Antioxidant Vitamins Attenuate Glyphosate-Induced Development of Type-2 Diabetes Through the Activation of Glycogen Synthase Kinase-3 β and Forkhead Box Protein O-1 in the Liver of Adult Male Rats

Abstract

Introduction: Glyphosate is a well-known broad-spectrum desiccant and herbicide. It is an active component used widely in popular weed control products like Roundup (BigHaat Agro Pvt Ltd, Bangalore, Karnataka, India), Rodeo (Corteva, Inc., Indianapolis, Indiana, United States), and PondMaster (PBI-Gordon Corporation, Shawnee, Kansas, United States). However, due to sustained presence, they tend to get deposited in the environmental resources and leach into the living system. It has been shown to develop various cancers and diabetes. However, its impact on GSK-3β (glycogen synthase kinase-3 beta) and FOXO-1 (forkhead box protein O1), both critical proteins involved in the regulation of glucose metabolism and insulin signaling, is unknown. Objective: The primary objective of this study was to check whether antioxidant vitamins (C and E) can reduce hyperglycemia and hyperinsulinemia in response to glyphosate exposure and the secondary objective was to investigate whether antioxidant vitamins have the capacity to downregulate GSK-3β and FOXO-1-mediated oxidative stress in the liver of glyphosate induced rats. Methods: We divided the experimental animals into three groups. Group 1 - control rats (animals were injected with olive oil (0.8ml) intraperitoneally), Group 2 - glyphosate-treated rats orally for ten weeks, Group 3 - glyphosate-treated rats received vitamin C and vitamin E. After 30 days of treatment, the animals were anesthetized, sera were separated and used for the biochemical analysis. Liver tissues from control and treated animals were dissected and stored at -20°C for further gene expression analysis. Fasting blood glucose (FBG) was assessed by calorimetric analysis, while serum insulin was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression studies of specific genes (FOXO1 and GSK3) were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

Results: The expression level of FOX01 and GSK3β genes was higher in glyphosate-induced animals compared with the control group but was reduced significantly (p＜0.05) upon treatment with antioxidant vitamins (C and E). Other biochemical parameters, including FBG, serum insulin, and antioxidant enzyme assays, also showed that antioxidant vitamins reduce glyphosate-induced insulin resistance and type-2 diabetes. Conclusion: The current study provides in vivo experimental evidence that antioxidant vitamins (C and E) reduce the glyphosate-mediated development of type-2 diabetes risk via the downregulation of FOX01 and GS-3β mRNA expression in the liver. Hence, vitamins C and E may be considered as therapeutics for the treatment of diabetes.

Keywords: antioxidant vitamins; diabetes; foxo; glyphosate; gsk3β.

Figure 1. Serum insulin level

Changes in the levels of serum insulin in normal, glyphosate, and vitamin C and E treated rats. Values are given as mean ± standard error of the mean. Significance at p<0.05. 'a': significantly different from the control group, 'ab': significantly different from the control and glyphosate-treated groups.

Figure 2. Fasting blood glucose (FBG) level

Changes in the FBG in normal, glyphosate, and vitamin C and E treated rats. Values are given as mean ± standard error of the mean. Significance at p<0.05. 'a': significantly different from the control group, 'ab': significantly different from the control and glyphosate-treated groups.

Figure 3. mRNA expression of glycogen synthase kinase-3 beta (GSK-3β)

Changes in the mRNA levels of GSK3β in normal, glyphosate, and vitamin C and E treated rats. Values are given as mean ± standard error of the mean. Significance at p<0.05. 'a': significantly different from the control group.

Figure 4. mRNA expression of forkhead box protein O1 (FOXO1)

Changes in the mRNA levels of FOXO1 in normal, glyphosate, and vitamin C and E treated rats. Values are given as mean ± standard error of the mean. Significance at p<0.05. 'a': significantly different from the control group.