Ameliorative effects of Japanese barnyard millet (Echinochloa esculenta H.Scholz) bran supplementation in streptozotocin-induced diabetic rats

Abstract

Japanese barnyard millet is gaining popularity in Japan. However, the utilization of bran is limited. Additionally, the role of Japanese barnyard millet bran in regulating glucose metabolism has not been fully investigated. In this study, we investigated changes in blood glucose levels in normal rats, as well as its ameliorative effects in streptozotocin-induced diabetic rats. The increase in blood glucose levels was lower in rats fed the bran diet than in those fed the control diet. In diabetic rats, a bran diet reduced diabetic polyuria, water intake, and HbA1c levels. The expression of heme oxygenase-1, an antioxidative enzyme, was higher in the liver of diabetic rats fed a bran diet. These results suggest that bran reduces the acute increase in blood glucose levels post feeding and has ameliorative and antioxidative effects in diabetic conditions. Thus, Japanese barnyard millet bran may be used a potential food material for maintaining glucose metabolism.

Introduction

Barnyard millet is an important food crop that is widely cultivated in Asian countries as food for human consumption and livestock feed. Among the many species of barnyard millet, Japanese barnyard millet (Echinochloa esculenta H. Scholz) is cultivated in Japan, Korea, and China (Renganathan et al., 2020). Recently, Japanese barnyard millet has become popular as a supplement to rice or as a staple food by substituting a part of rice in daily consumption because of the increased health awareness in Japan. Along with the prevalence of dietary needs for millet, the cultivation area and production have remained high. The production of Japanese barnyard millet in 2020 reached approximately 73 tons, almost 95% of which was produced in Iwate, located in the northeastern region of Japan ⁱ⁾. On refining, approximately half of the millet grain is obtained as bran, which is a by-product consisting primarily of millet hull, seed coat, aleurone layers, small amounts of embryos, and endosperm. The applications of bran, such as supplementation with livestock feed or compost material, are limited. However, bran is not necessarily unproductive. Bran contains many useful compounds such as lipids, minerals, phenolic compounds, and fibers. If suitably processed, bran can be used as a valuable source of oil or healthy food material for human consumption. This potential applicability also contributes to the reduction of agricultural waste (Bodie et al., 2019). Japanese barnyard millet can be grown virtually without agricultural chemicals and the resulting bran contains low levels of these chemicals; thus, to date, oil from Japanese barnyard millet bran (JMB) has been used for cosmetic applications ⁱⁱ⁾. We have previously proved that Japanese barnyard millet protein has ameliorative activity in diabetic mice (Nishizawa et al., 2009). However, the health benefits of bran are yet to be investigated.

Several studies have shown an inverse relationship between whole grain intake and the incidence of diabetes. In contrast, refined grain intake is not associated with a reduction in diabetic risk (Fung et al., 2002; Aune et al., 2013). One of the major differences between whole and refined grains is the retention levels of bran. Among the contents of the bran fraction, fiber is potentially responsible for the protective effect of whole grain (Slavin et al., 1999; Reynolds et al., 2020). Whole grains are also rich in phenolic compounds, which function as antioxidants. Rice bran contains several bioactive chemicals such as ferulic acid, p-coumaric acid, and caffeic acid with significant antioxidant activity (Saji et al., 2020a). Phenolic compound extracts of rice bran decrease the generation of lipid peroxides and intracellular reactive oxygen species in mouse macrophages stimulated with lipopolysaccharide and hydrogen peroxide (Saji et al., 2020b). Furthermore, these compounds upregulate the expression of some antioxidant-related genes, nuclear factor erythroid-2-related factor 2, NAD(P)H:quinone oxidoreductase 1 (NQO1), and heme oxygenase-1 (HO-1), and reduce inflammatory responses in endothelial cells under oxidative stress conditions (Saji et al., 2019).

Millet grains are also a rich source of fiber and phenolic compounds. The crude fiber content of barnyard millet is higher than that of other cereals (Ugare et al., 2014; Devi et al., 2014; Saleh et al., 2013). The fiber ratio ensures the slow absorption of sugars in blood, which is helpful for maintaining blood glucose levels. In addition, Japanese barnyard millet contains several antioxidant phenolic compounds (Watanabe, 1999). Among these compounds, luteolin inhibits α-glucosidase activity (Kim et al., 2000). Luteolin intake also suppresses renal and cardiac oxidative stress and induces superoxide dismutase (SOD) activity and the expression of HO-1 in streptozotocin (STZ)-induced diabetic animals (Wang et al., 2011; Wang et al., 2012). Considering these previous studies, JMB, which is rich in fiber and phenolic compounds, could be potentially useful against diabetic damage. However, to our knowledge, the role of JMB in regulating glucose metabolism and diabetic damage has not yet been investigated.

In this study, we aimed to explore the health benefit of JMB by investigating its nutritional value in growing rats, changes in blood glucose levels in rats fed a bran-containing diet, and its ameliorative effect on STZ-induced diabetic rats.

Materials and Methods

Materials and reagents    JMB was produced by refining Japanese barnyard millet at a local grain supplier (JA Green Service Hanamaki, Iwate, Japan). The bran was mechanically pressed to remove oil, and the resultant bran was used in the current study. The ingredients of the experimental diet were purchased from Oriental Yeast Co., Ltd (Tokyo, Japan) and Fujifilm Wako Pure Chemical (Osaka, Japan). Primary antibodies against HO-1 were obtained from Enzo Life Sciences (Farmingdale, NY, USA). Primary antibodies against NQO1 (ab80588) and γ-glutamylcysteine-synthetase (GCLC, ab190685) were obtained from Abcam (Cambridge, UK). Primary antibodies against β-actin (AC-15) were purchased from Novus Biologicals (Littleton, CO, USA). All other reagents used were of analytical grade.

Animals and experimental designs

Animals.    Male Wistar rats were purchased from Japan SLC. Inc. (Shizuoka, Japan). They were individually housed in stainless steel wired cages, maintained at 22 °C and 55% relative humidity, with a 12-h light/dark cycle (6:00-18:00). The animals were allowed ad libitum access to tap water and a 20% casein diet, based on AIN-93G. The animal care protocols used in this study were approved by the Iwate University Animal Research Committee under the Guidelines for Animal Experiments at Iwate University (approval number A202005).

Experiment 1.    Four-week-old rats were divided into two groups of five animals each and fed either the control or Japanese barnyard millet bran (JMB) diet ad libitum for 10 days. The ingredients of the experimental diets are listed in Table 1. In the last two days, feces from the rats were collected, freeze-dried, and weighed.

Table 1. Composition of experimental diets.

	Control	JMB
Ingredient (g/kg diet)
Casein	200	151.4
α-Corn starch	529.5	243.1
Sucrose	100	100
Cellulose	50	40
Soybean oil	70	70
AIN-93G mineral mixture	35	35
AIN-93 vitamin mixture	10	10
l-Cystine	3	3
Choline bitartrate	2.5	2.5
JMB	-	345

A composition of JMB was follows (%): moisture, 5.9; protein, 14.1; lipid, 7.1; ash, 5.6; fiber, 2.9; non-fibrous carbohydrate by difference, 64.4.

Experiment 2.    After 15-16 h of fasting, four-week-old rats were randomly divided into two groups (n = 9 in the control group, n = 10 in the JMB group) and initial blood glucose levels were measured on a glucometer (OneTouch VerioVue, LifeScan Japan, Tokyo, Japan), using a small amount of blood obtained by tail vein sampling. The rats were then fed either the control or the JMB diet for 30 min. Following this, the diets were terminated and blood glucose levels were immediately determined as the values at 30 min. The subsequent measurements were performed at 60, 120, and 180 min.

Experiment 3.    After 18 h of fasting, five-week-old rats were injected with streptozotocin (STZ, 50 mg/kg body weight) or sodium citrate buffer solution (0.1 M, pH 4.5) from the tail vein. Following STZ administration, a 10% glucose solution was provided for 24 h to prevent hypoglycemia. Two days after STZ injection, blood glucose levels were measured using a glucometer, and rats with blood sugar levels >200 mg/dL were considered diabetic. These rats were further divided into the following three groups: a control group (C group), which was administered citrate buffer and fed a casein diet (n = 5); a diabetic group (DC group), which was administered STZ and fed a casein diet (n = 7); and a JMB diet-fed diabetic group (DJ group), which was administered STZ and fed a JMB diet (n = 6). Animals in all groups were fed the respective diets ad libitum for 33 days. From days 27 to day 30, feces and urine samples were collected, and the quantity of drinking water consumed was measured. On day 32, HbA1c levels were measured using a small amount of blood obtained from the tail vein (DCA 2000 HbA1c cartridge, Siemens Healthcare Diagnostics, Tarrytown, NY, USA). On day 33, rats were euthanized after overnight fasting. Blood samples were collected from the inferior vena cava. The liver and kidneys were immediately removed, rinsed with cold saline, and weighed. All samples were stored at −80 °C until analysis.

Measurement of plasma glucose    Plasma glucose levels were determined by the glucose oxidase method using the Glucose CII Test Wako (Fujifilm Wako Pure Chemical).

Western blot analysis    For western blot analysis, samples of rat liver and kidney homogenates were prepared as previously described (Sato et al., 2018). Tissue homogenates were subjected to western blot analysis using specific antibodies, as described previously (Chiba et al., 2019).

Statistical analyses    All results are expressed as the mean ± standard error of the mean (SEM). Significant differences among multiple groups were analyzed using one-way analysis of variance with Tukey's post hoc tests (GraphPad Instat Software ver. 3.0a, GraphPad Software, Inc., San Diego, CA, USA). Pairwise comparisons were performed using unpaired t-tests and two-tailed p values were calculated. A value of p < 0.05 was considered statistically significant.

Results

Experiment 1:Assessment of the nutritional value of the bran diet in healthy rats    We first examined the nutritional properties of the JMB diet. As shown in Fig. 1A and Fig. 1B, there was no difference in body weight changes and food intake between rats fed the control diet and the bran diet (JMB diet). However, fecal excretion was higher in the JMB diet group than that in the control group (Fig. 1C). These results indicate that the JMB diet contains many indigestible components; however, its effect on growth is similar to that of the control diet.

Fig. 1.

Nutritional quality of Japanese barnyard millet bran (JMB) diet on growth in healthy rats.

(A) Changes in body weight of dietary groups (C: rats fed the control diet, J: rats fed JMB diet). (B) Total food intake. Data are expressed as means of total intake per each rat in the group during the experimental period. (C) Average of feces weight. Values are shown as the mean ± standard error of mean (SEM) (n = 5). Values with an asterisk are significantly different from each other (p < 0.05).

Experiment 2: Evaluation of the bran diet on carbohydrate absorption and digestibility in healthy rats    To determine the impact of supplementation with JMB on dietary carbohydrate absorption, we measured changes in blood glucose levels when rats were fed the control and the JMB diet. As shown in Fig. 2, blood glucose levels in rats fed the JMB diet were lower than those in rats fed the control diet, and the area under the curve (AUC) for glucose was significantly lower in the bran diet group than that in the control group, despite identical food intake by animals in both the groups (C group: 3.00 ± 0.12 g, J group: 3.01 ± 0.06 g during 30 min-feeding).

Fig. 2.

Changes in blood glucose in rats fed the control or JMB diet.

(A) Time course of blood glucose. Blood glucose levels were measured at 0, 30, 60, 120, and 180 min after rats were fed the control diet (C, n = 9) or JMB diet (J, n = 10). (B) The area under the curves for blood glucose levels. Values are shown as the mean ± SEM. Values with an asterisk in A are significantly different from each other at the same time point, and values with an asterisk in B are significantly different from each other (p < 0.05).

Experiment 3: Evaluation of anti-diabetic effect in streptozotocin-induced diabetic rats    Further, we investigated whether the JMB diet had an ameliorative effect in STZ-induced diabetic rats. Initial body weights, final body weights, body weight gains, and daily food intakes in the C, DC, and DJ groups during the experimental period are summarized in Table 2. There was no significant difference in body weights and food intake between diabetic groups. As shown in Fig. 3A and Fig. 3B, water intake and diabetic polyuria were reduced in DJ rats compared to DC rats, suggesting that diabetic dehydration might be suppressed by feeding the JMB diet. As shown in Fig. 3C, the daily food intake in the DC group was significantly higher than that in the DJ group during the feces collection period. In contrast, the amount of feces excreted (Fig. 3D) was higher in the DJ group than that in the DC group. In addition, HbA1c levels were significantly lower in the DJ group than those in the DC group, although plasma glucose levels at dissection were not significantly different among the diabetic groups (Fig. 3E and Fig. 3F).

Table 2. Body weight, weight gain, and food intake of the dietary groups in experiment 3.

Groups	C	DC	DJ
Initial body weight (g)	104.6 ± 2.3	98.9 ± 2.8	103.3 ± 4.6
Final body weight (g)	210.4 ± 8.3a	102.1 ± 10.0b	114.1 ± 7.1b
Body weight gain (g)	105.7 ± 6.9a	3.2 ± 8.5b	10.8 ± 8.8b
Daily food intake (g/day)	13.1 ± 0.6a	23.1 ± 0.7b	20.7 ± 1.0b

C: normal rats (n = 5), DC: diabetic control rats fed the control diet (n = 7), DJ: diabetic rats fed JMB diet (n = 6). Values are shown as the mean ± SEM. Significant differences between values are indicated with different letters (p < 0.05).

Fig. 3.

Effect of JMB diet on diabetic symptoms in STZ-induced diabetic rats.

Water intake (A), urine levels (B), the daily food intake (C), and the amount of feces excreted (D) from day 27 to day 30. HbA1c levels in the blood (E) on the day before sacrifice and the levels of plasma glucose (F) at sacrifice. C: normal rats (n = 5), DC: diabetic control rats fed the control diet (n = 7), and DJ: diabetic rats fed the JMB diet (n = 6). Values are shown as the mean ± SEM. Significant differences between values are indicated with different superscript letters (p < 0.05).

Effect of JMB on the expression levels of antioxidative enzymes in the liver and kidney of STZ-induced diabetic rats    We further examined the effect of the bran diet on antioxidative enzyme expression in the liver and kidney. The expression of NQO1 increased in the liver of diabetic control rats, and remained unchanged in the livers of diabetic rats fed the JMB diet (Fig. 4A). As shown in Fig. 4B, the JMB diet increased the expression of HO-1 in the liver. The expression of NQO1 was also higher in the kidneys of diabetic rats than in those of the control rats, and the JMB diet marginally reduced NQO1 expression (Fig 4C). In contrast, GCLC expression decreased in diabetic rats. However, GCLC expression was not restored by the JMB diet (Fig. 4D). These results suggest that the bran diet has the potential to modulate antioxidative activity in the liver and kidney of diabetic rats.

Fig. 4.

Effect of JMB diet on the expression of antioxidative enzymes in the liver and kidney of STZ-induced diabetic rats.

The levels of NQO1 (A) and HO-1 (B) expressions in the liver, and the levels of NQO1 (C) and GCLC (D) expressions in the kidney. C: normal rats (n = 5), DC: diabetic control rats fed the control diet (n = 7), DJ: diabetic rats fed JMB diet (n = 6). Expression levels were normalized with that of β-actin. Values are shown as the mean ± SEM. Significant differences between values are indicated with different superscript letters (p < 0.05).

Discussion

In the present study, we investigated the suppressive effect of bran from Japanese barnyard millet on carbohydrate absorption and its antidiabetic effect of bran in STZ-induced rats. We found that the JMB diet resulted in a lesser increase in blood glucose levels in normal rats than that with the control diet. However, the JMB diet did not have any adverse effects on growth and improved some diabetic symptoms, such as increased HbA1c levels, water intake, and urine volume in STZ-induced rats. To our knowledge, this is the first report to demonstrate the ameliorative effects of bran from Japanese barnyard millet on diabetic symptoms. Starch from barnyard millet results in a better decrease in blood glucose and lipid levels than rice and other millets do (Krishna et al., 1997). In an intervention study on patients with diabetes, Ugare et al. (2014) demonstrated that a barnyard millet diet significantly reduces glucose levels. However, the effect of JMB on diabetic symptoms remains unknown. Murtaza et al. (2014) showed that a diet containing finger millet bran reduces the buildup of body weight, glucose intolerance, and oxidative stress in high-fat diet-fed mice, and its effect was more pronounced than that of a diet containing whole grain. The current results are consistent with the results of these previous studies. Whole-grain consumption reduces the risk of diabetes, which is mediated by grain fiber (Fung et al., 2002; Aune et al., 2013; Reynolds et al., 2020). The crude fiber content of barnyard millet is higher than that of other cereals (Saleh et al., 2013), and millet bran is a promising substance for supplying fiber from the diet because of its high fiber content (Zhu et al., 2018). Thus, in the present study, the suppression of postprandial blood glucose increases and the antidiabetic effect may be attributed to the fiber fraction of JMB. In addition, our previous study showed that administration of the starch fraction prepared from refined Japanese millet, which includes 1.8% fiber, does not show any ameliorative effect on KK-Ay mice, a genetically type 2 diabetic mouse (Nishizawa et al., 2009). In the current study, the amount of fiber derived from JMB was approximately 1% in JMB diet, compared to approximately 0.86% in the previous study. Thus, in view of the past and present findings, the fiber fraction of bran might be more effective.

In addition to fibers, phenolic compounds may also be involved in reducing glucose absorption. The phenolic fraction from Korean barnyard millet exhibits strong inhibitory activity against α-glucosidase, comparable to that of the commonly used standard drug acarbose (Ofosu et al., 2020). Japanese barnyard millet contains N-p-coumaroyl serotonin (Watanabe, 1999). Takahashi and Miyazawa (2012) reported that two serotonin derivatives, N-p-coumaroyl serotonin and N-feruloyl serotonin, isolated from safflower seeds, show α-glucosidase inhibitory activity. In summary, the fiber and phenolic compounds in JMB might contribute to the reduction in glucose absorption.

Additionally, the bran prepared from Japanese barnyard millet contained approximately 14% protein, which is higher than the fiber content (Table 1). We previously showed that proteins from refined Japanese millet and Korean proso-millet reduce plasma glucose levels and improve glucose and lipid metabolism in KK-Ay type 2 diabetic mice (Nishizawa et al., 2009; Park et al., 2008). Similar results have been reported in an experiment with a high-fat diet and a STZ-induced diabetic mouse model for the protein fraction isolated from foxtail millet (Fu et al., 2021). Some millet proteins inhibit α-amylase and α-glucosidase (Karaś et al., 2019). In addition, some peptides derived from millet hydrolysates have antioxidative effects, based on in vitro analysis (Ji et al., 2019; Agrawal et al., 2019); the administration of a foxtail millet protein hydrolysate shows antioxidant effects in spontaneously hypertensive rats (Chen et al., 2017). These anti-hyperglycemic and antioxidative effects of millet proteins may contribute to the reduction in diabetic symptoms. However, millet protein levels in diets in previous experiments (Nishizawa et al., 2009; Park et al., 2008) have been approximately four times higher than those in the present study. Therefore, it is unlikely that the protein fraction in JMB is an active ingredient in the present study.

Millet bran is also rich in phytochemicals that have antioxidative properties, which may contribute to its antidiabetic effect in the present study (Ofosu et al., 2020; Liang and Liang, 2019). The administration of a diet containing a finger millet seed coat, which is rich in phenolic compounds, improves hyperglycemia and delays cataract development in STZ-induced diabetic rats (Shobana et al., 2010). The phenolic extract of barnyard millet scavenges radicals and reduces DNA damage and protein breakdown induced by hydroxyl radicals (Anis and Sreerama, 2020). Watanabe reported that Japanese barnyard millet contains several antioxidative phenolic compounds (Watanabe, 1999). Among these compounds, luteolin administration also reduces lipid oxidation and induces SOD activity and the expression of HO-1 in the kidney and myocardium of STZ-induced diabetic animals (Wang et al., 2011; Wang et al., 2012). The expression of NQO1 in the kidney is upregulated under hyperglycemic conditions (Moon et al., 2020) and its expression decreases along with the attenuation of diabetic oxidative stress (Guo et al., 2021). We observed that the expression of HO-1 increased in the liver of STZ-induced diabetic rats fed the JMB diet, and the increased expression of NQO1 in the kidney was marginally reduced by feeding the bran diet. Thus, in the current study, phenolic compounds in JMB might contribute to the reduction in oxidative stress and exhibit anti-diabetic effects. Further studies are required to identify the compounds responsible for this effect.

In conclusion, we revealed that JMB had preventive effects on postprandial hyperglycemia in normal rats and ameliorative effects in STZ-induced diabetic rats. The study findings contribute to identifying the potential application of bran from Japanese barnyard millet in maintaining health, along with reducing food waste from a refining process.

Acknowledgements    We would like to thank Kazuya Takahashi from JA Green Service Hanamaki Co., Ltd. for providing Japanese barnyard millet bran.

Conflict of interest    There are no conflicts of interest to declare.

Abbreviations

NQO1

NAD(P)H:quinone oxidoreductase 1

HO-1

heme oxygenase-1

SOD

superoxide dismutase

STZ

streptozotocin

GCLC

γ-glutamylcysteine-synthetase

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• i) https://www.pref.iwate.jp/sangyoukoyou/nourinsuisan/toukei/1018786/index.html (April 15, 2022) (in Japanese)
• ii) https://fermenstation.co.jp/en/ (April 15, 2022) (in Japanese)