Effect of Bitter-leaf (Vernonia amygdalina) Flavored Non-alcoholic Wheat Beer (NAWB) on, Insulin and GLUT-2 expression in Pancreas of High fat diet/Streptozotocin (HFD/STZ) Induced Diabetic Wistar Rats

Gbenga P Akerele; Bukola C Adedayo; Ganiyu Oboh; Opeyemi B Ogunsuyi

doi:10.1007/s40200-023-01216-2

. 2023 Mar 25;22(1):873–880. doi: 10.1007/s40200-023-01216-2

Effect of Bitter-leaf (Vernonia amygdalina) Flavored Non-alcoholic Wheat Beer (NAWB) on, Insulin and GLUT-2 expression in Pancreas of High fat diet/Streptozotocin (HFD/STZ) Induced Diabetic Wistar Rats

Gbenga P Akerele ^1,^✉, Bukola C Adedayo ¹, Ganiyu Oboh ¹, Opeyemi B Ogunsuyi ^1,²

PMCID: PMC10225370 PMID: 37255795

Abstract

Purpose

In the management of type 2 diabetes (T2D), dietary intervention has been proposed to be highly effective. This study, therefore, investigated the effect of bitter leaf-flavored non-alcoholic wheat beer (NAWB) on insulin secretion and GLUT-2 expression in the pancreas of STZ-induced diabetic rats.

Methods

In this study, the rats received a single intraperitoneal injection (i.p.) of STZ (35 mg/kg) after being fed a high-fat diet for 14 days to induce T2D. The rats were treated with bitter leaf flavored NAWB samples (100%HP- Hops only, 100%BL-Bitter leaf only, 75,25BL- 75% Hops, 25% Bitter Leaf, 50:50BL- 50% Hops:50% Bitter Leaf, and 25:75BL-25%Hops:75% Bitter Leaf) and Acarbose for 14 days. The superoxide dismutase, catalase, and glutathione peroxidase activity were also determined.

Results

The results from this study showed a correlation between GLUT-2 and Insulin expression. There was an upregulation of Insulin as GLUT-2 expression was upregulated. Furthermore, the treated groups showed better antioxidant activity when compared with the diabetic control.

Conclusion

Bitter leaf-flavored NAWB might thus be a good dietary intervention for type 2 diabetics.

Keywords: Diabetes Mellitus Type 2 (T2D), Glucose Transporter Type 2 (GLUT-2), Insulin, Antioxidant, Non-alcoholic beer

Introduction

Diabetes mellitus is a chronic disease that occurs due to raised levels of blood glucose [1]. Insulin is produced in the pancreas as an essential hormone that regulates blood glucose. A deficiency in its production by the body, or the body’s insensitivity to it, therefore, causes elevated blood glucose/hyperglycemia [2]. Insulin allows glucose uptake from the bloodstream into the cell, where it’s either converted into energy or stored. Type 2 diabetes (T2D) results from the ineffective use of insulin by the body. A larger percentage (95%) of diabetics suffer from T2D [3]. Glucose transporter −2 (GLUT-2) is a glucose transporter that plays a key role in the glucose-stimulated secretion of insulin from pancreatic β-cells and glucose metabolism in hepatocytes [4]. In the pancreatic β-cells, GLUT-2 acts as a glucose sensor that detects small changes in glucose levels leading to increased insulin secretion [5]. Oxidative stress has also been implicated in the pathogenesis of T2D as it has been reported to increase resistance to or the impairment of its secretion altogether [6]. In a bid to counter the effect of oxidative stress in T2D pathogenesis, research into the role of antioxidants has also been given priority.

The rapidly increasing prevalence of T2D has necessitated the need for continuous research into novel methods for the prevention, management, and treatment of this epidemic. One major approach in combating this chronic disease has been through dietary manipulation and intervention [7]. Research has revealed several plants and food products that are beneficial in the management of this global epidemic [8]. One such plant that has been researched severally for its efficacy in the management of T2D is bitter leaf (Vernonia amygdalina). V. amygdalina has been reported to lower blood glucose, and inhibit starch hydrolyzing enzymes and lipolysis [9]. Further research has also shown food products fortified with bitter leaf as effective means of dietary intervention in the management of T2D. One such product that we recently reported is non-alcholoic beer flavored with bitter leaf [10]. Beer is one of the oldest most consumed drinks globally known to man. It is typically made from malted cereals, hops, brewer’s yeast, and water [11]. Several reports have shown beer to be effective in stimulating insulin secretion and thus glucose reduction. However, the alcoholic content of beer has been tagged as a health risk to diabetics as it causes a rapid reduction in glucose leading to hypoglycemia in diabetics [12]. Acarbose is a synthetic antidiabetic drug that acts by inhibiting α-amylase and α-glucosidase activities, thus controlling the levels of blood glucose [13]. There is however a dearth of report on whether Acarbose acts beyond the inhibition of the activities of α-amylase and α-glucosidase at the gene level in managing T2D. While previous studies have reported production of non-alcoholic beer, there is dearth of information on their possible antidiababetic properties. Here, we present a novel approach at producing an antidiabetic alcohol-free beer flavored with bitter leaf (with reported antidiabetic properties) and evaluated their biochemical and molecular mechanisms of action by investigating its effect on NAWB on the antioxidant status, GLUT-2, and Insulin expression in the pancreas of STZ-induced diabetic rats It is hypothesized that this beer will exhibit significant antidiabetic properties due to non-alcoholic content and bitter leaf flavoring.

Materials and methods

Sample collection

Wheat was purchased from Oja –Oba, Akure, Ondo State, Southwest Nigeria while hop was obtained from a reputable brewery in Ilesha, Osun state, Southwest Nigeria. The hops were stored at 4 °C before use. Bitter leaf was sourced from Akure Metropolis and authenticated at the herbarium, CERAD, FUTA with voucher number FUTA/BIO/201. It was air-dried to a constant weight and pulverized. It was stored at room temperature in an airtight container for subsequent use.

Chemicals and reagents

All Chemicals and reagents used were of analytical grade. Streptozocin (STZ) (Art. No. S)-130), was procured from Sigma-Aldrich, Inc. (St Louis, MO, USA). Ethanol, acetic acid, sulfuric acid, sodium carbonate, methanol, potassium acetate, perchloric acid, phenol, and sodium hydroxide were sourced from BDH Chemicals Ltd., (Poole, Dorset, UK. Acarbose was procured from Matador Pharmaceutical Ltd. (Akure, Ondo State, Nigeria).

Beer brewing

With slight changes, the process outlined by Adenuga et al. [14] was used to create malted wheat. The wheat is steeped in water at 20 °C for 48 hours and the water is discarded every 8 hours. The wheat was germinated for 5 days on sterile trays covered with polythene nylon before being baked in a hot air oven at 70^oCelsius. The grain was cleaned of the rootlets, ground into a coarse texture, and kept at room temperature in sealed containers. The crushed malt was then placed into a strainer together with 5 L of warm water heated to 60 °C, and steeped for 30 minutes. The sieve was taken out to remove the used grit once the wort had come to a boil. In proportions of 100%, 75:25, 50:50, and 25:75 of hops to bitter leaf and 100% bitter leaf ratio, a portion of hop pellets and bitter leaf powder were added to the wort, and the remainder was added 10 minutes before the end of a 2-hour boil. Warm water was used to fill the wort to its initial capacity, and it was then cooled to 25 °C. Brewer’s yeast 5 g were added, and it was left to ferment for 14 days. After 14 days, the alcoholic content of the fermented beer was removed by distillation at 80^oCelsius, and the alcohol level was then determined using an alcohol meter. Thereafter, the beer was bottled and left to age for three months.

Animals

Wistar strain of Albino rats (Male; 180–240 g) was procured from the Animal House, Department of Biochemistry, Federal University of Technology, Akure. The experimental animals were acclimatized under standard laboratory conditions; water was given ad libitum with 12-h light and 12-h dark cycle.

Animal care and handling

The National Institutes of Health (NIH) manual was followed to the letter when handling the experimental animals. The FUTA-Committee for the Ethical Use of Research Animals, CERAD, Federal University of Technology Akure, accepted the protocols used for the animal study, and they were awarded the certificate number FUTA/ETH/20/25. The experiment was conducted at the Functional Foods and Nutraceuticals Unit of the Department of Biochemistry at the Federal University of Technology in Akure, Nigeria.

Induction of diabetes with STZ in HFD-fed Wistar rats (type 2 diabetic rat model)

Diabetes was induced using a modified procedure by Srinivasan and Ramarao [15]. Before giving adult Wistar rats a tailored diet, commercially available rat pellets (containing 15% crude protein, 4% crude fat, 1.1% crude fiber, 0.7% calcium, and 0.7% phosphorus) were given to the rats at Farm Support in Akure. Following acclimation, the rats were divided into two diet groups: the normal control (NC) and the high-fat diet (HFD). To induce diabetes in the rats fed the HFD, STZ was intraperitoneally injected into the rats after 2 weeks of dietary manipulation at a single dose of 35 mg kg⁻¹ body weight. After 72 hours of induction, the rats’ blood glucose levels were measured by tail vein puncture, and the fasting blood glucose levels were determined using an automatic auto-analyzer (Fine-test Auto-coding™). In the investigation, rats with fasting blood glucose levels ≥200 mg dL-1 were used. Intraperitoneally, 1 mL of 0.1molL citrate buffer was administered to the control group. Eight groups of five rats each were formed by randomly dividing the diabetic rats.

The various groups were then subjected to various treatment plans once per day for 14 days using the beer samples. The rats were grouped as follows:

Group I: Normal control (citrate buffer pH 4.5; 1 mL kg⁻¹ intraperitoneally). - NC
Group II: Type-2 diabetic control group - DC
Group III: Type-2 diabetic rats administered 25 mg kg⁻¹ body weight oral dose of Acarbose - Acarbose
Group IV: Type-2 diabetic rats administered 15 ml kg⁻¹ body weight oral dose of beer samples flavored with only hops - HP
Group V: Type-2 diabetic rats administered 15 ml kg⁻¹ body weight oral dose of beer samples flavored with only bitter leaf. - BL
Group VI: Type-2 diabetic rats administered 15 ml kg⁻¹ body weight oral dose of beer samples flavored with 75% hops and 25% bitter leaf - 75:25BL
Group VII: Type-2 diabetic rats administered 15 ml kg⁻¹ body weight oral dose of beer samples flavored with 50% hops and 50% bitter leaf – 50:50BL
Group VIII: Type-2 diabetic rats administered 15 ml kg⁻¹ body weight oral dose of beer samples flavored with 25% hops and 75% bitter leaf – 25:75 BL

Preparation of tissue homogenates

After 14 days of treatment, the rats were euthanized by cervical dislocation. The pancreas was isolated and rapidly placed on ice and weighed. This was subsequently homogenized in phosphate buffer (0.1 M, pH 7.4) with about ten up-and-down strokes at approximately 1200 rpm in a Teflon glass homogenizer. The homogenates were centrifuged for 10 min at 3000 rpm to yield pellets that were discarded and supernatant kept for analysis.

Estimation of superoxide dismutase (SOD) activity

The Fridovich method [16] was used to determine SOD activity. Briefly, 50 μL of samples were pipetted into test tubes and 1000 μL of 50 mM carbonate buffer was added to equilibrate in the spectrophotometer. The reaction started with the addition of 17 μL of freshly prepared 0.3 mM adrenaline to the mixture which was quickly mixed by inversion. Absorbance was read at 480 nm for 150 seconds at 30 seconds intervals and the activity of SOD was afterward calculated.

Estimation of catalase activity

Catalase activity was determined according to the method of Claiborne [17]. 1 ml of Phosphate buffer, 100 μL of the pancreatic tissue sample, and 400 μL of hydrogen peroxide were pipetted into test tubes and 2 mL of dichromate acetic acid was added at the point of reading. The change in absorbance was read at a wavelength of 620 nm for 3 minutes at minute intervals. The activity of catalase was afterward calculated.

Estimation of glutathione peroxidase (GPx) activity

GPx activity was determined according to the method of Rotruck et al., [18]. A reaction mixture containing 200 μL of 0.4 M phosphate buffer, 100 μL of 10 mM sodium azide (NaN₃), 200 μL of the sample, 200 μL of 10 mM glutathione, and 100 μL of 0.2 mM hydrogen peroxide were pipetted into test tubes. The whole mixture was incubated at 37 °C for 10 minutes after which 400 μL of TCA was added and thereafter centrifuge at 3000 g for 20 minutes. To 1 mL of each of the supernatant, 500 μL of Ellman’s reagent and 3 mL of phosphate buffer (0.2 M, pH 8.0) was added and the absorbance was read at 412 nm against the blank (2 ml of K₂HPO₄ + 1 ml of DTNB).

Lipid peroxidation and thiobarbituric acid reactions

The modified method of Ohkawa et al. [19] was used to perform the lipid peroxidation experiment. A reaction mixture containing 100𝜇L of the supernatant was combined with 30𝜇L of freshly generated 250 mM FeSO₄ and 30𝜇L of extract (0–100𝜇L). This combination was then incubated at 37 °C for 1 h. The reaction was started by adding 300𝜇L of 8.1% sodium dodecyl sulfate (SDS) to the mixture. Next, 500𝜇L of acetic acid (pH 3.4) and 500𝜇L of 0.6% TBA were added to the mixture. The production of thiobarbituric acid reactive species (TBARS) was measured at 532 nm after this combination was incubated at 100 °C for 1 h.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Using Trizol®, total RNA was isolated from the pancreas of the rats in each group. RNA contained in the samples was quantified using Nanodrop2000™, seen using 1.5% agarose gel, and subjected to DNase I treatment (Invitrogen). Using the iScript™ cDNA Synthesis kit, 1 μg of cDNA was synthesized. Three reference genes (Insulin, GLUT-2 and SOD 1) served as the standard for all expression levels. It took 40 thermal cycles of 15 seconds at 94 °C, 10 seconds at 60 °C, and 30 seconds at 72 °C to complete the reactions, which were carried out in a final volume of 20 μl with 2.5 ng/L of cDNA, 1x PCR buffer, 0.2 M of each primer, 0.2 mM dNTP, 2 mM MgCl₂, 0.1x SYBR® Green, and 0.25 U platinum Taq DNA [20]. To confirm the amplification of a single distinct product per reaction, dissociation curves were obtained using 1 cycle of 94 °C for 10 s, 55 °C for 1 min, and 94 °C for 15 s. The design and analysis program StepOne™ was used using SYBR fluorescence. Three to six separate experiments were run in triplicate to perform the reactions. The 2^−ΔΔCT method of Livak and Schmittgen [21] was used to establish the values of gene expression.

Data analysis

The results of triplicate experiments were pooled and expressed as mean ± standard deviation. Means were compared by one-way analysis of variance followed by Duncan’s multiple range test and least significant differences were carried out using SPSS (IBM version 20.0, SPSS Inc., Quarry bay, Hong kong) and accepted at p < 0.05 [22].

Results

The results for the antioxidant activity of SOD, Catalase, GPx, and MDA are presented in Table 1. The group HP treated with NAWB flavoured with hops alone had the highest expression of SOD activity (6.64 μmol/min/mg protein), when compared to the rest. All the treated groups were significantly different (p < 0.05) from the NC and DC. 50:50BL had the lowest expression of SOD activity (2.08 μmol/min/mg protein). When compared to the NC, the SOD activity in the DC is upregulated, however, several of the treated groups had a significantly different (p < 0.05) elevated activity of SOD higher than the DC. The groups, Acarbose, HP and BL are the only groups that had higher expressed SOD activity when compared to the DC. The trend seen in the expression of SOD activity was also seen in Catalase activity in the groups, HP had the highest expression of catalase activity (15.61 μmol/mg protein) while 50:50BL had the lowest (6.13 μmol/mg protein). Furthermore, the groups Acarbose, HP and BL were the only groups that had a higher expression of Catalase activity when compared to all the groups. The results from this study also indicated that the GPx activity of all the groups were significantly different (P < 0.05) from the DC. The DC had the lowest expression of GPx activity (0.07 U/mg protein). However, there was no significant difference in the GPx activity between the remaining treated groups. Also, although not significantly different (p > 0.05) from the rest of the groups asides BL, the trend noticed in the expression of SOD and Catalase activity was also seen where the groups Acarbose, HP and BL had the highest expressed GPx activity (0.17, 0.17, 0.15 U/mg protein). The DC group showed the highest level (5.77 mm/g protein) of the lipid peroxidation marker, TBARS produced in the MDA assay. All the treated groups had significantly different (P < 0.05) levels of TBARS produced when compared to the DC.

Table 1.

SOD, CATALASE, GPx, and MDA levels in the pancreas of diabetic rats

SAMPLE	SOD (μmol/min/mg protein)	Catalase (μmol/mg protein)	GPx (U/mg protein)	MDA (mm/g protein)
NC	2.78 ± 0.69^a	6.79 ± 1.23^a	0.13 ± 0.00^b	2.94 ± 0.04^a
DC	3.86 ± 0.32^b	9.01 ± 2.08^b	0.07 ± 0.01^a	5.77 ± 1.57^c
Acarbose	3.57 ± 0.89^b	9.33 ± 1.15^b	0.17 ± 0.01^b	3.47 ± 0.39^b
HP	6.64 ± 0.84^c	15.61 ± 3.80^c	0.17 ± 0.02^b	3.40 ± 0.13^b
BL	4.13 ± 1.11^b	11.28 ± 3.08^c	0.15 ± 0.02^b	2.15 ± 0.42^a
75:25 BL	3.52 ± 1.14^b	6.51 ± 0.30^a	0.12 ± 0.00^b	2.08 ± 0.21^a
50:50 BL	2.08 ± 1.72^a	6.13 ± 0.42^a	0.11 ± 0.00^b	2.11 ± 0.26^a
25:75 BL	3.19 ± 1.00^a	7.32 ± 0.87^a	0.11 ± 0.01^b	2.94 ± 0.37^a

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Mean (±SEM) with different alphabetical superscripts in the same column are significantly different at p < 0.05

Key: NC Normal Control, DC Diabetic control, Acarbose = STZ+ Acarbose HP = STZ+ 100% Hops alone; 100%BL = STZ + Bitter Leaf alone; 75:25BL = STZ + 75% Hops: 25% Bitter Leaf; 50:50BL = STZ + 50% Hops: 50% Bitter Leaf; 25:75BL = STZ + 25% Hops: 75% Bitter Leaf

At the molecular level, Fig. 1 shows the SOD activity in the pancreas of the diabetic rats treated with NAWB. The NC had the lowest SOD activity expressed. However, all the treated groups except HP were significantly different in the expression of SOD activity when compared to the DC group.

Fig. 2 shows the GLUT-2 expression in the pancreas of diabetic wistar rats treated with NAWB. 75:25BL had the highest expressed GLUT-2 significantly different from DC. The study revealed that the higher the percentage inclusion of BL in the NAWB, the lower the expression of GLUT-2; with group BL having the lowest GLUT-2 expression. Fig. 3 showing the expression of insulin however does not reveal any significant difference (p > 0.05) however, it follows the trend of the GLUT-2 expression among all the groups.

Fig. 3 — Effect of NAWB on mRNA expression of Insulin in the pancreas of Diabetic rats. Bars represent mean ± standard deviation (n = 5); *^#Values are not significantly (p > 0.05) different from normal and diabetic control group; Key: NC = Normal Control; DC = Diabetic control; Acarbose = STZ+ Acarbose HP = STZ+ 100% Hops alone; 100%BL = STZ + Bitter Leaf alone; 75:25BL = STZ + 75% Hops: 25% Bitter Leaf; 50:50BL = STZ + 50% Hops: 50% Bitter Leaf; 25:75BL = STZ + 25% Hops: 75% Bitter Leaf

Discussion

In cells, the first line of defense against oxidative assault is the antioxidant enzymes present in such cells [23]. However, there is a low expression and activity of antioxidant enzymes in pancreatic β- cells thus making them prone to oxidative damage [24]. Superoxide dismutase (SOD) an antioxidant enzyme catalyzes the conversion of superoxide anions to hydrogen peroxide (H₂0₂) which is subsequently detoxified by catalase or glutathione peroxidase to oxygen and water [25]. In this study, the diabetic rats showed an up-regulation in the expression of SOD in the pancreas. Studies have reported overexpression of SOD to be toxic to the pancreas as it catalyzes a continuous production of H₂0₂ which could overwhelm catalase that detoxifies H₂0₂ into water and oxygen [26]. The up-regulation of SOD in diabetic rats might thus be a mechanism through which the body is raising its defense against oxidative stress. Catalase activity also shows a positive correlation with the SOD activity as it was also up-regulated in the diabetic rats as evidenced in this study. The upregulation of Catalase might be a way to counter the oxidative damage that H₂0₂ might cause to the pancreatic cells. This observation is further reinforced in this study as the group treated with NAWB flavored only with hops that has the highest expressed SOD activity also had the highest expressed Catalase activity. This upregulation of SOD and catalase is in tandem with previous reports that suggested that there is an upregulation of antioxidant enzymes in diabetic rats as an adaptive/defense mechanism [27, 28]. However, in this study, the treated groups also showed an up-regulation in SOD activity. The up-regulation of the antioxidant enzymes SOD and Catalase however is in negative correlation to the glutathione peroxidase (GPx) activity. Whereas the treated group showed an upregulation of the GPx activity, the diabetic group showed a downregulation. The downregulation of GPx activity in the diabetic group might be a reason why the adaptive/defense mechanism of the cell against oxidative stress in diabetic rats collapses. Continuous production of H₂0₂ by an upregulated SOD activity could have been responsible for the upregulation of Catalase activity in the treated groups. An increase in the GPx activity in the treated groups might also be a way to prevent catalase from being overwhelmed. In the diabetic groups, however, the downregulation of GPx activity could be an indicator of how the natural defense mechanism of the cell collapses in diabetic patients. The low level of lipid peroxidation marker, TBARS in the treated samples, however, shows that the upregulation of SOD, Catalase, and GPx is a response to protect against oxidative stress. Furthermore, the high level of TBARS observed in the untreated diabetic rats showed that the upregulation of the antioxidant enzymes is an adaptive/defense mechanism in combating oxidative stress. Diabetes has been reported to cause a breakdown in the cells defense against oxidative damage; however, this study revealed that in responding to this oxidative damage, these samples upregulates both SOD and Catalase activity in diabetic rats treated with NAWB thus preventing a situation where the constant production of H₂O₂ will overwhelm Catalase in managing oxidative assault arising from hyperglycemia. Furthermore, in the treated groups, GPx activity was upregulated. This might further reveal a mechanism of action of the NAWB in ensuring that excess H₂O₂ arising from the overexpression of SOD that may likely overwhelm Catalase is being taken care of by GPx.

Hyperglycemia frequently results in increased ROS production and impaired antioxidant defenses because chronic hyperglycemia encourages the formation of ROS from multiple sources [29–31]. When blood sugar levels are raised, the endothelial cells produce more O₂⁻. Glyceraldehyde-3-phosphate dehydrogenase, a crucial enzyme in the glycolytic process, has been reported to be inhibited by an excess of O₂⁻. This results in a switch to other alternative glucose metabolism routes, the buildup of glucose and other intermediate metabolites of this pathway, and an increase in the synthesis of advanced glycation end products [32, 33]. SOD catalyzes the conversion of O₂⁻ to H₂O₂, therefore in response to raised levels of O₂⁻, there is an upregulation of SOD as an adaptive/defense mechanism. The result from this study showed the mRNA expression of the SOD gene to be upregulated in diabetic rats which corresponds to the pattern recorded in in vivo assay for SOD activity. However, in the animal groups treated with acarbose and bitter leaf-flavored beer, there was a significant upregulation of the SOD gene when compared to the diabetic control. This clearly shows that the SOD upregulation was beyond just an adaptive mechanism but rather a defense mechanism against oxidative damage arising from hyperglycemia.

A crucial part of the glucose-stimulated insulin production from pancreatic beta-cells and glucose metabolism in hepatocytes is played by the glucose transporter GLUT-2. GLUT-2 functions as a glucose sensor in pancreatic beta-cells, detecting even minute variations in blood glucose levels and triggering an increase in insulin release [5]. This study shows a relationship between the mRNA expression of GLUT-2 and Insulin expression. Although there was no significant difference in the expression of Insulin among the groups, there was however an expression of Insulin among the groups that followed the pattern with which GLUT-2 was expressed. Among the groups treated with various inclusions of bitter leaf flavored NAWB, the group (75:25BL) with the highest level of expressed mRNA of GLUT-2 had the highest level of Insulin expressed while 25:75BL which had the lowest expression of GLUT-2 also had the lowest expression of Insulin. This is in line with previous reports [34, 35] that have indicated that the sensory function of GLUT-2 to variations in blood glucose levels is responsible for the secretion of insulin in response to the blood glucose. An upregulated expression of GLUT-2, therefore, leads to an upregulated expression of Insulin. This study has shown the antioxidative potentials and effect of bitter leaf flavoured NAWB on GLUT-2 and Insulin expression in diabetic rats. However, lack of immediate clinical evaluation data of the samples in humans could be identified as one of the limitations of this study. Therefore, clinical observations of diabetic patients treated with bitter leaf flavoured NAWB to provide better insight into the potency and mechanisms of action of these samples is highly recommended.

Conclusion

From the results of this study, the group HP also had a good antioxidative potential in managing T2D, however, the samples with bitter leaf inclusions had better performance. This observation thus makes NAWB flavoured with bitter leaf a viable potential intervention in helping diabetics satiate their thirst for their favourite drink while still making it therapeutic. Furthermore, its effect on GLUT-2 and subsequently, Insulin expression at the molecular level shows a possible mechanism through which the beer samples could serve as a dietary intervention in T2D management.

Authors’ contribution

Prof. G. Oboh – Experimental research design.

G. P. Akerele – Beer production, Methodology & Result analysis.

B.C. Adedayo – Result analysis and supervision.

O.B. Ogunsuyi – Methodology and PCR analysis.

Funding

This research is funded by NRF-Tertiary Education Trust Fund (TETFUND). Grant number: TETF/ES/DR&D-CE/NRF 2020/SETI/40/VOL. 1.

Data availability

Datasets for this study will only be made available on request.

Declarations

Competing interests

We declare that there are no competing interests either financial or non-financial for this research.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Gbenga P. Akerele, Email: akereleg@gmail.com

Bukola C. Adedayo, Email: chrisadeb2013@yahoo.com

Ganiyu Oboh, Email: goboh2001@yahoo.com.

Opeyemi B. Ogunsuyi, Email: obogunsuyi@futa.edu.ng

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16.Fridovich I. Superoxide dismutases: an adaptation to a paramagnetic gas. J Biol Chem. 1989;264(14):7761–7764. doi: 10.1016/S0021-9258(18)83102-7. [DOI] [PubMed] [Google Scholar]
17.Claiborne AL. CRC handbook of methods for oxygen radical research. CRC press; 2018. Catalase activity; pp. 283–284. [Google Scholar]
18.Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra W. Selenium: biochemical role as a component of glutathione peroxidase. Science. 1973;179(4073):588–590. doi: 10.1126/science.179.4073.588. [DOI] [PubMed] [Google Scholar]
19.Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351–358. doi: 10.1016/0003-2697(79)90738-3. [DOI] [PubMed] [Google Scholar]
20.Olagoke OC, Afolabi BA, Rocha JB. Streptozotocin induces brain glucose metabolic changes and alters glucose transporter expression in the Lobster cockroach; Nauphoeta cinerea (Blattodea: Blaberidae) Mol Cell Biochem. 2021;476(2):1109–1121. doi: 10.1007/s11010-020-03976-4. [DOI] [PubMed] [Google Scholar]
21.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25(4):402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
22.Zar JH. Simple linear regression. Biostat Analysis. 1984;4:324–359. [Google Scholar]
23.Ighodaro OM, Akinloye OA. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alexandria J Med. 2018;54(4):287–293. doi: 10.1016/j.ajme.2017.09.001. [DOI] [Google Scholar]
24.Wang J, Wang H. Oxidative stress in pancreatic beta cell regeneration. Oxid Med Cell Longev. 2017;2017 [DOI] [PMC free article] [PubMed]
25.Andrés CM, de la Lastra JMP, Juan CA, Plou FJ, Pérez-Lebeña E. Chemistry of Hydrogen Peroxide Formation and Elimination in Mammalian Cells, and Its Role in Various Pathologies. Stresses. 2022;2(3):256–274. doi: 10.3390/stresses2030019. [DOI] [Google Scholar]
26.Gardner R, Salvador A, Moradas-Ferreira P. Why does SOD overexpression sometimes enhance, sometimes decrease, hydrogen peroxide production? A minimalist explanation. Free Radic Biol Med. 2002;32(12):1351–1357. doi: 10.1016/S0891-5849(02)00861-4. [DOI] [PubMed] [Google Scholar]
27.Sahana A, Agarwal S. Carbaryl and Human Health: A Review. J Sci. 2018;2(5)
28.Harikrishnan R, Devi G, Van Doan H, Balasundaram C, Esteban MÁ, Abdel-Tawwab M. Impact of grape pomace flour (GPF) on immunity and immune-antioxidant-anti-inflammatory genes expression in Labeo rohita against Flavobacterium columnaris. Fish Shellfish Immunol. 2021;111:69–82. doi: 10.1016/j.fsi.2021.01.011. [DOI] [PubMed] [Google Scholar]
29.Yan LJ. Pathogenesis of chronic hyperglycemia: from reductive stress to oxidative stress. J Diabetes Res. 2014;2014 [DOI] [PMC free article] [PubMed]
30.McLellan AC, Thornalley PJ, Benn J, Sonksen PH. Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin Sci. 1994;87(1):21–29. doi: 10.1042/cs0870021. [DOI] [PubMed] [Google Scholar]
31.Saxena AK, Srivastava P, Kale RK, Baquer NZ. Impaired antioxidant status in diabetic rat liver: effect of vanadate. Biochem Pharmacol. 1993;45(3):539–542. doi: 10.1016/0006-2952(93)90124-F. [DOI] [PubMed] [Google Scholar]
32.Graier WF, Posch K, Fleischhacker E, Wascher TC, Kostner GM. Increased superoxide anion formation in endothelial cells during hyperglycemia: an adaptive response or initial step of vascular dysfunction? Diabetes Res Clin Pract. 1999;45(2–3):153–160. doi: 10.1016/S0168-8227(99)00045-5. [DOI] [PubMed] [Google Scholar]
33.Du X, Matsumura T, Edelstein D, Rossetti L, Zsengellér Z, Szabó C, Brownlee M. Inhibition of GAPDH activity by poly (ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J Clin Invest. 2003;112(7):1049–1057. doi: 10.1172/JCI18127. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Fournel A, Marlin A, Abot A, Pasquio C, Cirillo C, Cani PD, Knauf C. Glucosensing in the gastrointestinal tract: impact on glucose metabolism. Am J Physiol. 2016;310(9):G645–G658. doi: 10.1152/ajpgi.00015.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Holst JJ, Gribble F, Horowitz M, Rayner CK. Roles of the gut in glucose homeostasis. Diabetes Care. 2016;39(6):884–892. doi: 10.2337/dc16-0351. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Datasets for this study will only be made available on request.

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[CR15] 15.Srinivasan K, Ramarao P. Animal model in type 2 diabetes research: An overview. Indian J Med Res. 2007;125(3):451. [PubMed] [Google Scholar]

[CR16] 16.Fridovich I. Superoxide dismutases: an adaptation to a paramagnetic gas. J Biol Chem. 1989;264(14):7761–7764. doi: 10.1016/S0021-9258(18)83102-7. [DOI] [PubMed] [Google Scholar]

[CR17] 17.Claiborne AL. CRC handbook of methods for oxygen radical research. CRC press; 2018. Catalase activity; pp. 283–284. [Google Scholar]

[CR18] 18.Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra W. Selenium: biochemical role as a component of glutathione peroxidase. Science. 1973;179(4073):588–590. doi: 10.1126/science.179.4073.588. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351–358. doi: 10.1016/0003-2697(79)90738-3. [DOI] [PubMed] [Google Scholar]

[CR20] 20.Olagoke OC, Afolabi BA, Rocha JB. Streptozotocin induces brain glucose metabolic changes and alters glucose transporter expression in the Lobster cockroach; Nauphoeta cinerea (Blattodea: Blaberidae) Mol Cell Biochem. 2021;476(2):1109–1121. doi: 10.1007/s11010-020-03976-4. [DOI] [PubMed] [Google Scholar]

[CR21] 21.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25(4):402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]

[CR22] 22.Zar JH. Simple linear regression. Biostat Analysis. 1984;4:324–359. [Google Scholar]

[CR23] 23.Ighodaro OM, Akinloye OA. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alexandria J Med. 2018;54(4):287–293. doi: 10.1016/j.ajme.2017.09.001. [DOI] [Google Scholar]

[CR24] 24.Wang J, Wang H. Oxidative stress in pancreatic beta cell regeneration. Oxid Med Cell Longev. 2017;2017 [DOI] [PMC free article] [PubMed]

[CR25] 25.Andrés CM, de la Lastra JMP, Juan CA, Plou FJ, Pérez-Lebeña E. Chemistry of Hydrogen Peroxide Formation and Elimination in Mammalian Cells, and Its Role in Various Pathologies. Stresses. 2022;2(3):256–274. doi: 10.3390/stresses2030019. [DOI] [Google Scholar]

[CR26] 26.Gardner R, Salvador A, Moradas-Ferreira P. Why does SOD overexpression sometimes enhance, sometimes decrease, hydrogen peroxide production? A minimalist explanation. Free Radic Biol Med. 2002;32(12):1351–1357. doi: 10.1016/S0891-5849(02)00861-4. [DOI] [PubMed] [Google Scholar]

[CR27] 27.Sahana A, Agarwal S. Carbaryl and Human Health: A Review. J Sci. 2018;2(5)

[CR28] 28.Harikrishnan R, Devi G, Van Doan H, Balasundaram C, Esteban MÁ, Abdel-Tawwab M. Impact of grape pomace flour (GPF) on immunity and immune-antioxidant-anti-inflammatory genes expression in Labeo rohita against Flavobacterium columnaris. Fish Shellfish Immunol. 2021;111:69–82. doi: 10.1016/j.fsi.2021.01.011. [DOI] [PubMed] [Google Scholar]

[CR29] 29.Yan LJ. Pathogenesis of chronic hyperglycemia: from reductive stress to oxidative stress. J Diabetes Res. 2014;2014 [DOI] [PMC free article] [PubMed]

[CR30] 30.McLellan AC, Thornalley PJ, Benn J, Sonksen PH. Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin Sci. 1994;87(1):21–29. doi: 10.1042/cs0870021. [DOI] [PubMed] [Google Scholar]

[CR31] 31.Saxena AK, Srivastava P, Kale RK, Baquer NZ. Impaired antioxidant status in diabetic rat liver: effect of vanadate. Biochem Pharmacol. 1993;45(3):539–542. doi: 10.1016/0006-2952(93)90124-F. [DOI] [PubMed] [Google Scholar]

[CR32] 32.Graier WF, Posch K, Fleischhacker E, Wascher TC, Kostner GM. Increased superoxide anion formation in endothelial cells during hyperglycemia: an adaptive response or initial step of vascular dysfunction? Diabetes Res Clin Pract. 1999;45(2–3):153–160. doi: 10.1016/S0168-8227(99)00045-5. [DOI] [PubMed] [Google Scholar]

[CR33] 33.Du X, Matsumura T, Edelstein D, Rossetti L, Zsengellér Z, Szabó C, Brownlee M. Inhibition of GAPDH activity by poly (ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J Clin Invest. 2003;112(7):1049–1057. doi: 10.1172/JCI18127. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Fournel A, Marlin A, Abot A, Pasquio C, Cirillo C, Cani PD, Knauf C. Glucosensing in the gastrointestinal tract: impact on glucose metabolism. Am J Physiol. 2016;310(9):G645–G658. doi: 10.1152/ajpgi.00015.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Holst JJ, Gribble F, Horowitz M, Rayner CK. Roles of the gut in glucose homeostasis. Diabetes Care. 2016;39(6):884–892. doi: 10.2337/dc16-0351. [DOI] [PubMed] [Google Scholar]

PERMALINK

Effect of Bitter-leaf (Vernonia amygdalina) Flavored Non-alcoholic Wheat Beer (NAWB) on, Insulin and GLUT-2 expression in Pancreas of High fat diet/Streptozotocin (HFD/STZ) Induced Diabetic Wistar Rats

Gbenga P Akerele

Bukola C Adedayo

Ganiyu Oboh

Opeyemi B Ogunsuyi

Abstract

Purpose

Methods

Results

Conclusion

Introduction

Materials and methods

Sample collection

Chemicals and reagents

Beer brewing

Animals

Animal care and handling

Induction of diabetes with STZ in HFD-fed Wistar rats (type 2 diabetic rat model)

Preparation of tissue homogenates

Estimation of superoxide dismutase (SOD) activity

Estimation of catalase activity

Estimation of glutathione peroxidase (GPx) activity

Lipid peroxidation and thiobarbituric acid reactions

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Data analysis

Results

Table 1.

Fig. 1.

Fig. 2.

Fig. 3.

Discussion

Conclusion

Authors’ contribution

Funding

Data availability

Declarations

Competing interests

Footnotes

Contributor Information

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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