Gut microbial degradation of organophosphate insecticides-induces glucose intolerance via gluconeogenesis

Abstract

Background: Organophosphates are the most frequently and largely applied insecticide in the world due to their biodegradable nature. Gut microbes were shown to degrade organophosphates and cause intestinal dysfunction. The diabetogenic nature of organophosphates was recently reported but the underlying molecular mechanism is unclear. We aimed to understand the role of gut microbiota in organophosphate-induced hyperglycemia and to unravel the molecular mechanism behind this process.

Results: Here we demonstrate a high prevalence of diabetes among people directly exposed to organophosphates in rural India (n = 3080). Correlation and linear regression analysis reveal a strong association between plasma organophosphate residues and HbA1c but no association with acetylcholine esterase was noticed. Chronic treatment of mice with organophosphate for 180 days confirms the induction of glucose intolerance with no significant change in acetylcholine esterase. Further fecal transplantation and culture transplantation experiments confirm the involvement of gut microbiota in organophosphate-induced glucose intolerance. Intestinal metatranscriptomic and host metabolomic analyses reveal that gut microbial organophosphate degradation produces short chain fatty acids like acetic acid, which induces gluconeogenesis and thereby accounts for glucose intolerance. Plasma organophosphate residues are positively correlated with fecal esterase activity and acetate level of human diabetes.

Conclusion: Collectively, our results implicate gluconeogenesis as the key mechanism behind organophosphate-induced hyperglycemia, mediated by the organophosphate-degrading potential of gut microbiota. This study reveals the gut microbiome-mediated diabetogenic nature of organophosphates and hence that the usage of these insecticides should be reconsidered.

Keywords: Acetic acid; Diabetes; Fecal transplantation; Gluconeogenesis; Glucose intolerance; Gut microbiota; Metabolomics; Metatranscriptomics; Organophosphates.

Fig. 1

Blood plasma OP residues correlate with self-reported exposure and diabetic status. a Prevalence of diabetics among humans exposed to OP (n =1686) and not directly exposed to OP (n = 1394). The percentage of diabetic prevalence and non-prevalence are mentioned in the bars. b Plasma acetylcholine esterase (AChE) of people indirectly exposed (n = 303) and directly exposed (n = 499) to OP. Dotted lines represent the reference values for males (green) and females (pink). Regression plot of plasma OP residues vs. blood HbA_1c indirectly exposed (n = 303) and directly exposed (n = 499) to (c) MCP, (d) CHL, (e) MAL, (f) MPAR. Horizontal lines represent mean; error bars represent s.e.m; *P < 0.05 Rank sum, Mann–Whitney U Test (b). The hollow circle represents individual values and straight line represents the trend line. *P < 0.05; **P < 0.01. PCC Pearson correlation coefficient, β regression coefficient (c–f)

Fig. 2

Chronic intake of OP-induces hyperglycemia and glucose intolerance leading to oxidative stress a Periodical fasting blood glucose of animals drinking pure water or MCP mixed water (n = 09). b Periodical plasma AChE level of animals drinking pure water or MCP mixed water (n = 10). c Oral glucose tolerance test (OGTT) of animals drinking pure water or MCP mixed water after 180 days (n = 09). d Serum lipid peroxidation level of animals after 180 days drinking pure water or MCP mixed water (n = 09). Horizontal lines or symbols represent mean; error bars represent s.e.m; ****P < 0.0001, **P < 0.01, P < 0.05. Unpaired two-sided student-t test. Experiments were repeated twice/thrice

Fig. 3

OP-induced glucose intolerance is mediated by gut microbiome a OGTT of animals following transplant of microbiota for seven days from pure water or MCP mixed water drinking mice (n = 08). b OGTT of animals following seven days of transplantation of fecal cultures grown in presence of OP (n = 06). Horizontal lines or symbols represent mean; error bars represent s.e.m; ****P < 0.0001, **P < 0.01, P < 0.05. Unpaired two-sided student-t test (a) or two-way ANOVA with Bonferroni correction (b). Experiments were repeated twice

Fig. 4

Chronic OP exposure activates the gut microbiome xenobiotic metabolism genes. a Percentage of normalized counts assigned to each KEGG category module. b Expression profile of OP degrading genes expressed as reads per kilomillion counts (RPKM) (n = 3). c OGTT of animals fed with fecal culture/suspended cells/supernatant grown in the presence or absence of MCP (n = 10). d Fecal esterase activity of the animals fed with fecal culture/suspended cells/supernatant grown in the presence or absence of MCP (n = 6). Horizontal lines, bars, or symbols represent mean; error bars represent s.e.m; ***P < 0.001, *P < 0.05. Unpaired two-sided student-t test (b, d) or two-way ANOVA with Bonferroni correction (c). Experiments were repeated twice

Fig. 5

Gut microbial degradation of OP-induces gluconeogenesis. a Top five metabolic pathways represented by quantitative MSEA. The P values of Q-statistics are mentioned at the end of bars. b Expression of metabolites associated with gluconeogenesis expressed as normalized peak area (n = 3). c Intestinal and (d) Hepatic glucose-6 phosphatase activity of animals fed with fecal whole culture or suspended cells or culture supernatant grown in the presence or absence of MCP (n = 06). e Fecal acetate level of the animals fed with fecal whole culture or suspended cells or culture supernatant grown in the presence or absence of MCP (n = 03). f OGTT of animals treated with sodium acetate (NaAc) orally and by rectal infusion (RI) (n = 08). g Intestinal and hepatic glucose-6 phosphatase activity of NaAc treated animals (n = 08). Bars, horizontal lines, or symbols represent mean; error bars represent s.e.m; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 Two-way ANOVA with Bonferroni correction (f) or one-way ANOVA with Tukey post-hoc analysis (g) or unpaired two-sided Student’s t-test (b–e). Experiments were repeated twice

Fig. 6

Plasma OP residues correlate with fecal esterase activity and fecal acetate. Fecal samples were collected from control (n = 60) and diabetic (n = 60) humans. a Fecal esterase activity of non-diabetic vs. diabetic individuals. b Regression plot of plasma total OPs vs. fecal esterase activity. c Fecal acetate level of non-diabetic vs. diabetic. d Regression plot of plasma total OPs vs. fecal acetate content. Horizontal lines represent mean; error bars represent s.e.m; *P < 0.05 Rank sum, Mann–Whitney U Test (a, c). The hollow circle represents individual values and straight line represents the trend line. PCC Pearson correlation coefficient, β regression coefficient. *P < 0.05; **P < 0.01

Fig. 7

Schematic summary of the molecular mechanism behind gut microbiome mediated OP-induced glucose intolerance. OPs (star) enter the human digestive system via food and are metabolized into acetic acid (trapezoid) by the gut microbiota (oval). Subsequently, acetic acid was absorbed by the intestinal cells and the majority of them were transported to the liver through the periportal vein. Eventually, acetic acid was converted into glucose (hexagon) by gluconeogenesis in the intestine and liver and thus accounts for glucose intolerance