The pesticide chlorpyrifos promotes obesity by inhibiting diet-induced thermogenesis in brown adipose tissue

Obesity results from a caloric imbalance between energy intake, absorption and expenditure. In both rodents and humans, diet-induced thermogenesis contributes to energy expenditure and involves the activation of brown adipose tissue (BAT). We hypothesize that environmental toxicants commonly used as food additives or pesticides might reduce BAT thermogenesis through suppression of uncoupling protein 1 (UCP1) and this may contribute to the development of obesity. Using a step-wise screening approach, we discover that the organophosphate insecticide chlorpyrifos suppresses UCP1 and mitochondrial respiration in BAT at concentrations as low as 1 pM. In mice housed at thermoneutrality and fed a high-fat diet, chlorpyrifos impairs BAT mitochondrial function and diet-induced thermogenesis, promoting greater obesity, non-alcoholic fatty liver disease (NAFLD) and insulin resistance. This is associated with reductions in cAMP; activation of p38MAPK and AMPK; protein kinases critical for maintaining UCP1 and mitophagy, respectively in BAT. These data indicate that the commonly used pesticide chlorpyrifos, suppresses diet-induced thermogenesis and the activation of BAT, suggesting its use may contribute to the obesity epidemic.

. CPF intake and the Effects of CPF on AChE activity. a, Chlorpyrifos (CPF) intake of C57BL/6J male mice fed with CD (10 kcal% fat) or HFD (45 kcal% fat) supplemented with 0 (Control), 0.5 mg/kg/BW (CPF-0.5) or 2.0 mg/kg/BW (CPF-2.0) at room temperature (RT,22 °C) or thermoneutrality (TN, 30 °C), n = 5. b, AChE activity assessed in hindlimb skeletal muscle of CPF treated mice, n = 12. c, ChE activity in serum of C57BL/6J mice at 4h post gavage of 100 mg/kg CPF (n = 6) or after treatment with 2.0 mg/kg/day CPF for 3 weeks (n = 10), relative to basal. Significant differences between 3 or more mean values were determined by one-way ANOVA with the post hoc Bonferroni's multiple comparisons test; differences between 2 mean values were determined by two-tailed Student's t-test. Data presented are mean ± SEM.

Supplementary Figure 3. Effects of CPF on body weight, fat mass and glucose metabolism of mice fed a control diet.
C57BL/6J male mice were treated with CD (10 kcal% fat) supplemented with 0 (Control), 0.5 mg/kg/BW (CPF-0.5) or 2.0 mg/kg/BW (CPF-2.0) at room temperature (RT, 22 °C) or thermoneutrality (TN, 30 °C). a-b, Body weight of mice at RT (a) or TN (b). c-d, Fat mass of mice at RT (c) or TN (d). e-f, Glucose tolerance test (GTT) of mice at RT (e) or TN (f). gh, Insulin tolerance test (ITT) of mice at RT (g) or TN (h). Significant differences between mean values were determined by one-way ANOVA with Tukey's multiple comparisons test. Significant differences between mean values were determined by one-way ANOVA with the post hoc Bonferroni's multiple comparisons test. Data presented are mean ± SEM, n = 10.

Supplementary Figure 4. Effects of CPF on body weight and glucose metabolism of mice fed a high-fat diet at room temperature.
C57BL/6J male mice were treated with HFD (45 kcal% fat) supplemented with 0 (Control), 0.5 mg/kg/BW (CPF-0.5) or 2.0 mg/kg/BW (CPF-2.0) at room temperature (RT, 22 °C). a, Body weight (BW). b, Fat mass. c, Glucose tolerance test (GTT). d, Insulin tolerance test (ITT). e, Fat tissues weight. f, Representative images of inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) adipocytes and the quantification of adipocyte size. Significant differences between mean values were determined by one-way ANOVA with the post hoc Bonferroni's multiple comparisons test. Data presented are mean ± SEM, n = 10, * p < 0.05. Scale bar = 100 μm.

Supplementary Figure 5. Effects of CPF on NAFLD in mice fed a high-fat diet at room temperature.
C57BL/6J male mice were treated with HFD (45 kcal% fat) supplemented with 0 (Control), 0.5 mg/kg/BW (CPF-0.5) or 2.0 mg/kg/BW (CPF-2.0) at room temperature (RT, 22 °C). a, Liver weight. b, Liver TG. c, Serum alanine aminotransferase (ALT) level. d, aspartate aminotransferase (AST) level. e, Representative images of Oil Red O stained liver lipids. Significant differences between mean values were determined by one-way ANOVA with the post hoc Bonferroni's multiple comparisons test. Data presented are mean ± SEM, n = 10. Scale bar = 100 μm.

Supplementary Figure 6. Effects of CPF on NAFLD in mice fed a control diet.
C57BL/6J male mice were treated with CD (10 kcal% fat) supplemented with 0 (Control), 0.5 mg/kg/BW (CPF-0.5) or 2.0 mg/kg/BW (CPF-2.0) at room temperature (RT, 22 °C) or thermoneutrality (TN, 30 °C). a-b, Liver TG of mice at RT (a) or TN (b). c-d, Serum alanine aminotransferase (ALT) level of mice at RT (c) or TN (d). e-f, serum free fatty acid (FFA) of mice at RT (e) or TN (f). g-h, serum triacylglycerol (TG) of mice at RT (g) or TN (h). i-j, mRNA level of brown adipose genes in BAT of mice at RT (i) or TN (j). k, Representative images of Oil Red O stained liver lipids Significant differences between mean values were determined by one-way ANOVA with the post hoc Bonferroni's multiple comparisons test. Data presented are mean ± SEM, n = 10. Scale bar = 100 μm.

Supplementary Figure 7. Effects of CPF on BAT of mice fed a HFD at room temperature.
C57BL/6J male mice were treated with HFD (45 kcal% fat) supplemented with two doses of CPF at room temperature (RT). a, UCP1 protein content in BAT. b, Representative immunohistochemistry images showing UCP1 in BAT and H&E images, Scale bar = 100 μm. c, Representative electron micrographs for mitochondria, scale bar = 2 μm. Significant differences between mean values were determined by one-way ANOVA with the post hoc Bonferroni's multiple comparisons test. Data presented are mean ± SEM, n = 10.