperglycemic subjects.
The present study also demonstrated that the supplementation with PT significantly decreased the plasma total cholesterol, non-HDL cholesterol, and FFA levels; the significant interaction effect of FFA was also observed between the groups. Non-HDL cholesterol is regarded as an important indicator of cardiovascular risk [40]. In addition, soy leaves have been reported to reduce non-HDL cholesterol, although serum total cholesterol showed a trend of lowering effects in hamsters [26]. An increase in the plasma FFA level is associated with metabolic disorders and insulin resistance, while FFA reduction partially leads to improved glucose metabolism through a decrease of gluconeogenesis and glycogenolysis [41][42][43]. Taken together, these results support that PT supplementation contributes to improving hyperglycemia and insulin sensitivity by decreasing the plasma FFA concentration.
Obesity is linked to chronic low-grade inflammation and elevates the production of proinflammatory adipokines, which can cause the development of type 2 diabetes and obesity-related comorbidities [44,45]. In general, proinflammatory plasma markers such as TNF-α, IL-6, and MCP-1 are present in an early inflammatory stage of obesity. On the other hand, macrophage inflammatory protein-1β (MIP-1β) levels increase when obesity is well established [46]. TNF-α is produced in an insulin-resistant state and reduces the expression level of plasma adiponectin [47]. PAI-1 is not only associated with thrombosis and fibrosis but also with obesity, metabolic syndrome and insulin resistance [48]. Furthermore, there is a positive correlation between the levels of TNF-α, which has been reported to be a potent PAI-1 inducer, and PAI-1 [49]. In this trial, the plasma TNF-α, IL-6 and PAI-1 levels were significantly decreased in the PT group after the 12 weeks of supplementation, although the interaction effect of time and group showed only the TNF-α level between the groups. In addition, a previous study showed that pterocarpan-enriched soy leaf supplementation led to inhibiting the gene expression of TNF-α and IL-6 in the white adipose tissue in type 2 diabetic mice [27]. Taken together, these observations indicate that the decreased TNF-α level may be induced by improving the plasma PAI-1 level. Moreover, decreased cytokine levels are partially linked to enhanced insulin sensitivity and glucose homeostasis.
Phosphoinositide-dependent protein kinase-1 (PDPK-1) is the downstream kinase of phosphatidylinositol-3 kinase and is stimulated by insulin. It also targets protein kinase B, the main effector of PDPK-1 [50]. In addition, Tawaramoto et al. [51] reported that the knockout of vascular endothelial PDPK-1 improved obesity and insulin sensitivity in HFD-fed knockout mice by reducing the visceral fat accumulation compared to that in the wild-type mice. Our mRNA sequencing analysis in PBMCs revealed downregulated mRNA expression of PDPK-1 in PT-supplemented subjects, possibly owing to a decreased insulin level.
Chemokines are considered therapeutic targets for inflammatory diseases [52]. In the current study, the mRNA expression of CC chemokine receptor (CCR) 6, CCR4, and CC chemokine ligand 4-like 2 (CCL4L2), which is similar to CCL4, was downregulated. In obesity, inflammatory mediators such as IL-1β, TNF-α, and interferon gamma can elevate the CCR6 expression level [53]. In addition, CCR4 and CCR6 are more expressed in type 2 diabetes patients than in non-diabetic persons [54]. CCL4, also known as MIP-1β, is expressed at two-fold higher levels in obese and metabolic syndrome subjects compared with those in healthy individuals [55]. Our data suggest that PT has the potential to regulate insulin resistance and metabolic syndrome by lowering the mRNA expression of PDPK-1, CCR4, CCR6, and CCL4L2 in PBMCs.
There are some limitations of this trial. Participants of the present study were overweight or obese subjects with mild metabolic syndrome. However, the impact of the study would be higher when lean or different types of subjects are used to identify clear interpretation.

Conclusions
In conclusion, the present study indicates that the supplementation with PT (2 g/day) can reduce the risk factors associated with diabetes and dyslipidemia in obese subjects with mild metabolic syndrome and simultaneously decrease the inflammatory-related gene expression in PBMCs.