1. Introduction
Dietary fiber can be classified as insoluble (cellulose, hemicellulose, and lignin) or soluble (pectin and gum), depending on its solubility in water. It has been well established that dietary fiber contributes to multiple health benefits [
1] by reducing caloric absorption [
2,
3] and increasing the fecal excretion of energy, protein, and fat [
1,
4]. Interestingly, ingesting insoluble fiber has been found to reduce energy digestibility, body weight gain, or blood lipid levels in animals, e.g., feeding a dietary fiber-rich by-product of the soy milk industry to healthy rats for 4 weeks showed decreases in weight gain and serum total cholesterol level [
5]. In addition, we previously found that feeding the outer bran fractions of rice (OBFR) to rats for 3 weeks enhanced fecal lipid excretion, and consequently reduced abdominal lipid accumulation [
6].
Rice bran is a by-product of the rice milling process, and consists of several cellular layers, including the pericarp, tegmen, and aleurone, and is thus rich in fiber [
7]. Eating rice bran can substantially improve bowel movement and fecal excretion [
8], and reduce plasma triacylglycerol and total cholesterol levels in rats [
9]. Feeding rats rice bran oil also increased fecal weight and fecal bile acid excretion [
10], and eating rice bran significantly improved bowel movement in humans [
11], although the effects of dietary rice bran on plasma lipid profiles were reportedly small [
12]. Rice bran contains functional components including ferulic acid and γ-oryzanol, which contribute to the inhibition of cholesterol absorption in the intestines and its excretion via the feces [
13,
14]. In addition, phytic acid reportedly inhibited the activity of porcine pancreatic lipase [
15], and this inhibition reduced the availability of dietary fat in the intestine [
16].
Multi-break milling systems have recently become prevalent in the rice industry. In such systems, multiple milling machines are used to remove more bran from the rice kernels after the initial break. The outer layer of rice bran mainly consists of the pericarp, which is richer in lignin than the inner layer, while the amounts of pectic substances, hemicellulose, and α-cellulose are approximately equal in the inner and outer layers [
17]. In a previous study, we collected the OBFR from the first and second breaks of a commercial quadruple-break milling system and found that the OBFR contains γ-oryzanol and phytic acid at 1.2-fold higher abundance than whole rice bran [
6]. The aim of the current study is to examine the effects of dietary OBFR on the intestinal environment and plasma, hepatic, and cecal metabolite profiles of rats.
4. Discussion
Feeding the OBFR diet affected neither mean body weight nor mean body weight gain. In addition, it had no significant effect on the weight of the individual organs or tissue types (i.e., heart, liver, kidney, abdominal fat, and soleus muscle). However, although the OBFR diet had no significant effect on feed intake, it significantly reduced the feed efficiency and the digestibilities of CP, EE, and NFE of the rats, compared with their counterparts fed the control diet. The decreases in the feed efficiency and digestibility may have been due to the high dietary fiber content of the OBFR. It has been well established that eating dietary fiber reduces the digestibility of ingested nutrients by disrupting nutrient absorption [
2,
3]. More specifically, feeding insoluble fiber to rats reduced the digestibility of CP and EE [
4,
5,
26]. Given that OBFR is rich in insoluble fiber (i.e., lignin, hemicellulose and pectic substance) [
16], these results suggest that the OBFR diet reduced feed efficiency by reducing the digestibility of CP, EE, and NFE.
However, as was found for the body weight, body weight gain, and individual organ and tissue weights, there were no significant differences in the measured blood parameters (glucose, triacylglycerol, and total cholesterol) between the treatment groups. In addition, the plasma 3-MeHis concentration, which serves as an index for muscle protein degradation [
27], did not differ between the two groups, indicating that feeding the OBFR diet did not affect the muscle protein degradation rate. However, the digestibility of the NDF in the rats fed the OBFR diet was double that of their control diet counterparts. These findings suggest that the OBFR diet may have been fermented and used in the rats’ intestines.
Since the OBFR diet significantly reduced the pH of the rats’ cecal contents, we performed untargeted GC/MS-based metabolomics analysis to evaluate the impact of feeding the OBFR diet on the cecal metabolites. A total of 64 metabolites were detected in the rats’ cecal contents. The principal component analysis score scatter-plot based on all the identified metabolites in the cecal contents (
Figure S1A) suggested that the cecal metabolites were distinctly distinguishable between these two groups. In addition, feeding the OBFR diet markedly increased the concentrations of some organic compounds with lower pKa (i.e., 3-hydroxyphenylacetic acid, adenine, glutaric acid, glyceric acid, and 3-hydroxybenzoic acid), suggesting that these compounds may have contributed to the lower pH of the cecal contents of the rats fed the OBFR diet.
Dietary fiber in the intestinal tract is converted into short-chain fatty acids (SCFAs) by some species of enteric bacteria, such as
Lactobacillus,
Streptococcus and
Bifidobacterium, and eating a fiber-enriched diet has reportedly resulted in a lower pH in the intestinal tract in humans [
28]. We therefore determined the organic acid concentrations involving SCFA in the intestinal contents, and found that feeding the OBFR diet markedly increased the concentrations of acetic acid, butyric acid, and propionic acid. This is in agreement with the earlier studies showed that feeding diet containing rice bran markedly increases in intestinal SCFA in rats [
29,
30]. Furthermore, 95% of the SCFAs produced in the cecum and large intestine are rapidly absorbed by colonocytes [
31,
32,
33,
34], and SCFAs have reportedly been found in hepatic, portal, and peripheral blood [
35,
36]. It has therefore been suggested that SCFAs provide 10% of the daily caloric requirements in humans [
37]. In this study, the concentrations of acetic acid, butyric acid, and propionic acid in the cecal content were 1.5–2.8-fold higher in the rats in the OBFR group, and this may have affected the metablites profiles of these rats.
Untargeted GC/MS-based metabolomics analysis was therefore performed to investigate the effects of the OBFR diet on the metabolites profiles of the plasma and liver of the rats. Although 35 metabolites were detected in both the plasma and liver tissue, 19 and 38 metabolites were detected only in the plasma and liver, respectively. Because the liver plays a major role in metabolism and has a number of functions, a relatively large number of metabolites were confirmed in the liver compared to the plasma under the same condition for detection. On the other hand, since metabolites in plasma reflect the state of the rat’s whole body, the 19 metabolites detected only in the plasma might be derived from tissues except for the liver. As was found for the cecal content, the principal component analysis score scatter plots for the plasma and liver (
Figure S2B,C, respectively) suggested that the two treatment groups were distinctly distinguishable.
In this study, 64, 54, and 73 metabolites were identified in the rats’ cecal contents, plasma, and liver, respectively. The numbers of identified metabolites were relatively less than other studies using an untargeted metabolomics approach, e.g., 140, 167, and 207 metabolites were identified in the rat’s plasma [
38], liver [
39], and feces [
40], respectively. The reason for this might be due to the condition for metabolite identification shown in
Table S1. In the GC/MS untargeted metabolomics of this study, the annotation reached level 2 on the scale of confidence in metabolite identification, as defined by the Chemical Analysis Working Group of the Metabolomics Standards Initiative [
41]. Indeed, GC/MS untargeted metabolomics studies used similar conditions to ours to identify metabolites have reported that 78 metabolites were identified in serum [
42] and liver [
43], respectively.
In the plasma, seven of these 11 metabolic pathways were involved in lipid metabolism (fatty acid metabolism, fatty acid elongation in mitochondria, steroid biosynthesis, bile acid biosynthesis, glycerolipid metabolism, fatty acid biosynthesis, and phosphatidylinositol phosphate metabolism), three were involved in carbohydrate metabolism (inositol metabolism and inositol phosphate metabolism), and one was involved in amino acid metabolism (valine, leucine, and isoleucine degradation). In contrast, in the liver, seven of the nine metabolic pathways were involved in carbohydrate metabolism (amino sugar metabolism, glycolysis, fructose and mannose degradation, gluconeogenesis, the Warburg effect, the pentose phosphate pathway, and pyruvate metabolism), and the remaining two were involved in amino acid metabolism (glutamate metabolism) and nucleotide metabolism (pyrimidine metabolism).
Although the metabolites profiles of the plasma indicated seven metabolic pathways involved in lipid metabolism, neither of them was indicated in the liver. In this study, both plasma and liver tissue were extracted by methanol–chloroform–water, and the aqueous layer was used for GC/MS untargeted metabolomics. Although fatty acids are difficult to be distributed in the aqueous layer, they are often reported to be identified in plasma [
44]. In agreement with this, we identified 9 fatty acids in the plasma, of which 4 fatty acids (i.e., nonanoic acid, myristic acid, palmitic acid, eicosanoic acid) were significantly increased. On the other hand, in the liver, only palmitic acid was identified, and there was no significant difference between the two groups. Although the reason why fatty acids are seldom identified in the liver remains unclear, such few fatty acids identified in the liver might indicate that no metabolic pathway is involved in lipid metabolism in the liver.
As mentioned above, because the liver plays a major role in metabolism and has a number of functions, including lipid metabolism, glucose metabolism, drug detoxification, and plasma protein synthesis, we explored the expression levels of genes encoding enzymes related to metabolic processes. The changes at the metabolites and gene expression levels induced by feeding the OBFR diet identified in this study are summarized in
Figure 3.
Although there was no significant difference, feeding the OBFR diet tended to increase palmitic acid compared with their control diet counterparts (
p = 0.09). In addition, the OBFR diet increased the mRNA expressions of SREBP1C, FAS, and ACC, suggesting that fatty acid synthesis may be enhanced. The OBFR diet also increased the mRNA expression of SREBP2, which is the master regulator of cholesterol synthesis and metabolism [
45], whereas HMGR, the rate-limiting enzyme for cholesterol synthesis, did not differ between the two groups. Furthermore, neither CYP7A1, the rate-limiting enzyme for bile acid synthesis, nor CPT1a, the rate-limiting enzyme for fatty acid β-oxidation in the liver, were altered by the OBFR diet.
As the enrichment analysis by using the metabolites profiles of the liver indicated that changes in pyruvate metabolism and the pentose phosphate pathway had taken place, we examined the expression levels of genes involved in these processes in the rats’ livers. Feeding the OBFR diet increased the mRNA expression of PDH, but did not affect the mRNA expressions of PC, LDHa, or LDHb. In addition, the level of lactic acid was significantly lower in the rats fed the OBFR diet than in their control diet counterparts (
Table 6). Furthermore, feeding the OBFR diet increased the mRNA expression of G6PD, which is a rate-limiting enzyme involved in the pentose phosphate pathway. These results suggest that feeding the OBFR diet may have enhanced either the conversion from pyruvate into acetyl-CoA, or NAPDH production, and consequently contributed to fatty acid synthesis by increasing substrate production in the livers of the rats.
The enrichment analysis by using the metabolites profiles of the liver also identified changes in either glycolysis or gluconeogenesis, and we therefore examined the expression levels of genes encoding rate-limiting enzymes for these two processes in the livers of rats. Feeding the OBFR diet did not affect the expression levels of the genes encoding GCK and PFK, whereas it significantly increased the expression levels of the genes encoding PEPCK mRNA and F1,6BP mRNA. In addition, G6Pase mRNA expression tended to be higher in the livers of the rats fed the OBFR diet. Furthermore, feeding the OBFR diet significantly increased the level of fructose 6-phosphate, which is an intermediate metabolite of gluconeogenesis, in the rats’ livers (
Table 6). These results therefore suggest that feeding the OBFR diet may have enhanced hepatic gluconeogenesis than glycolysis. Our findings suggest that the OBFR diet-induced fatty acid synthesis and/or gluconeogenesis may be reasons that rats fed the OBFR diet could maintain their blood glucose and triacylglycerol levels, despite the fact that OBFR diet reduced the digestibility of ingested nutrients.
Certain non-carbohydrate organic compounds (e.g., glucogenic amino acids, pyruvate, lactic acid, and propionate) are known to be used as substrates for gluconeogenesis. In the liver, glucogenic amino acids are converted into pyruvate, succinyl-CoA, or fumaric acid. Feeding the OBFR diet decreased the levels of some glucogenic amino acids (tryptophan, isoleucine, and phenylalanine), and increased the fumaric acid and malic acid levels in the rats’ livers (
Table 6). In addition, feeding the OBFR diet markedly increased the cecal propionic acid concentration. Propionic acid is found in the hepatic, portal, and peripheral blood [
46,
47], and is converted into succinyl-CoA. Furthermore, feeding the OBFR diet increased the expressions of the genes encoding MCEE, PCC, and MUT in the livers of the rats. These results suggest that either glucogenic amino acids or propionate may have been converted into pyruvate, succinyl-CoA, or fumaric acid in the livers of the rats fed the OBFR diet. Although feeding the OBFR diet did not change the mRNA expressions of CS, IDH, and OGDH, it significantly increased the mRNA expressions of SCS, SDH, FH, and MDH. These data support the hypothesis that feeding the OBFR diet increased the use of glucogenic amino acids and propionate as substrates for gluconeogenesis in the livers of the rats. Furthermore, in this study, since we found that feeding the OBFR diet markedly increased the concentrations of propionic acid in the intestinal contents, the increased propionic acid may have been converted to glucose via hepatic gluconeogenesis and contributed as an energy source in rats fed the OBFR diet.
Interestingly, the level of isoleucine in the cecal content was five-fold higher in the rats fed the OBFR diet than in the control rats (
Table 4). In contrast, the levels of isoleucine in the liver and plasma were significantly lower in the rats fed the OBFR diet than in the control rats (
Table 5 and
Table 6). The enrichment analysis by using the metabolites profiles of the plasma identified a change in valine, leucine, and isoleucine degradation. Branched-chain amino acid (BCAA) is known to be metabolized solely in the liver [
46,
47], while it can be metabolized mainly in skeletal muscle by branched-chain aminotransferase (BCAT) to produce energy. We therefore examined the expression levels of the genes encoding BCAT2, BCKDCα, and BCKDCβ in the rats’ soleus muscles, and found that feeding the OBFR diet significantly increased the expression of all three genes (
Figure S3A). These results suggest that isoleucine may have been metabolized in the skeletal muscles of the rats fed the OBFR diet. However, as feeding the OBFR diet did not change the expression levels of the genes encoding enzymes involved in glycolysis, β-oxidation, or the citric acid cycle in the soleus muscle (
Figure S3B,C), the OBFR diet may not have affected the metabolic state of the skeletal muscle of the rats. It has been suggested that BCAA are converted into branched chain α-keto acids by BCAT in the skeletal muscle and that these branched chain α-keto acids are then metabolized by BCKDC and used as an energy source in the liver [
47]. In this study, feeding the OBFR diet did not change the BCAT2 mRNA expression level in the liver, while it significantly increased the BCKDCα mRNA level (
Figure 2G). These results support the hypothesis that feeding the OBFR diet enhanced isoleucine degradation in the skeletal muscle, and consequently contributed to energy production via branched chain α-keto acid metabolism in the livers of the rats.
However, the enrichment analysis by using the metabolites profiles of either the plasma or the liver also indicated that changes in the remaining metabolic pathways involved in carbohydrate metabolisms, amino acid metabolism, or nucleotide metabolism. One possible explanation for the reasons for changes in them might be partially due to changes in cecal metabolites. In cecal contents of the rats fed the OBFR diet, some sugar and sugar acid compounds (i.e., sucrose, mannitol, and glyceric acid) were increased compared with their control diet counterparts (
Table 4), suggesting that these sugar and sugar acid compounds affects carbohydrate metabolisms either in liver or in the whole body of rats. In addition, although the enrichment analysis by using the metabolites profiles of the liver indicated that change in glutamate metabolism, it was a result from an increase in one compound (i.e., fructose 6-phosphate) in the liver. Therefore, it raised the possibility that cecal sugar metabolites might also affect fructose and mannose degradation, and consequently impact glutamate metabolism. Furthermore, the OBFR diet increased some nucleic acid metabolites (i.e., adenine and hypoxanthine) in rats’ cecal content (
Table 4), suggesting that these metabolites might affect nucleotide metabolism. However, the reason why only pyrimidine metabolism was indicated in rats fed the OBFR diet, although the OBFR increased purine bases in rats’ cecal content, is unclear. Further studies are needed to gain insight into the effects of feeding the OBFR diet on nucleotide metabolisms in the liver and whole body of rats.