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Chronic exposure to the herbicide, atrazine, causes mitochondrial dysfunction and insulin resistance.

Lim S, Ahn SY, Song IC, Chung MH, Jang HC, Park KS, Lee KU, Pak YK, Lee HK - PLoS ONE (2009)

Bottom Line: ATZ blocked the activities of oxidative phosphorylation complexes I and III, resulting in decreased oxygen consumption.It also suppressed the insulin-mediated phosphorylation of Akt.These results suggest that long-term exposure to the herbicide ATZ might contribute to the development of insulin resistance and obesity, particularly where a high-fat diet is prevalent.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
There is an apparent overlap between areas in the USA where the herbicide, atrazine (ATZ), is heavily used and obesity-prevalence maps of people with a BMI over 30. Given that herbicides act on photosystem II of the thylakoid membrane of chloroplasts, which have a functional structure similar to mitochondria, we investigated whether chronic exposure to low concentrations of ATZ might cause obesity or insulin resistance by damaging mitochondrial function. Sprague-Dawley rats (n = 48) were treated for 5 months with low concentrations (30 or 300 microg kg(-1) day(-1)) of ATZ provided in drinking water. One group of animals was fed a regular diet for the entire period, and another group of animals was fed a high-fat diet (40% fat) for 2 months after 3 months of regular diet. Various parameters of insulin resistance were measured. Morphology and functional activities of mitochondria were evaluated in tissues of ATZ-exposed animals and in isolated mitochondria. Chronic administration of ATZ decreased basal metabolic rate, and increased body weight, intra-abdominal fat and insulin resistance without changing food intake or physical activity level. A high-fat diet further exacerbated insulin resistance and obesity. Mitochondria in skeletal muscle and liver of ATZ-treated rats were swollen with disrupted cristae. ATZ blocked the activities of oxidative phosphorylation complexes I and III, resulting in decreased oxygen consumption. It also suppressed the insulin-mediated phosphorylation of Akt. These results suggest that long-term exposure to the herbicide ATZ might contribute to the development of insulin resistance and obesity, particularly where a high-fat diet is prevalent.

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Decrease in endogenous oxygen consumption by ATZ in L6 cells.L6 rat skeletal muscle cells were treated for 48 hours with or without ATZ (100 µg/mL). (A) Endogenous cellular (coupled), FCCP-uncoupled (FCCP) and KCN-insensitive respiration (KCN) of trypsinized intact cells were measured in phenol red-free media using an Oxygraph-2K apparatus (*P<0.05; n = 3). (B) Western analysis of nDNA- and mtDNA-encoded OXPHOS complex subunit proteins. Complex I (ND9), complex II (SDHA), complex III (UQCRC2) and complex IV (COXI, mtDNA-encoded, COXIV, nDNA-encoded) were examined. β-actin was used as an equal loading control. (C) Oxygen consumption by each complex in digitonin-permeabilized cells was measured using an Oxygraph-2K apparatus (P<0.05; n = 4). (D) Enzyme activities of complex II or III were determined by spectrophotometry in ATZ-treated L6 muscle cells (*P<0.05; n = 3).
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pone-0005186-g005: Decrease in endogenous oxygen consumption by ATZ in L6 cells.L6 rat skeletal muscle cells were treated for 48 hours with or without ATZ (100 µg/mL). (A) Endogenous cellular (coupled), FCCP-uncoupled (FCCP) and KCN-insensitive respiration (KCN) of trypsinized intact cells were measured in phenol red-free media using an Oxygraph-2K apparatus (*P<0.05; n = 3). (B) Western analysis of nDNA- and mtDNA-encoded OXPHOS complex subunit proteins. Complex I (ND9), complex II (SDHA), complex III (UQCRC2) and complex IV (COXI, mtDNA-encoded, COXIV, nDNA-encoded) were examined. β-actin was used as an equal loading control. (C) Oxygen consumption by each complex in digitonin-permeabilized cells was measured using an Oxygraph-2K apparatus (P<0.05; n = 4). (D) Enzyme activities of complex II or III were determined by spectrophotometry in ATZ-treated L6 muscle cells (*P<0.05; n = 3).

Mentions: Changes in cellular respiration and differences in the respiratory effects of inhibitors are important indicators of mitochondrial functional defects that result from damaged mitochondrial proteins or DNA (mtDNA), or substantial alterations to mitochondrial signaling cascades. To test if ATZ impaired mitochondrial function directly or indirectly, we monitored both cellular and mitochondrial respiration levels in ATZ-treated, cultured L6 muscle cells and isolated mitochondria. Incubation of L6 muscle cells with ATZ decreased both endogenous coupled and FCCP-uncoupled oxygen consumption by 30% and 40%, respectively (Fig. 5A). ATZ had no effect on the levels of nuclear DNA (nDNA)- or mtDNA-encoded OXPHOS complex proteins (Fig. 5B). When oxygen consumption was measured using digitonin-permeabilized cells, respiration levels in complex II plus III were inhibited by ATZ (Fig. 5C), similar to the in vivo results (see Fig. 4C). Enzyme activities of complex III (cytochrome bc1) were also decreased by ATZ treatment (Fig. 5D). Direct treatment of isolated mouse liver mitochondria with ATZ also reduced the OCR of complex I and complex II plus III by 49% and 37%, respectively (Fig. 6A); the magnitude of these ATZ effects were greater in isolated mitochondria than in cells. Complex IV activities were not significantly changed. Again, consistent with the results of the in vivo study, SDH enzymatic activity was not changed, but the activity of the cytochrome bc1 complex was decreased significantly by ATZ treatment (Fig. 6B). These results strongly suggest that ATZ itself, not ATZ metabolites, intervenes directly at Q binding sites between complex I and III, or II and III.


Chronic exposure to the herbicide, atrazine, causes mitochondrial dysfunction and insulin resistance.

Lim S, Ahn SY, Song IC, Chung MH, Jang HC, Park KS, Lee KU, Pak YK, Lee HK - PLoS ONE (2009)

Decrease in endogenous oxygen consumption by ATZ in L6 cells.L6 rat skeletal muscle cells were treated for 48 hours with or without ATZ (100 µg/mL). (A) Endogenous cellular (coupled), FCCP-uncoupled (FCCP) and KCN-insensitive respiration (KCN) of trypsinized intact cells were measured in phenol red-free media using an Oxygraph-2K apparatus (*P<0.05; n = 3). (B) Western analysis of nDNA- and mtDNA-encoded OXPHOS complex subunit proteins. Complex I (ND9), complex II (SDHA), complex III (UQCRC2) and complex IV (COXI, mtDNA-encoded, COXIV, nDNA-encoded) were examined. β-actin was used as an equal loading control. (C) Oxygen consumption by each complex in digitonin-permeabilized cells was measured using an Oxygraph-2K apparatus (P<0.05; n = 4). (D) Enzyme activities of complex II or III were determined by spectrophotometry in ATZ-treated L6 muscle cells (*P<0.05; n = 3).
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Related In: Results  -  Collection

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pone-0005186-g005: Decrease in endogenous oxygen consumption by ATZ in L6 cells.L6 rat skeletal muscle cells were treated for 48 hours with or without ATZ (100 µg/mL). (A) Endogenous cellular (coupled), FCCP-uncoupled (FCCP) and KCN-insensitive respiration (KCN) of trypsinized intact cells were measured in phenol red-free media using an Oxygraph-2K apparatus (*P<0.05; n = 3). (B) Western analysis of nDNA- and mtDNA-encoded OXPHOS complex subunit proteins. Complex I (ND9), complex II (SDHA), complex III (UQCRC2) and complex IV (COXI, mtDNA-encoded, COXIV, nDNA-encoded) were examined. β-actin was used as an equal loading control. (C) Oxygen consumption by each complex in digitonin-permeabilized cells was measured using an Oxygraph-2K apparatus (P<0.05; n = 4). (D) Enzyme activities of complex II or III were determined by spectrophotometry in ATZ-treated L6 muscle cells (*P<0.05; n = 3).
Mentions: Changes in cellular respiration and differences in the respiratory effects of inhibitors are important indicators of mitochondrial functional defects that result from damaged mitochondrial proteins or DNA (mtDNA), or substantial alterations to mitochondrial signaling cascades. To test if ATZ impaired mitochondrial function directly or indirectly, we monitored both cellular and mitochondrial respiration levels in ATZ-treated, cultured L6 muscle cells and isolated mitochondria. Incubation of L6 muscle cells with ATZ decreased both endogenous coupled and FCCP-uncoupled oxygen consumption by 30% and 40%, respectively (Fig. 5A). ATZ had no effect on the levels of nuclear DNA (nDNA)- or mtDNA-encoded OXPHOS complex proteins (Fig. 5B). When oxygen consumption was measured using digitonin-permeabilized cells, respiration levels in complex II plus III were inhibited by ATZ (Fig. 5C), similar to the in vivo results (see Fig. 4C). Enzyme activities of complex III (cytochrome bc1) were also decreased by ATZ treatment (Fig. 5D). Direct treatment of isolated mouse liver mitochondria with ATZ also reduced the OCR of complex I and complex II plus III by 49% and 37%, respectively (Fig. 6A); the magnitude of these ATZ effects were greater in isolated mitochondria than in cells. Complex IV activities were not significantly changed. Again, consistent with the results of the in vivo study, SDH enzymatic activity was not changed, but the activity of the cytochrome bc1 complex was decreased significantly by ATZ treatment (Fig. 6B). These results strongly suggest that ATZ itself, not ATZ metabolites, intervenes directly at Q binding sites between complex I and III, or II and III.

Bottom Line: ATZ blocked the activities of oxidative phosphorylation complexes I and III, resulting in decreased oxygen consumption.It also suppressed the insulin-mediated phosphorylation of Akt.These results suggest that long-term exposure to the herbicide ATZ might contribute to the development of insulin resistance and obesity, particularly where a high-fat diet is prevalent.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
There is an apparent overlap between areas in the USA where the herbicide, atrazine (ATZ), is heavily used and obesity-prevalence maps of people with a BMI over 30. Given that herbicides act on photosystem II of the thylakoid membrane of chloroplasts, which have a functional structure similar to mitochondria, we investigated whether chronic exposure to low concentrations of ATZ might cause obesity or insulin resistance by damaging mitochondrial function. Sprague-Dawley rats (n = 48) were treated for 5 months with low concentrations (30 or 300 microg kg(-1) day(-1)) of ATZ provided in drinking water. One group of animals was fed a regular diet for the entire period, and another group of animals was fed a high-fat diet (40% fat) for 2 months after 3 months of regular diet. Various parameters of insulin resistance were measured. Morphology and functional activities of mitochondria were evaluated in tissues of ATZ-exposed animals and in isolated mitochondria. Chronic administration of ATZ decreased basal metabolic rate, and increased body weight, intra-abdominal fat and insulin resistance without changing food intake or physical activity level. A high-fat diet further exacerbated insulin resistance and obesity. Mitochondria in skeletal muscle and liver of ATZ-treated rats were swollen with disrupted cristae. ATZ blocked the activities of oxidative phosphorylation complexes I and III, resulting in decreased oxygen consumption. It also suppressed the insulin-mediated phosphorylation of Akt. These results suggest that long-term exposure to the herbicide ATZ might contribute to the development of insulin resistance and obesity, particularly where a high-fat diet is prevalent.

Show MeSH
Related in: MedlinePlus