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Genistein enhances the secretion of glucagon-like peptide-1 (GLP-1) via downregulation of inflammatory responses
Biomedicine & Pharmacotherapy 112 (2019) 108670
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Genistein enhances the secretion of glucagon-like peptide-1 (GLP-1) via downregulation of inflammatory responses Kanwal Rehmana, Mehwish Bagh Alia, Muhammad Sajid Hamid Akashb, a b
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Department of Pharmacy, University of Agriculture, Faisalabad, Pakistan Department of Pharmaceutical Chemistry, Government College University Faisalabad, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: GLP-1 Diabetes mellitus Genistein Metformin Inflammatory responses Incretin hormone
Glucagon-like peptide-1 (GLP-1) an incretin hormone, is known to regulate the glucose-mediated insulin secretion. However, reduction in the level of GLP-1 is considered to be a major cause for the reduction of GLP-1dependent insulin secretory response. Genistein an isoflavone, is an important polyphenol and has wide range of therapeutic potentials, but its therapeutic effects alone and/or in combination with metformin on GLP-1 secretion have not been investigated yet. Hence, we aimed to investigate the stimulatory action of genistein in combination with metformin on GLP-1 via downregulation of inflammatory mediators, hyperlipidemia and hyperglycemia in alloxan-induced diabetic rats. Diabetes was induced in experimental rats by single administration of alloxan intraperitoneally. Metformin (50 mg/kg/day), genistein (20 mg/kg/day) and combination of genistein and metformin was administered in alloxan-induced diabetic rats. We found that genistein alone and/ or in combination with metformin significantly increased the serum level (P < 0.01) and tissue content (P < 0.05) of GLP-1 in intestine when compared with that of metformin-treated animals. Similarly, genistein alone and/or in combination with metformin also resulted in normoglycemia (P < 0.001), glucose tolerance (P < 0.01), insulin sensitivity (P < 0.0001), hyperlipidemia (P < 0.01), liver and kidney function biomarkers (P < 0.01) as compared to that of metformin-treated experimental animals. Moreover, genistein alone and/or in combination with metformin also downregulated the inflammatory responses by decreasing the levels of interleuin-6, tumor necrosis factor-α and C-reactive protein in serum (P < 0.05) and intestine (P < 0.001) more efficiently as compared to that of metformin-treated experimental animals. The downregulation of inflammatory responses in intestine, was positively associated with increased secretion of GLP-1 from intestine. Histopathology of pancreas and intestine also showed that genistein significantly improved the deleterious effects of alloxan on pancreas and intestine. Hence, our work provides new insights on the synergistic effects of genistein and metformin on GLP-1 secretion. This may significantly improve the perception for proposing new GLP-1-based synergistic approaches for the treatment of diabetes mellitus.
1. Introduction Diabetes mellitus (DM), with its growing occurrence is predictable to affect about 8% of the global population till 2030 [1,2]. DM is a metabolic disease during which glucose homeostasis becomes irregular. Further, it is characterized by impaired carbohydrate metabolism, insulin resistance and impaired insulin secretion [3,4]. The world is facing an estimated lifetime risk of 25% due to growing prevalence of DM. Different factors are considered to be responsible for the cause of insulin resistance and failure of β-cell to secrete insulin on demand. One of them is alteration in the pathways or the factors that regulate the glucose metabolism for instance incretin hormone like glucagon-like peptide-1 (GLP-1) [5,6]. GLP-1 is secreted as an incretin hormone from ⁎
the gut in response to intake of food [7,8] and is known to have number of functions like it potentiates insulin secretion from pancreas and regulates the metabolism of glucose [7,8]. In the past few years, metformin, which is a well-recognized commercially available anti-diabetic drug, has shown to increase the levels of GLP-1 [7,9,10]. Preprandial metformin prescribed in diabetic patients, have shown increase the plasma levels of GLP-1 [11–13]. The results of these studies suggest that GLP-1-specific effects involving L-cells of gut endocrine system may be influenced by the inhibition of dipeptidyl peptidase-IV (DPP-IV) [14] as the inhibition of DPP-IV may reduce the inactivation of GLP-1. It has also been reported that the effects of incretin may be impaired because of unresponsiveness of pancreatic β-cells in diabetic patients [15]. Hence, researchers are now focusing on improving insulin secretion, or
Corresponding author. E-mail addresses: [email protected], [email protected] (M.S.H. Akash).
https://doi.org/10.1016/j.biopha.2019.108670 Received 13 January 2019; Received in revised form 31 January 2019; Accepted 4 February 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.3. Biochemical analysis
maintaining the glucose homeostasis using various therapeutic approaches. Moreover, studies have also suggested that improving the proliferation and survival of β-cells using drugs like GLP-1 receptor agonists might be helpful in treating DM [4,16,17]. Mechanism of actions of GLP-1 including stimulation of insulin secretion, suppression of glucagon secretion, delay in gastric emptying, and inhibition of small bowel motility, have been suggested to contribute for its anti-diabetogenic effects. Thereby, different approaches have been used to focus on the cost-effectiveness of anti-diabetic agents to maintain the glucose homeostasis with improved glucose metabolism. This may help in decreasing the prevalence and burden of DM along with its associated complications globally. Polyphenols present in diet have been considered to play important role in regulating the metabolism of carbohydrates [18–24]. This is done by controlling the postprandial hyperglycemia by reducing the absorption of digested carbohydrates [18–20]. Extracts of different berries having polyphenols have also known to inhibit the level of carbohydrate metabolizing enzymes [25]. Hence, we tried to focus on similar therapeutic approach, which involves the use of natural product being cost-effective, less toxic, and having ability to target the major pathways known for controlling and/or regulating the normal glucose metabolism. For this, we opted to use an isoflavone; genistein (4,5,7trihydroxyisoflavone) that is abundantly found in soybean. However, phytoestrogens are also known as rich source of genistein. Genistein has shown to have therapeutic potential for improving body weight, food intake along with maintaining the adipose tissue mass in ovariectomized mice [26]. Moreover, genistein has also shown to inhibit the synthesis and/or secretion of leptin from adipocytes [27,28]. In addition, genistein has also shown beneficial effects against hyperglycemic state because of its potential of stimulating glucose-mediated secretion of insulin from pancreatic islets [29]. Consistently, in chemically-induced diabetic rats, dietary medications of isoflavones have given better results by decreasing the sugar levels in blood and by releasing the glucose controlling hormone [30,31]. To the best of our knowledge, until now, the potential role of genistein alone and/or in combination with metformin on GLP-1 remains to be elucidated. Therefore, we opted to use genistein and investigated its stimulatory action on GLP-1 via downregulation of inflammatory responses in alloxan-induced diabetic rats.
2.3.1. Estimation of glycemic control biomarkers The effect of treatment on glycemia was estimated on predefined time points. To estimate the effect of treatment on glycemic levels of animals, the fasting blood glucose (FBG) and random blood glucose (RBG) levels were recorded with the help of glucometer twice in a week. To evaluate the tendency of glucose tolerance in experimentallyinduced diabetic animals, oral glucose tolerance test (OGTT) was performed after an overnight starvation. Fasting blood glucose level of each animal was measured with the help of glucometer. Using the technique of oral gavage, 2 gm/kg of glucose solution was administered to each rat and blood glucose levels were measured at predefined time points (30, 60, 90 and 120 min). We also used homeostasis model assessment for insulin resistance (HOMA-IR) to predict the effect of treatment on insulin sensitivity. For HOMA-IR, we used the fasting values of insulin and glucose that were recorded before the administration of glucose to perform OGTT. 2.3.2. Estimation of GLP-1 in serum and intestine GLP-1 stimulates the release of insulin from the β-cells of pancreatic islets and have an important role in regulating the glucose homeostasis. GLP-1 was measured in the serum before, during and the end of treatment period, however, at the end of treatment period, the level of GLP-1 was also estimated in collected samples of intestinal tissue homogenates using its corresponding ELISA kit. 2.3.3. Estimation of inflammatory biomarkers Inflammatory mediators including C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured in the serum using their corresponding ELISA kits before, during and at the end of treatment period, however, at the end of treatment period, the levels of IL-6 and TNF-α were also estimated in collected samples of the intestinal tissue homogenates using their corresponding ELISA kits. 2.3.4. Estimation of lipid profile biomarkers Lipid profile biomarkers namely; triglycerides (TGs), low-density lipoproteins (LDL), high-density lipoproteins (HDL) and cholesterol were estimated before, during and at the end of treatment period using their assay reagent kits.
2. Material and methods 2.3.5. Estimation of kidney and liver function biomarkers To evaluate the effect of treatment on alloxan-induced DM, liver function biomarkers including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and kidney function biomarkers notably, blood urea nitrogen (BUN) and creatinine were also estimated before, during and at the end of treatment period using their assay reagent kits.
2.1. Experimentally-induced diabetic animal model About 30 albino rats of same age having body weight 150–210 g were kept at room temperature (25.5 ± 2 ᴼC) in the animal house of University of Agriculture, Faisalabad, Pakistan. All rats were fed on normal diet with water ad libitum. Animals were divided into 5 groups; group-1: normal control having non-diabetic rats (NDC); group-2: positive control having alloxan induced-diabetic rats (DC); group-3: alloxan-induced diabetic rats treated with metformin (50 mg/kg/day) (MET); group-4: alloxan-induced diabetic rats treated with genistein (20 mg/kg/day) (GEN); group-5: alloxan-induced diabetic rats treated with combination of genistein and metformin (20 mg/kg/day + 50 mg/ kg/day) (GEN + MET). The ethical procedures to experimental animals were followed in accordance with the ethical committee of University of Agriculture, Faisalabad, Pakistan.
2.3.6. Collection of tissues for protein analysis and histopathological examination At the end of treatment period, blood was collected from the abdominal vein of rats from all groups for the purpose of biochemical analysis. Tissue samples of intestine and pancreas were collected for histopathological examination. For the process of homogenization, firstly, the lysis buffer was prepared to help deteriorate fatty and nuclei membranes surrounding the cells and within the cells. About 0.1 M phosphate buffered saline (PBS) was used for the homogenization of tissue. Tissue sample was then taken in falcon tube already having 0.1 M PBS. The tube was placed under tissue homogenizer for homogenization at 3000 rpm. Homogenized tissue was stored at -20 °C till the analysis of content of GLP-1, IL-6 and TNF-α. Moreover, for histopathological examination, tissues from the intestine and pancreas were also collected and followed the procedure of fixation, tissue embedding, sectioning, mounting and staining for histopathological analysis.
2.2. Blood sampling Blood samples from each group were collected before the start, during and at the end of treatment period. For biochemical analysis, blood samples were centrifuged at 3000 × g for 15 min and stored at -20 ᴼC till further analysis. 2
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Fig. 1. Effect of treatment on glycemia: The level of fasting (A) and random (B) blood glucose was measured in all experimental groups at 1st, 14th and 28th day of the treatment period. The level of significant difference was estimated by bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.01 when compared with DCgroup. c represents P < 0.001 when compared with DC- group. d represents P < 0.001 when compared with MET- group. e represents P < 0.01 when compared with MET- group. Abbreviations| NDC: non-diabetic control, DC: diabetic control, MET: metformin.
Fig. 2. Effect of treatment on glucose tolerance (A) and insulin resistance (B): To estimate the effect of treatment on glucose tolerance, we performed OGTT (A) before the end of treatment period. The level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.001 when compared with DC- group. We also estimated the effect of treatment on insulin resistance with the help of HOMA-IR (B). a represent P < 0.0001 when compared with NDC- group. b represents P < 0.0001 when compared with DCgroup. c represents P < 0.0001 when compared with MET- group. Abbreviations| OGTT: oral glucose tolerance test, HOMA-IR: homeostatic model assessment for insulin resistance, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
3. Results 3.1. Effect of treatment on glycemia Therapeutic effect of genistein alone and/or in combination with metformin on glycemic profile including FBG and RBG of alloxan-induced diabetic rats were evaluated and compared with that of nontreated (DC) and metformin-treated groups. It was observed that alloxan increased the levels of both FBG (P < 0.001) and RBG (P < 0.001) as shown in Fig. 1A and B. After the start of treatment, we found that metformin and genistein significantly decreased the levels of FBG (P < 0.01 and P < 0.001, respectively) and RBG (P < 0.01 and P < 0.001, respectively) when compared with that of non-treated alloxan-induced diabetic rats. However, at the end of treatment period, genistein alone (P < 0.01) and/or in combination with metformin (P < 0.01) showed progressively more hypoglycemic eff ;ects as compared to that of metformin-treated rats (Fig. 1B). Similarly, to evaluate the effect of treatment on glucose tolerance, we also performed OGTT (Fig. 2A) and predicted the effect of treatment on insulin sensitivity (Fig. 2B) with the help of HOMA-IR. To perform OGTT, rats from all groups were kept on overnight fasting and blood samples were collected to measure the fasting levels of glucose and insulin, followed by the administration of glucose solution and later, the blood samples were collected to measure the blood glucose level at predefined time
points. As shown in Fig. 2A, alloxan-induced diabetic rats had maximum level of glucose (P < 0.001) at 30 min as compared to that of NDC-group (P < 0.001) and remained persistently high (P < 0.001) at all-time points when compared with NDC- and treated-groups (Fig. 2A). However, a significant decline in the level of blood glucose was observed with the passage of time in experimental groups that were either receiving genistein (P < 0.001) and/or its combination with metformin (P < 0.001) when compared with that of alloxan-induced diabetic rats (Fig. 2A). Whereas, genistein alone and/or in combination with metformin improved insulin sensitivity more efficiently (P < 0.0001) when compared with that of metformin-treated experimental animals (Fig. 2B).
3.2. Effect of treatment on GLP-1 We investigated the stimulatory effects of genistein and its combination with metformin (GEN + MET) on GLP-1. Before the start of treatment with genistein and metformin, alloxan significantly decreased (P < 0.001) the level of GLP-1 in serum (Fig. 3A) and in tissue homogenate of intestine (Fig. 3B) when compared with that of NDC3
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DC-group (P < 0.001) and metformin-treated experimental animals (Fig. 4). 3.4. Effect of treatment on lipidemia We also estimated the therapeutic effects of genistein on lipid profile biomarkers by estimating the serum levels of TGs, LDL, HDL and cholesterol (Fig. 5). Before the start treatment, rats receiving alloxan showed significantly elevated serum levels of TGs, LDL and cholesterol except the serum level of HDL which was observed to be low, when compared (P < 0.001) with that of NDC-experimental rats. While at the end of treatment period, genistein alone and/or in combination with metformin significantly improved hyperlipidemia by regulating the serum levels of TGs, LDL, HDL and cholesterol when compared (P < 0.01) with that of metformin-treated experimental animals (Fig. 5). 3.5. Effect of treatment on liver and kidney function biomarkers We measured the serum level of liver (AST and ALT) and kidney (creatinine and BUN) function biomarkers (Fig. 6) before, during and at the end of treatment period. We observed that alloxan significantly elevated the serum levels of liver (Fig. 6 A and B) and kidney (Fig. 6 C and D) function biomarkers in comparison (P < 0.001) to NDC-experimental rats. The treatment with genistein alone and/or in combination with metformin improved the liver and kidney function biomarkers when compared (P < 0.001) with that of alloxan-induced alteration in liver and kidney function biomarkers (Fig. 6). 3.6. Effect of treatment of histopathology of pancreas and intestine The non-diabetic control group of rats had normal proportion and structure of islets of langerhans with normal histological architecture. The acinar cells were well arranged with prominent nuclei. However, the rats exposed to alloxan, showed damaging effects to the pancreatic β-cells including the islets and acini with appearance of vacuoles. The treatment with metformin, genistein alone or in combination showed reduced β-cell damage with or without partial restoration (Fig. 7 A–E). We also examined the histology of intestine at the end of treatment period. The intestinal cells were found to be normal in all groups in terms of histological architecture and structure except for some minor disruption in villi epithelium and increased blood vessels of alloxan exposed group. However, genistein and metformin significantly improved the histology of intestine (Fig. 7 F–J).
Fig. 3. Effect of treatment on GLP-1: To estimate the effect of treatment on the serum level of GLP-1 (A) at 1st, 15th, and 30th day of the treatment period and tissue content of GLP-1 (B) from intestine at the end of treatment period. The level of significant difference for serum level of GLP-1 was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.01 when compared with DCgroup. c represents P < 0.001 when compared with DC- group. d represent P < 0.05 when compared with DC- group. d represent P < 0.01 when compared with MET- group. For tissue content of GLP-1 was estimated by NewmanKeuls Multiple Comparison Test by one-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.01 when compared with DC- group. c represents P < 0.05 when compared with MET- group. Abbreviations| GLP-1: glucagon like peptide-1, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
4. Discussion
experimental rats. However, the level of GLP-1 was significantly regained in experimental rats when treated with genistein and metformin, but genistein alone and/or in combination with metformin enhanced the secretion of GLP-1 more efficiently when compared with that of metformin-treated experimental rats (Fig. 3). Moreover, genistein alone and/or in combination form also increased the levels of GLP-1 in tissue homogenates of intestine when compared with that of metformintreated group (P < 0.05) as shown in Fig. 3B.
In current study, we evaluated the effect of genistein on incretin hormone; GLP-1 via downregulation of inflammatory responses, hyperglycemia and hyperlipidemia, and compared its therapeutic effects with that of metformin. Metformin is considered as standard anti-diabetic agent and first-line oral glucose-lowering agent that has been previously reported to have an efficient potential of increasing the secretion of GLP-1 [32,33]. β-cell atrophy is a serious condition; however, no drug so far has been clinically approved to possess potential for its potential except for GLP-1 analogues [34]. Hence, recent research work focuses on exploring the new therapeutic members of drugs; polyphenols that are known to have maximal efficacy with limited side effects. These plant-oriented compounds have shown potential for reversing or at least preventing the progression of diseases like DM by partially regulating the functioning of pancreatic β-cells [35]. DPP-IV inhibitors are known to prevent the inactivation of incretin hormones [36]. The combination of these inhibitors with metformin has also been evaluated against the treatment of DM in many of the scientific works [37,38]. This combination provides effective probably by either augmenting the secretion of incretin or reducing the degradation of these
3.3. Effect of treatment on inflammatory responses To estimate the influence of treatment on inflammatory responses, we measured the levels of CRP, IL-6, and TNF-α in serum and in tissue homogenate of intestine (Fig. 4). Before the start of treatment, alloxan significantly increased (P < 0.001) the serum levels of IL-6, TNF-α and CRP as compared to that of NDC-experimental rats. However, at the end of the treatment, genistein alone and/or in combination with metformin downregulated the expression levels of these inflammatory mediators in tissues homogenates as evidenced by the decreased levels these inflammatory mediators in serum when compared with that of 4
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Fig. 4. Effect of treatment on inflammatory biomarkers: To estimate the effect of treatment on serum level of IL-6 (A), TNF-α (C) and CRP (E) at 1st, 15th, and 30th day of the treatment period and on tissue contents of IL-6 (B) and TNF-α (D) at the end of treatment period. For serum levels of IL-6, TNF-α and CRP, the level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.001 when compared with DC- group. c represents P < 0.05 when compared with MET- group. For tissue content of GLP-1 was estimated by Newman-Keuls Multiple Comparison Test by one-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.001 when compared with DC- group. c represents P < 0.001 when compared with MET- group. Abbreviations| IL-6; interleukin-6, TNF-α: tumor necrosis factor-alpha, CRP: C-reactive protein, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
significant decline in the levels of FBG and RBG with either genistein (P < 0.001) and/or in combination with metformin (P < 0.01) as compared to that of metformin-treated animals (Fig. 1). These results are in accordance with already published results elsewhere [40–42]. Similarly, the tolerance for regulating the blood glucose in terms of metabolizing and up taking of glucose was estimated by OGTT in which genistein alone and/or in combination with metformin proved to be significantly effective (P < 0.001) by lowering the blood glucose levels in response to exogenously administered glucose when compared with that of metformin-treated experimental animals (Fig. 2A). These results are in accordance with already published findings [43].
hormones [33,39]. Correspondingly, the combination of a potent phytonutrient commonly available to patients through daily diet and food with metformin might have a fruitful combination for elevating the levels of GLP-1 and regulating incretin-dependent secretion of insulin from pancreatic cells. Hence, we utilized genistein alone and/or in combination with metformin for evaluating their influence on GLP-1. Till know, to the best of our knowledge, no work has been previously done to estimate the potential of this combination in the above-mentioned aspect of GLP-1 secretion and maintain its downstream function of secreting insulin in response to the elevated blood glucose concentration. The surprising fact observed in current work was the 5
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Fig. 5. Effect of treatment on lipidemia: To estimate the effect of treatment on serum level of cholesterol (A), HDL (B), LDL (C) and TGs (D) at 1st, 15th, and 30th day of the treatment period. The level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.05 when compared with DC- group. c represents P < 0.001 when compared with DC- group. d represent P < 0.01 when compared with MET- group. Abbreviations| HDL: high density lipoprotein, LDL: low density lipoprotein, TGs: triglycerides, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
with impaired secretion of GLP-1 [55,56]. We also estimated the serum levels of lipid profile biomarkers and found that genistein significantly improved the lipotoxicity by regulating the serum levels of lipid biomarkers when compared with that of metformin-treated experimental animals (Fig. 5). The antihyperlipidemic effects of genistein in this study are also associated with increased levels of GLP-1 in serum and tissue homogenates of intestine. These Results supports the findings of other studies which exhibit the potential of genistein having antihyperlipidemic effects [57]. Correspondingly, as previously published [58,59], our study also verified the reno- and hepato-protective effects of genistein particularly in combination with metformin (Fig. 6). Moreover, The results of histological examination of pancreas and intestine (Fig. 7) were also found to be in accordance with the biochemical results of this study. Alloxan has shown prominent degeneration of pancreatic cells including islets and acinar cells as revealed on histological study, whereas, genistein protected the pancreatic islets and acinar cells from the deleterious effects of alloxan. The significant findings of this study would help to recognize the therapeutic potential of genistein in combination with metformin as stimulatory enhancer of GLP-1 secretion via downregulation of inflammatory responses and glucolipotoxicity. This combination therapy may provide considerable guideline for future drug design of such types of combination.
We mainly focused to determine the impact of genistein and/or its combination with metformin (GEN + MET) on GLP-1 (Fig. 3). Alloxan significantly reduced the level of GLP-1 in serum (P < 0.001) and tissue homogenates (P < 0.001), while, the treatment with genistein alone and/or in combination with metformin was depicted to be significant in regaining the levels of GLP-1 in tissue homogenates of intestine even when compared (P < 0.05) with metformin-treated experimental animals (Fig. 3B). These results were found to be in accordance with the results of the other studies supporting the efficacy of genistein for having potential to enhance GLP-1 secretion [44]. This potential of genistein may in turn help in GLP-1-mediated release of insulin for the regulation of glucose homeostasis. Tissue-specific inflammatory responses have also been reported to play their role in altering the survival and functioning of β-cells [45–49]. Moreover, studies have also found that increased inflammatory responses have been found to be associated with impaired secretion of GLP-1 [50–52]. We also focused to evaluate the effect of genistein on inflammatory mediators including IL-6, TNF-α and CRP. We found that genistein alone and/or in combination with metformin downregulated the expression of these inflammatory mediators more efficiently when compared with that of metformin-treated experimental animals (Fig. 4). The anti-inflammatory potential of genistein exhibits that genistein enhances the secretion of GLP-1 by decreasing the tissuespecific inflammatory responses as evidenced by the decreased levels of IL-6 (Fig. 4B) and TNF-α (Fig. 4D) in tissue homogenates of intestine. These Results supports the findings of other studies which exhibit the potential of genistein against inflammation [53,54]. In recent studies, it has been found that lipotoxicity is associated
5. Conclusion and future perspective Exploration of pathways by which the phytonutrients may exert their role in ameliorating the symptoms and underlying causes for pathogenesis of DM, is becoming the foremost interest in drug discovery 6
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Fig. 6. Effect of treatment on liver and kidney function biomarkers: To estimate the effect of treatment on liver and kidney function biomarkers, we measured the serum level of AST (A), ALT (B) creatinine (C) and BUN (D) at 1st, 15th, and 30th day of the treatment period. The level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.05 when compared with DC- group. c represents P < 0.001 when compared with DC- group. d represents P < 0.01 when compared with MET- group. Abbreviations| ALT: alanine aminotransferase, AST: aspartate aminotransferase, BU: blood urea nitrogen, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
Fig. 7. Effect of treatment of histopathology of pancreas and intestine: In the histopathology of pancreas (A–E), NDC-group; β-cells are normal in appearance and normal in numbers. Acinar cells (AC) are normal in appearance and darkly stained. DC-group; appearance of β-cells is not normal and photomicrography indicating increase in number of β-cells. And at few places increase of blood vessels (BV) seen. MET-group; acinar cells (AC) are normal in appearance and darkly stained. Congestion (BC) is present at few places. Blood vessels (BV) seen normal in appearance. GEN-group; acinar cells (AC) normal in appearance and β-cells normally distributed and increase number of blood vessels (BV) are seen. MET + GEN- group; β-cells are normal in appearance and normal in numbers. Acinar cells (AC) are also normal in appearance and darkly stained and a few places blood vessels (BV) are seen normal. Congestion (BC) is present at few places. In the histopathology of intestine (F–J), NDC-group; enterocytes (EC) are normal in appearance seen and apical length of villi (AV) are normal. Tips of villi (TOV) are also normal observed, having intact epithelium (IE). DC-group; appearance of enterocytes (EC) are not normal and at few places increase of blood vessels (BV) seen. Villi (AV) not reach to its full length. MET-group; enterocytes are normal but with disruption of villi epithelium (VE) is minor. Villi have intact epithelium and reach to its full length (AV). GEN-group; villi reach up to its full length (AV). Enterocytes (EC) are normal in appearance and villi have intact epithelium (IE). MET + GEN- group; enterocytes (EC) are normal and villi are full in length (AV) and have intact epithelium (IE) and presence of goblet cells (GC) are also seen. Abbreviations| NDC: non-diabetic control, DC: diabetic control, MET: metformin. 7
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Fig. 8. Proposed mechanism of Genistein in improving glucose uptake via GLP-1 secretion. Genistein may appears to improve the glucose uptake and insulin resistance via secretion of GLP-1 from enteroendocrine L-cells of intestine. The probable mechanism for this improvement is its potent inhibitory effect on proinflammatory mediators (IL-6, TNF-α and CRP). Which in turn help in reducing the inflammation-induced consequences including oxidative stress, damage of intestinal L-cells. Similarly, the increased oxidative stress may also result in β-cell dysfunction. Genistein tends to improve the secretion of GLP-1, its binding to GLP-1 receptor on pancreas, insulin secretion and glucose metabolism. Abbreviations| GLP-1: Glucagon like peptide-1, IL-6: Interleukin 6, TNF-α: Tumor necrosis factor alpha, CRP: C-reactive protein.
and development. In current work, we have also focused on similar potential of genistein in combination with metformin which exhibited significantly better therapeutic effects for restoring the altered biochemical parameters and indicators of glucolipotoxicity and augmented levels of inflammatory biomarkers. Furthermore, this combination was also found to have potential to enhance the secretion GLP-1 via downregulation of inflammatory responses, and glucolipotoxicity. It is proposed by the results of our study (Fig. 8) that genistein alone and/or in combination with metformin increases the levels of GLP-1 in tissue homogenates of intestine with a simultaneous decline in the tissue levels of inflammatory mediators. This depicts that alloxan-induced oxidative stress in enteroendocrine L-cells as evident by increase in the level of tissue inflammatory mediators and histopathological examination, was responsible for L-cell damage which resulted in reduction of GLP-1 secretion/expression. However, the L-cell damage was prevented by the treatment with genistein and its combination which resulted in partial restoration of GLP-1 expression. Hence, depending on the multiple targeting effects of genistein in combination with metformin, we would suggest future studies for the pharmacokinetic evaluation of this combination for a better future therapeutic mean for managing and curing DM.
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Genistein enhances the secretion of glucagon-like peptide-1 (GLP-1) via downregulation of inflammatory responses Kanwal Rehmana, Mehwish Bagh Alia, Muhammad Sajid Hamid Akashb, a b
T
⁎
Department of Pharmacy, University of Agriculture, Faisalabad, Pakistan Department of Pharmaceutical Chemistry, Government College University Faisalabad, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: GLP-1 Diabetes mellitus Genistein Metformin Inflammatory responses Incretin hormone
Glucagon-like peptide-1 (GLP-1) an incretin hormone, is known to regulate the glucose-mediated insulin secretion. However, reduction in the level of GLP-1 is considered to be a major cause for the reduction of GLP-1dependent insulin secretory response. Genistein an isoflavone, is an important polyphenol and has wide range of therapeutic potentials, but its therapeutic effects alone and/or in combination with metformin on GLP-1 secretion have not been investigated yet. Hence, we aimed to investigate the stimulatory action of genistein in combination with metformin on GLP-1 via downregulation of inflammatory mediators, hyperlipidemia and hyperglycemia in alloxan-induced diabetic rats. Diabetes was induced in experimental rats by single administration of alloxan intraperitoneally. Metformin (50 mg/kg/day), genistein (20 mg/kg/day) and combination of genistein and metformin was administered in alloxan-induced diabetic rats. We found that genistein alone and/ or in combination with metformin significantly increased the serum level (P < 0.01) and tissue content (P < 0.05) of GLP-1 in intestine when compared with that of metformin-treated animals. Similarly, genistein alone and/or in combination with metformin also resulted in normoglycemia (P < 0.001), glucose tolerance (P < 0.01), insulin sensitivity (P < 0.0001), hyperlipidemia (P < 0.01), liver and kidney function biomarkers (P < 0.01) as compared to that of metformin-treated experimental animals. Moreover, genistein alone and/or in combination with metformin also downregulated the inflammatory responses by decreasing the levels of interleuin-6, tumor necrosis factor-α and C-reactive protein in serum (P < 0.05) and intestine (P < 0.001) more efficiently as compared to that of metformin-treated experimental animals. The downregulation of inflammatory responses in intestine, was positively associated with increased secretion of GLP-1 from intestine. Histopathology of pancreas and intestine also showed that genistein significantly improved the deleterious effects of alloxan on pancreas and intestine. Hence, our work provides new insights on the synergistic effects of genistein and metformin on GLP-1 secretion. This may significantly improve the perception for proposing new GLP-1-based synergistic approaches for the treatment of diabetes mellitus.
1. Introduction Diabetes mellitus (DM), with its growing occurrence is predictable to affect about 8% of the global population till 2030 [1,2]. DM is a metabolic disease during which glucose homeostasis becomes irregular. Further, it is characterized by impaired carbohydrate metabolism, insulin resistance and impaired insulin secretion [3,4]. The world is facing an estimated lifetime risk of 25% due to growing prevalence of DM. Different factors are considered to be responsible for the cause of insulin resistance and failure of β-cell to secrete insulin on demand. One of them is alteration in the pathways or the factors that regulate the glucose metabolism for instance incretin hormone like glucagon-like peptide-1 (GLP-1) [5,6]. GLP-1 is secreted as an incretin hormone from ⁎
the gut in response to intake of food [7,8] and is known to have number of functions like it potentiates insulin secretion from pancreas and regulates the metabolism of glucose [7,8]. In the past few years, metformin, which is a well-recognized commercially available anti-diabetic drug, has shown to increase the levels of GLP-1 [7,9,10]. Preprandial metformin prescribed in diabetic patients, have shown increase the plasma levels of GLP-1 [11–13]. The results of these studies suggest that GLP-1-specific effects involving L-cells of gut endocrine system may be influenced by the inhibition of dipeptidyl peptidase-IV (DPP-IV) [14] as the inhibition of DPP-IV may reduce the inactivation of GLP-1. It has also been reported that the effects of incretin may be impaired because of unresponsiveness of pancreatic β-cells in diabetic patients [15]. Hence, researchers are now focusing on improving insulin secretion, or
Corresponding author. E-mail addresses: [email protected], [email protected] (M.S.H. Akash).
https://doi.org/10.1016/j.biopha.2019.108670 Received 13 January 2019; Received in revised form 31 January 2019; Accepted 4 February 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.3. Biochemical analysis
maintaining the glucose homeostasis using various therapeutic approaches. Moreover, studies have also suggested that improving the proliferation and survival of β-cells using drugs like GLP-1 receptor agonists might be helpful in treating DM [4,16,17]. Mechanism of actions of GLP-1 including stimulation of insulin secretion, suppression of glucagon secretion, delay in gastric emptying, and inhibition of small bowel motility, have been suggested to contribute for its anti-diabetogenic effects. Thereby, different approaches have been used to focus on the cost-effectiveness of anti-diabetic agents to maintain the glucose homeostasis with improved glucose metabolism. This may help in decreasing the prevalence and burden of DM along with its associated complications globally. Polyphenols present in diet have been considered to play important role in regulating the metabolism of carbohydrates [18–24]. This is done by controlling the postprandial hyperglycemia by reducing the absorption of digested carbohydrates [18–20]. Extracts of different berries having polyphenols have also known to inhibit the level of carbohydrate metabolizing enzymes [25]. Hence, we tried to focus on similar therapeutic approach, which involves the use of natural product being cost-effective, less toxic, and having ability to target the major pathways known for controlling and/or regulating the normal glucose metabolism. For this, we opted to use an isoflavone; genistein (4,5,7trihydroxyisoflavone) that is abundantly found in soybean. However, phytoestrogens are also known as rich source of genistein. Genistein has shown to have therapeutic potential for improving body weight, food intake along with maintaining the adipose tissue mass in ovariectomized mice [26]. Moreover, genistein has also shown to inhibit the synthesis and/or secretion of leptin from adipocytes [27,28]. In addition, genistein has also shown beneficial effects against hyperglycemic state because of its potential of stimulating glucose-mediated secretion of insulin from pancreatic islets [29]. Consistently, in chemically-induced diabetic rats, dietary medications of isoflavones have given better results by decreasing the sugar levels in blood and by releasing the glucose controlling hormone [30,31]. To the best of our knowledge, until now, the potential role of genistein alone and/or in combination with metformin on GLP-1 remains to be elucidated. Therefore, we opted to use genistein and investigated its stimulatory action on GLP-1 via downregulation of inflammatory responses in alloxan-induced diabetic rats.
2.3.1. Estimation of glycemic control biomarkers The effect of treatment on glycemia was estimated on predefined time points. To estimate the effect of treatment on glycemic levels of animals, the fasting blood glucose (FBG) and random blood glucose (RBG) levels were recorded with the help of glucometer twice in a week. To evaluate the tendency of glucose tolerance in experimentallyinduced diabetic animals, oral glucose tolerance test (OGTT) was performed after an overnight starvation. Fasting blood glucose level of each animal was measured with the help of glucometer. Using the technique of oral gavage, 2 gm/kg of glucose solution was administered to each rat and blood glucose levels were measured at predefined time points (30, 60, 90 and 120 min). We also used homeostasis model assessment for insulin resistance (HOMA-IR) to predict the effect of treatment on insulin sensitivity. For HOMA-IR, we used the fasting values of insulin and glucose that were recorded before the administration of glucose to perform OGTT. 2.3.2. Estimation of GLP-1 in serum and intestine GLP-1 stimulates the release of insulin from the β-cells of pancreatic islets and have an important role in regulating the glucose homeostasis. GLP-1 was measured in the serum before, during and the end of treatment period, however, at the end of treatment period, the level of GLP-1 was also estimated in collected samples of intestinal tissue homogenates using its corresponding ELISA kit. 2.3.3. Estimation of inflammatory biomarkers Inflammatory mediators including C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured in the serum using their corresponding ELISA kits before, during and at the end of treatment period, however, at the end of treatment period, the levels of IL-6 and TNF-α were also estimated in collected samples of the intestinal tissue homogenates using their corresponding ELISA kits. 2.3.4. Estimation of lipid profile biomarkers Lipid profile biomarkers namely; triglycerides (TGs), low-density lipoproteins (LDL), high-density lipoproteins (HDL) and cholesterol were estimated before, during and at the end of treatment period using their assay reagent kits.
2. Material and methods 2.3.5. Estimation of kidney and liver function biomarkers To evaluate the effect of treatment on alloxan-induced DM, liver function biomarkers including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and kidney function biomarkers notably, blood urea nitrogen (BUN) and creatinine were also estimated before, during and at the end of treatment period using their assay reagent kits.
2.1. Experimentally-induced diabetic animal model About 30 albino rats of same age having body weight 150–210 g were kept at room temperature (25.5 ± 2 ᴼC) in the animal house of University of Agriculture, Faisalabad, Pakistan. All rats were fed on normal diet with water ad libitum. Animals were divided into 5 groups; group-1: normal control having non-diabetic rats (NDC); group-2: positive control having alloxan induced-diabetic rats (DC); group-3: alloxan-induced diabetic rats treated with metformin (50 mg/kg/day) (MET); group-4: alloxan-induced diabetic rats treated with genistein (20 mg/kg/day) (GEN); group-5: alloxan-induced diabetic rats treated with combination of genistein and metformin (20 mg/kg/day + 50 mg/ kg/day) (GEN + MET). The ethical procedures to experimental animals were followed in accordance with the ethical committee of University of Agriculture, Faisalabad, Pakistan.
2.3.6. Collection of tissues for protein analysis and histopathological examination At the end of treatment period, blood was collected from the abdominal vein of rats from all groups for the purpose of biochemical analysis. Tissue samples of intestine and pancreas were collected for histopathological examination. For the process of homogenization, firstly, the lysis buffer was prepared to help deteriorate fatty and nuclei membranes surrounding the cells and within the cells. About 0.1 M phosphate buffered saline (PBS) was used for the homogenization of tissue. Tissue sample was then taken in falcon tube already having 0.1 M PBS. The tube was placed under tissue homogenizer for homogenization at 3000 rpm. Homogenized tissue was stored at -20 °C till the analysis of content of GLP-1, IL-6 and TNF-α. Moreover, for histopathological examination, tissues from the intestine and pancreas were also collected and followed the procedure of fixation, tissue embedding, sectioning, mounting and staining for histopathological analysis.
2.2. Blood sampling Blood samples from each group were collected before the start, during and at the end of treatment period. For biochemical analysis, blood samples were centrifuged at 3000 × g for 15 min and stored at -20 ᴼC till further analysis. 2
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Fig. 1. Effect of treatment on glycemia: The level of fasting (A) and random (B) blood glucose was measured in all experimental groups at 1st, 14th and 28th day of the treatment period. The level of significant difference was estimated by bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.01 when compared with DCgroup. c represents P < 0.001 when compared with DC- group. d represents P < 0.001 when compared with MET- group. e represents P < 0.01 when compared with MET- group. Abbreviations| NDC: non-diabetic control, DC: diabetic control, MET: metformin.
Fig. 2. Effect of treatment on glucose tolerance (A) and insulin resistance (B): To estimate the effect of treatment on glucose tolerance, we performed OGTT (A) before the end of treatment period. The level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.001 when compared with DC- group. We also estimated the effect of treatment on insulin resistance with the help of HOMA-IR (B). a represent P < 0.0001 when compared with NDC- group. b represents P < 0.0001 when compared with DCgroup. c represents P < 0.0001 when compared with MET- group. Abbreviations| OGTT: oral glucose tolerance test, HOMA-IR: homeostatic model assessment for insulin resistance, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
3. Results 3.1. Effect of treatment on glycemia Therapeutic effect of genistein alone and/or in combination with metformin on glycemic profile including FBG and RBG of alloxan-induced diabetic rats were evaluated and compared with that of nontreated (DC) and metformin-treated groups. It was observed that alloxan increased the levels of both FBG (P < 0.001) and RBG (P < 0.001) as shown in Fig. 1A and B. After the start of treatment, we found that metformin and genistein significantly decreased the levels of FBG (P < 0.01 and P < 0.001, respectively) and RBG (P < 0.01 and P < 0.001, respectively) when compared with that of non-treated alloxan-induced diabetic rats. However, at the end of treatment period, genistein alone (P < 0.01) and/or in combination with metformin (P < 0.01) showed progressively more hypoglycemic eff ;ects as compared to that of metformin-treated rats (Fig. 1B). Similarly, to evaluate the effect of treatment on glucose tolerance, we also performed OGTT (Fig. 2A) and predicted the effect of treatment on insulin sensitivity (Fig. 2B) with the help of HOMA-IR. To perform OGTT, rats from all groups were kept on overnight fasting and blood samples were collected to measure the fasting levels of glucose and insulin, followed by the administration of glucose solution and later, the blood samples were collected to measure the blood glucose level at predefined time
points. As shown in Fig. 2A, alloxan-induced diabetic rats had maximum level of glucose (P < 0.001) at 30 min as compared to that of NDC-group (P < 0.001) and remained persistently high (P < 0.001) at all-time points when compared with NDC- and treated-groups (Fig. 2A). However, a significant decline in the level of blood glucose was observed with the passage of time in experimental groups that were either receiving genistein (P < 0.001) and/or its combination with metformin (P < 0.001) when compared with that of alloxan-induced diabetic rats (Fig. 2A). Whereas, genistein alone and/or in combination with metformin improved insulin sensitivity more efficiently (P < 0.0001) when compared with that of metformin-treated experimental animals (Fig. 2B).
3.2. Effect of treatment on GLP-1 We investigated the stimulatory effects of genistein and its combination with metformin (GEN + MET) on GLP-1. Before the start of treatment with genistein and metformin, alloxan significantly decreased (P < 0.001) the level of GLP-1 in serum (Fig. 3A) and in tissue homogenate of intestine (Fig. 3B) when compared with that of NDC3
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DC-group (P < 0.001) and metformin-treated experimental animals (Fig. 4). 3.4. Effect of treatment on lipidemia We also estimated the therapeutic effects of genistein on lipid profile biomarkers by estimating the serum levels of TGs, LDL, HDL and cholesterol (Fig. 5). Before the start treatment, rats receiving alloxan showed significantly elevated serum levels of TGs, LDL and cholesterol except the serum level of HDL which was observed to be low, when compared (P < 0.001) with that of NDC-experimental rats. While at the end of treatment period, genistein alone and/or in combination with metformin significantly improved hyperlipidemia by regulating the serum levels of TGs, LDL, HDL and cholesterol when compared (P < 0.01) with that of metformin-treated experimental animals (Fig. 5). 3.5. Effect of treatment on liver and kidney function biomarkers We measured the serum level of liver (AST and ALT) and kidney (creatinine and BUN) function biomarkers (Fig. 6) before, during and at the end of treatment period. We observed that alloxan significantly elevated the serum levels of liver (Fig. 6 A and B) and kidney (Fig. 6 C and D) function biomarkers in comparison (P < 0.001) to NDC-experimental rats. The treatment with genistein alone and/or in combination with metformin improved the liver and kidney function biomarkers when compared (P < 0.001) with that of alloxan-induced alteration in liver and kidney function biomarkers (Fig. 6). 3.6. Effect of treatment of histopathology of pancreas and intestine The non-diabetic control group of rats had normal proportion and structure of islets of langerhans with normal histological architecture. The acinar cells were well arranged with prominent nuclei. However, the rats exposed to alloxan, showed damaging effects to the pancreatic β-cells including the islets and acini with appearance of vacuoles. The treatment with metformin, genistein alone or in combination showed reduced β-cell damage with or without partial restoration (Fig. 7 A–E). We also examined the histology of intestine at the end of treatment period. The intestinal cells were found to be normal in all groups in terms of histological architecture and structure except for some minor disruption in villi epithelium and increased blood vessels of alloxan exposed group. However, genistein and metformin significantly improved the histology of intestine (Fig. 7 F–J).
Fig. 3. Effect of treatment on GLP-1: To estimate the effect of treatment on the serum level of GLP-1 (A) at 1st, 15th, and 30th day of the treatment period and tissue content of GLP-1 (B) from intestine at the end of treatment period. The level of significant difference for serum level of GLP-1 was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.01 when compared with DCgroup. c represents P < 0.001 when compared with DC- group. d represent P < 0.05 when compared with DC- group. d represent P < 0.01 when compared with MET- group. For tissue content of GLP-1 was estimated by NewmanKeuls Multiple Comparison Test by one-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.01 when compared with DC- group. c represents P < 0.05 when compared with MET- group. Abbreviations| GLP-1: glucagon like peptide-1, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
4. Discussion
experimental rats. However, the level of GLP-1 was significantly regained in experimental rats when treated with genistein and metformin, but genistein alone and/or in combination with metformin enhanced the secretion of GLP-1 more efficiently when compared with that of metformin-treated experimental rats (Fig. 3). Moreover, genistein alone and/or in combination form also increased the levels of GLP-1 in tissue homogenates of intestine when compared with that of metformintreated group (P < 0.05) as shown in Fig. 3B.
In current study, we evaluated the effect of genistein on incretin hormone; GLP-1 via downregulation of inflammatory responses, hyperglycemia and hyperlipidemia, and compared its therapeutic effects with that of metformin. Metformin is considered as standard anti-diabetic agent and first-line oral glucose-lowering agent that has been previously reported to have an efficient potential of increasing the secretion of GLP-1 [32,33]. β-cell atrophy is a serious condition; however, no drug so far has been clinically approved to possess potential for its potential except for GLP-1 analogues [34]. Hence, recent research work focuses on exploring the new therapeutic members of drugs; polyphenols that are known to have maximal efficacy with limited side effects. These plant-oriented compounds have shown potential for reversing or at least preventing the progression of diseases like DM by partially regulating the functioning of pancreatic β-cells [35]. DPP-IV inhibitors are known to prevent the inactivation of incretin hormones [36]. The combination of these inhibitors with metformin has also been evaluated against the treatment of DM in many of the scientific works [37,38]. This combination provides effective probably by either augmenting the secretion of incretin or reducing the degradation of these
3.3. Effect of treatment on inflammatory responses To estimate the influence of treatment on inflammatory responses, we measured the levels of CRP, IL-6, and TNF-α in serum and in tissue homogenate of intestine (Fig. 4). Before the start of treatment, alloxan significantly increased (P < 0.001) the serum levels of IL-6, TNF-α and CRP as compared to that of NDC-experimental rats. However, at the end of the treatment, genistein alone and/or in combination with metformin downregulated the expression levels of these inflammatory mediators in tissues homogenates as evidenced by the decreased levels these inflammatory mediators in serum when compared with that of 4
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Fig. 4. Effect of treatment on inflammatory biomarkers: To estimate the effect of treatment on serum level of IL-6 (A), TNF-α (C) and CRP (E) at 1st, 15th, and 30th day of the treatment period and on tissue contents of IL-6 (B) and TNF-α (D) at the end of treatment period. For serum levels of IL-6, TNF-α and CRP, the level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.001 when compared with DC- group. c represents P < 0.05 when compared with MET- group. For tissue content of GLP-1 was estimated by Newman-Keuls Multiple Comparison Test by one-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.001 when compared with DC- group. c represents P < 0.001 when compared with MET- group. Abbreviations| IL-6; interleukin-6, TNF-α: tumor necrosis factor-alpha, CRP: C-reactive protein, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
significant decline in the levels of FBG and RBG with either genistein (P < 0.001) and/or in combination with metformin (P < 0.01) as compared to that of metformin-treated animals (Fig. 1). These results are in accordance with already published results elsewhere [40–42]. Similarly, the tolerance for regulating the blood glucose in terms of metabolizing and up taking of glucose was estimated by OGTT in which genistein alone and/or in combination with metformin proved to be significantly effective (P < 0.001) by lowering the blood glucose levels in response to exogenously administered glucose when compared with that of metformin-treated experimental animals (Fig. 2A). These results are in accordance with already published findings [43].
hormones [33,39]. Correspondingly, the combination of a potent phytonutrient commonly available to patients through daily diet and food with metformin might have a fruitful combination for elevating the levels of GLP-1 and regulating incretin-dependent secretion of insulin from pancreatic cells. Hence, we utilized genistein alone and/or in combination with metformin for evaluating their influence on GLP-1. Till know, to the best of our knowledge, no work has been previously done to estimate the potential of this combination in the above-mentioned aspect of GLP-1 secretion and maintain its downstream function of secreting insulin in response to the elevated blood glucose concentration. The surprising fact observed in current work was the 5
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Fig. 5. Effect of treatment on lipidemia: To estimate the effect of treatment on serum level of cholesterol (A), HDL (B), LDL (C) and TGs (D) at 1st, 15th, and 30th day of the treatment period. The level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.05 when compared with DC- group. c represents P < 0.001 when compared with DC- group. d represent P < 0.01 when compared with MET- group. Abbreviations| HDL: high density lipoprotein, LDL: low density lipoprotein, TGs: triglycerides, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
with impaired secretion of GLP-1 [55,56]. We also estimated the serum levels of lipid profile biomarkers and found that genistein significantly improved the lipotoxicity by regulating the serum levels of lipid biomarkers when compared with that of metformin-treated experimental animals (Fig. 5). The antihyperlipidemic effects of genistein in this study are also associated with increased levels of GLP-1 in serum and tissue homogenates of intestine. These Results supports the findings of other studies which exhibit the potential of genistein having antihyperlipidemic effects [57]. Correspondingly, as previously published [58,59], our study also verified the reno- and hepato-protective effects of genistein particularly in combination with metformin (Fig. 6). Moreover, The results of histological examination of pancreas and intestine (Fig. 7) were also found to be in accordance with the biochemical results of this study. Alloxan has shown prominent degeneration of pancreatic cells including islets and acinar cells as revealed on histological study, whereas, genistein protected the pancreatic islets and acinar cells from the deleterious effects of alloxan. The significant findings of this study would help to recognize the therapeutic potential of genistein in combination with metformin as stimulatory enhancer of GLP-1 secretion via downregulation of inflammatory responses and glucolipotoxicity. This combination therapy may provide considerable guideline for future drug design of such types of combination.
We mainly focused to determine the impact of genistein and/or its combination with metformin (GEN + MET) on GLP-1 (Fig. 3). Alloxan significantly reduced the level of GLP-1 in serum (P < 0.001) and tissue homogenates (P < 0.001), while, the treatment with genistein alone and/or in combination with metformin was depicted to be significant in regaining the levels of GLP-1 in tissue homogenates of intestine even when compared (P < 0.05) with metformin-treated experimental animals (Fig. 3B). These results were found to be in accordance with the results of the other studies supporting the efficacy of genistein for having potential to enhance GLP-1 secretion [44]. This potential of genistein may in turn help in GLP-1-mediated release of insulin for the regulation of glucose homeostasis. Tissue-specific inflammatory responses have also been reported to play their role in altering the survival and functioning of β-cells [45–49]. Moreover, studies have also found that increased inflammatory responses have been found to be associated with impaired secretion of GLP-1 [50–52]. We also focused to evaluate the effect of genistein on inflammatory mediators including IL-6, TNF-α and CRP. We found that genistein alone and/or in combination with metformin downregulated the expression of these inflammatory mediators more efficiently when compared with that of metformin-treated experimental animals (Fig. 4). The anti-inflammatory potential of genistein exhibits that genistein enhances the secretion of GLP-1 by decreasing the tissuespecific inflammatory responses as evidenced by the decreased levels of IL-6 (Fig. 4B) and TNF-α (Fig. 4D) in tissue homogenates of intestine. These Results supports the findings of other studies which exhibit the potential of genistein against inflammation [53,54]. In recent studies, it has been found that lipotoxicity is associated
5. Conclusion and future perspective Exploration of pathways by which the phytonutrients may exert their role in ameliorating the symptoms and underlying causes for pathogenesis of DM, is becoming the foremost interest in drug discovery 6
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Fig. 6. Effect of treatment on liver and kidney function biomarkers: To estimate the effect of treatment on liver and kidney function biomarkers, we measured the serum level of AST (A), ALT (B) creatinine (C) and BUN (D) at 1st, 15th, and 30th day of the treatment period. The level of significant difference was estimated by Bonferroni post-test using two-way ANOVA. a represent P < 0.001 when compared with NDC- group. b represents P < 0.05 when compared with DC- group. c represents P < 0.001 when compared with DC- group. d represents P < 0.01 when compared with MET- group. Abbreviations| ALT: alanine aminotransferase, AST: aspartate aminotransferase, BU: blood urea nitrogen, NDC: non-diabetic control, DC: diabetic control, MET: metformin.
Fig. 7. Effect of treatment of histopathology of pancreas and intestine: In the histopathology of pancreas (A–E), NDC-group; β-cells are normal in appearance and normal in numbers. Acinar cells (AC) are normal in appearance and darkly stained. DC-group; appearance of β-cells is not normal and photomicrography indicating increase in number of β-cells. And at few places increase of blood vessels (BV) seen. MET-group; acinar cells (AC) are normal in appearance and darkly stained. Congestion (BC) is present at few places. Blood vessels (BV) seen normal in appearance. GEN-group; acinar cells (AC) normal in appearance and β-cells normally distributed and increase number of blood vessels (BV) are seen. MET + GEN- group; β-cells are normal in appearance and normal in numbers. Acinar cells (AC) are also normal in appearance and darkly stained and a few places blood vessels (BV) are seen normal. Congestion (BC) is present at few places. In the histopathology of intestine (F–J), NDC-group; enterocytes (EC) are normal in appearance seen and apical length of villi (AV) are normal. Tips of villi (TOV) are also normal observed, having intact epithelium (IE). DC-group; appearance of enterocytes (EC) are not normal and at few places increase of blood vessels (BV) seen. Villi (AV) not reach to its full length. MET-group; enterocytes are normal but with disruption of villi epithelium (VE) is minor. Villi have intact epithelium and reach to its full length (AV). GEN-group; villi reach up to its full length (AV). Enterocytes (EC) are normal in appearance and villi have intact epithelium (IE). MET + GEN- group; enterocytes (EC) are normal and villi are full in length (AV) and have intact epithelium (IE) and presence of goblet cells (GC) are also seen. Abbreviations| NDC: non-diabetic control, DC: diabetic control, MET: metformin. 7
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Fig. 8. Proposed mechanism of Genistein in improving glucose uptake via GLP-1 secretion. Genistein may appears to improve the glucose uptake and insulin resistance via secretion of GLP-1 from enteroendocrine L-cells of intestine. The probable mechanism for this improvement is its potent inhibitory effect on proinflammatory mediators (IL-6, TNF-α and CRP). Which in turn help in reducing the inflammation-induced consequences including oxidative stress, damage of intestinal L-cells. Similarly, the increased oxidative stress may also result in β-cell dysfunction. Genistein tends to improve the secretion of GLP-1, its binding to GLP-1 receptor on pancreas, insulin secretion and glucose metabolism. Abbreviations| GLP-1: Glucagon like peptide-1, IL-6: Interleukin 6, TNF-α: Tumor necrosis factor alpha, CRP: C-reactive protein.
and development. In current work, we have also focused on similar potential of genistein in combination with metformin which exhibited significantly better therapeutic effects for restoring the altered biochemical parameters and indicators of glucolipotoxicity and augmented levels of inflammatory biomarkers. Furthermore, this combination was also found to have potential to enhance the secretion GLP-1 via downregulation of inflammatory responses, and glucolipotoxicity. It is proposed by the results of our study (Fig. 8) that genistein alone and/or in combination with metformin increases the levels of GLP-1 in tissue homogenates of intestine with a simultaneous decline in the tissue levels of inflammatory mediators. This depicts that alloxan-induced oxidative stress in enteroendocrine L-cells as evident by increase in the level of tissue inflammatory mediators and histopathological examination, was responsible for L-cell damage which resulted in reduction of GLP-1 secretion/expression. However, the L-cell damage was prevented by the treatment with genistein and its combination which resulted in partial restoration of GLP-1 expression. Hence, depending on the multiple targeting effects of genistein in combination with metformin, we would suggest future studies for the pharmacokinetic evaluation of this combination for a better future therapeutic mean for managing and curing DM.
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