1. Introduction
Glucose homeostasis is regulated by a sequence of events within the pancreatic β-cells, which result in the secretion of insulin [
1]. Typically, in the postprandial state, increased levels of glucose in plasma can initiate pancreatic β-cells to secrete insulin, consequently suppressing hepatic glucose output and increasing peripheral tissue glucose uptake [
2]. However, impairment of glucose-stimulated insulin secretion as a result of oxidative stress and inflammation can result in β-cell dysfunction and insulin resistance, subsequently leading to the pathogenesis of type 2 diabetes mellitus (T2DM) [
3]. There are several essential genes involved in insulin secretion pathways that are specifically expressed in pancreatic β-cells. They are known to be involved in the processes leading to insulin release from the initial glucose entry into the β-cells followed by mitochondrial adenosine triphosphate (ATP) generation and potassium and calcium membrane depolarization leading to exocytosis events [
4]. They include, glucose transporter 2 (
Glut2) [
5], pancreatic and duodenal homeobox 1 (
Pdx1) [
6], sirtuin 1 (
Sirt1) [
7], mitochondrial transcription factor A (
Tfam) [
8], and insulin 1 (
Ins1) [
9].
The
Glut2 gene is located in the pancreatic plasma membrane and functions as a glucose transporter as part of the glucose-sensing mechanism for the stimulation of insulin secretion [
2]. The
Pdx1 gene plays an important role in mitochondrial embryonic development and β-cell differentiation and is known to regulate the expression of a variety of different pancreatic endocrine genes, including
Glut2 [
10]. The
Sirt1 gene is known to serve as a key energy redox sensor involved in generating ATP that helps promote glucose-stimulated insulin secretion in pancreatic β-cells and potentially contribute to β-cell adaptation in response to insulin resistance [
1]. In the liver, skeletal muscles, and white adipose tissues, the
Sirt1 gene has key functions that include regulation of glucose production, improvement in insulin sensitivity via fatty acid oxidation, and control of the production of adipokines [
7]. The
Tfam gene plays an essential role in the maintenance of mitochondrial DNA (mtDNA) and replication [
11]. Altered mitochondrial function is known to result in a defective oxidative metabolism, which seems to be involved in visceral fat gain and the development of insulin resistance [
12]. Moreover, the
Tfam gene is also involved in insulin exocytosis events by maintaining appropriate ADP/ATP ratio [
4]. The
Ins1 gene and its transcription factors are regulated by the circulating levels of glucose [
9]. It encodes the production of insulin that plays a vital role in the regulation of carbohydrate and lipid metabolism [
13]. Therefore, a disruption in the function of these genes (
Glut2,
Pdx1,
Sirt1,
Tfam, and
Ins1) in the pancreatic β-cells is known to impair insulin secretion and result in the development of T2DM.
Recent studies have shown the potential of plant-derived phenolic compounds in ameliorating β-cell dysfunction via their antioxidant and free radical scavenging properties [
14,
15,
16]. Exposure of polyphenols to β-cells has also been responsible for the modulation of several signalling proteins, including transcription factors, protein kinase, and ion channels [
17].
Rice bran (RB), a by-product of the rice milling process, is usually discarded or used as animal feed [
18]. However, the bran layer is composed of several bioactive phytochemicals, including polyphenols and phenolic acids [
19]. Although RB phenolic extracts are believed to target metabolic pathways associated with T2DM, the mechanisms behind its effect on gene expression under normal and diabetic conditions have not been investigated. This study aimed to determine the effect of RB phenolic extracts on the expression of genes (
Glut2,
Pdx1,
Sirt1,
Tfam, and
Ins1) associated with insulin secretion pathways and on glucose-stimulated insulin secretion under normal and high glucose conditions.
4. Discussion
Prolonged exposure of pancreatic β-cells to a high glucose environment is known to result in oxidative stress, consequently leading to the downregulation of pancreatic genes, in turn causing impaired β-cell function and insulin secretion [
16]. Plant-derived phenolic compounds via their antioxidant, free radical scavenging and metal chelating properties have been observed to target metabolic pathways associated with the pathogenesis of T2DM [
14]. The present study demonstrated that RB phenolic extracts effectively alter β-cell function in insulin-secreting cells by modulating the expression of genes and insulin secretion. It was observed that RB phenolic extracts upregulated the expression of key genes associated with β-cell function, including
Glut2,
Pdx1,
Sirt1,
Tfam, and
Ins1 both under normal and high glucose-induced stress conditions (
Figure 2,
Figure 3,
Figure 4,
Figure 5 and
Figure 6).
The
Glut2 gene primarily acts as a glucose transporter and the decreased expression of the
Glut2 gene is directly proportional to the loss of glucose-stimulated insulin secretion [
5]. In this study, a significant increase in the expression of the
Glut2 gene was observed under normal conditions compared to that in high glucose conditions. This may have been caused by the increase in glucotoxic stress created by the high glucose environment, resulting in a reduced ability to maintain normal functioning as a glucose transporter. Nevertheless, a significant up-regulation of the
Glut2 gene was observed under both conditions compared to those of the respective controls after treatment with varying concentrations of RB extract (
Figure 2). Similarly, studies in which phenolic compounds derived from
M. pumilum var. alata extracts and purified phenolic compounds such as resveratrol were tested improved β-cell function, and insulin signalling was observed as a result of increased expression of the
Glut2 gene in the pancreas [
21,
23]. This is most likely due to the polyphenols targeting the exchange of calcium ions resulting in the exocytosis of insulin-containing granules, thereby favourably modulating β-cell function [
5,
24].
Pdx1 gene expression is essential for the homeostatic regulation of the glucose-sensing system in β-cells [
6]. It is also essential for survival and differentiation of β-cells as it primarily acts by upregulating the transcription of several β-cell-specific genes, including the
Ins and
Glut2 genes [
25]. Results obtained from this study show that under both normal and high glucose conditions, a significant upregulation of the
Pdx1 gene was evident after treatment with RB phenolic extracts (
Figure 3). Upregulation of the
Pdx1 gene has been observed elsewhere, in which administration of
Teucrium polium extract, known to contain phenolic compounds with strong antioxidant and anti-inflammatory effects, was found to reverse the symptoms of streptozotocin-induced diabetes in rats [
26]. Another study, wherein the effect of gallic acid against glucolipotoxicity and insulin secretion was examined, showed that pre-treatment with different concentrations of gallic acid was found to increase insulin secretion and resulted in the upregulation of the
Pdx1 gene in RINm5F β-cells [
27]. Reduction in insulin secretion has been attributed to the c-Jun N-terminal kinase (JNK) pathway activation under oxidative stress conditions. JNK activation as a result of oxidative stress results in forkhead box protein O1 (FOXO1) phosphorylation, and the nuclear localization of the FOXO1protein leads to a reduction in the expression of the
Pdx1 gene [
28]. As an adequate expression of the pancreatic
Pdx1 gene is essential to maintain the proper function of insulin-producing β-cells, inhibition of the JNK pathway is crucial. As phenolic compounds are recognized to modulate the regulation of the JNK pathway [
26], it is likely that the observed upregulation of the
Pdx1 gene by RB-derived phenolic extracts resulted from an inhibition of the JNK pathway.
The
Sirt1 gene is known to be a major contributor to the metabolic regulation of a cell via lipid metabolism and insulin secretion [
7]. In the current study, under high glucose conditions, a significant increase in the expression of the
Sirt1 gene was observed after treatment with RB phenolic extracts (
Figure 4). Sun, Zhang [
29] demonstrated that resveratrol improved insulin sensitivity by repressing the protein tyrosine phosphatase (PTP) constitute and PTP
1B transcription at the chromatin level (on the
Sirt1 gene) under normal versus insulin-resistant conditions. Hence, it is believed that upregulation of the
Sirt1 gene as a result of treatment with RB phenolics can potentially target PTP
1B ranscription consequently improving insulin sensitivity.
Any disruption to the
Tfam gene in the pancreatic β-cell is known to result in impaired insulin secretion, reduced β-cell mass, and, consequently, the development of T2DM [
8]. The current study shows a significant increase in the expression of the
Tfam gene under normal and high glucose conditions post-treatment with RB phenolic extracts (
Figure 5). In an in vivo study where rats were gavaged with pterostilbene,
Tfam gene expression was significantly increased in addition to improvements to glycaemic control and insulin resistance [
30]. Furthermore, the treatment of INS-1E cells with resveratrol also displayed marked potentiation of glucose-stimulated insulin secretion as a result of the up-regulation of
Tfam [
21]. From the above studies, it is believed that RB phenolics have the potential to enhance the efficiency of mitochondrial function via interaction with transcription factors such as
Tfam.
Appropriate regulation of the
Ins1 gene is essential for central insulin signalling as it is an anorectic gene that encodes for the production of the insulin hormone that plays a vital role in the regulation of carbohydrate and lipid metabolism [
31]. Chronic exposure to high glucose conditions can reduce the expression of the
Ins1 gene in β-cells and is often accompanied by the decreased binding activity of the β-cell-specific transcription factor,
Pdx1 [
32]. In the current study, although there was no significant increase in
Ins1 gene expression after RB extract treatment under normal glucose conditions, the expression of the
Ins1 gene was significantly upregulated under high glucose conditions (
Figure 6). Similarly, an in vivo study by the author of [
33] also demonstrated blueberry-leaf extract rich in chlorogenic acid and flavonol glycosides attenuates glucose homeostasis and improves pancreatic β-cell function by increasing the expression of several genes including
Ins1. Polyphenols present in common spices, such as cinnamon, cloves, turmeric, and bay leaves, due to their doubly-linked procyanidin type-A polymers, have also shown an insulin-like activity in vitro [
34]. The mechanism of cinnamon’s insulin-like activity may be in part due to increases in the amounts of insulin receptor β and
Glut4 expression [
34]. Some of the polyphenols present in cinnamon include caffeic, ferulic,
p-coumaric, protocatechuic, and vanillic acids [
35], a similar phenolic profile observed in the RB samples used in this study [
18]. Therefore, it is likely that the effects observed in this study may be due to the insulin-like activity displayed by the polyphenols present in RB individually or via synergistic bioactivity.
Hormones such as insulin and amylin are co-secreted by β-cells in a fixed molecular ratio that provides circulating energy in the form of glucose and stored energy in the form of visceral adipose tissue [
36]. However, conditions such as obesity, T2DM, and pancreatic cancer result in an increase in the amount of amylin relative to the insulin, which can disturb the regulation of energy homeostasis [
36]. It was observed that under normal and high glucose-induced conditions, RB phenolic extracts significantly increased glucose-stimulated insulin secretion (
Figure 7). Bhattacharya, Oksbjerg [
15] also observed a similar trend where caffeic acid, naringenin, and quercetin significantly increased glucose-stimulated insulin secretion under hyperglycaemic and glucotoxic conditions in INS-1E cells. Similarly, several other phenolic compounds such as ferulic acid [
37] and
p-coumaric acid [
38] have also been shown to increase insulin secretion both in vitro and in vivo, respectively. In this study, it was observed that RB phenolic compounds increase the expression of both the
Ins1 gene and the secretion of insulin in INS-1E cells under high glucose conditions. Since the
Ins1 gene is known to encode for the production of insulin hormone, this may indicate that there may be a correlation between insulin secretion and the expression of the
Ins1 gene.
Furthermore, it was observed that lower doses of the RB extract used in this study favourably modulated β-cell function associated gene expression and insulin secretion when compared to the higher doses in vitro. This phenomenon may be explained through the effect of hormesis, a biphasic dose-response to an environmental agent, wherein glucose-stimulated insulin secretion was observed to have a stimulatory or beneficial effect at low doses and an inhibitory or toxic effect at high doses of RB extract [
39]. Dietary polyphenols are known to have strong cytoprotective effects, however, the hormetic role of dietary antioxidants in free radical-related diseases have demonstrated that under uncontrolled nutritional supplementation, gene induction effects and the interaction with detoxification responses can result in a negative response by generating more reactive and harmful intermediates [
40].
As a result of hindrance by cereal matrices, most of the bound phenolic compounds present in cereal grains are usually not readily accessible by digestive enzymes, leading to low bioavailability [
41]. Studies have demonstrated that this could be improved by increasing their accessibility through suitable processing techniques, for example, thermal treatments [
18,
41]. The RB sample examined in this study was previously studied with respect to several thermal treatments. Of the treatments studied, drum drying resulted in the optimal antioxidant activity and was therefore selected for the current investigation [
18]. The drum-dried RB samples resulted in a total free phenolic content of 362.17 ± 34.16 gallic acid equivalent (GAE)/100 g of RB with antioxidant activity of 975.33 ± 20.24 Fe
2+/100 g of RB and a total bound phenolic content of 160.65 ± 5.52 GAE/100 g of RB with antioxidant activity of 551.91 ± 8.82 Fe
2+/100 g of RB. This was much higher compared to that of a non-treated sample that had a total free phenolic content of 238.26 ± 30.34 GAE/100 g of RB with antioxidant activity of 621.76 ± 26.76 Fe
2+/100 g of RB and a total bound phenolic content of 222.94 ± 3.74 GAE/100 g of RB with antioxidant activity of 712.37 ± 14.57 Fe
2+/100 g of RB [
18].