The high-production volume fungicide pyraclostrobin induces triglyceride accumulation associated with mitochondrial dysfunction, and promotes adipocyte differentiation independent of PPARγ activation, in 3T3-L1 cells

Abstract

Pyraclostrobin is one of the most heavily used fungicides, and has been detected on a variety of produce, suggesting human exposure occurs regularly. Recently, pyraclostrobin exposure has been linked to a variety of toxic effects, including neurodegeneration and triglyceride (TG) accumulation. As pyraclostrobin inhibits electron transport chain complex III, and as mitochondrial dysfunction is associated with metabolic syndrome (cardiovascular disease, type II diabetes, obesity), we designed experiments to test the hypothesis that mitochondrial dysfunction underlies its adipogenic activity. 3T3-L1 cells were differentiated according to standard protocols in the presence of pyraclostrobin, resulting in TG accumulation. However, TG accumulation occurred without activation of the peroxisome proliferator activated nuclear receptor gamma (PPARγ), the canonical pathway mediating adipogenesis. Furthermore, cells failed to express many markers of adipogenesis (PPARγ, lpl, CEBPα), while co-exposure to pyraclostrobin and two different PPARγ antagonists (GW9662, T0070907) failed to mitigate TG accumulation, suggesting TG accumulation occurred through a PPARγ-independent mechanism. Instead, pyraclostrobin reduced steady-state ATP, mitochondrial membrane potential, basal mitochondrial respiration, ATP-linked respiration, and spare respiratory capacity, demonstrating mitochondrial dysfunction, while reduced expression of genes involved in glucose transport (Glut-4), glycolysis (Pkm, Pfkl, Pfkm), fatty acid oxidation (Cpt-1b), and lipogenesis (Fasn, Acacα, Acacβ) further suggested a disruption of metabolism. Finally, inhibition of cAMP responsive element binding protein (CREB), a PPARγ coactivator, partially mitigated pyraclostrobin-induced TG accumulation, suggesting TG accumulation is occurring through a CREB-driven mechanism. In contrast, rosiglitazone, a known PPARγ agonist, induced TG accumulation in a PPARγ-dependent manner and enhanced mitochondrial function. Collectively, these results suggest pyraclostrobin-induced mitochondrial dysfunction inhibits lipid homeostasis, resulting in TG accumulation. Exposures that disrupt mitochondrial function may have the potential to contribute to the rising incidence of metabolic syndrome, and thus more research is needed to understand the human health impact of pyraclostrobin exposure.

Keywords: Adipogenesis; Fungicide; Metabolic disruption; Mitochondrial toxicity; Pyraclostrobin.

Figure 1. Representative images of pyraclostrobin-induced triglyceride accumulation in 3T3-L1 cells

Lipid and DNA content is visualized with AdipoRed and NucBlue staining, respectively, in Zenbio 3T3-L1 cells differentiated according to standard protocols for ten days in the presence of 0.1% DMSO (vehicle control), or various concentrations of rosiglitazone or pyraclostrobin. Bright-field images were merged with a DAPI filter (to visualize NucBlue, blue) and a Green Fluorescent Protein filter (AdipoRed, green). Quantitative determination of lipid accumulation is shown in Fig. S1.

Figure 2. Pyraclostrobin-induced TG accumulation is independent of PPARγ activity

(A) Rosiglitazone (RSG) (one-way ANOVA (p<0.0001), N=4), but not pyraclostrobin (PYRA) (one-way ANOVA (p=0.89), N=4), increased PPARγ activity in a dose-dependent manner utilizing the Invitrogen GeneBLAzer PPARγ FRET reporter assay. Exposure to pyraclostrobin reduced (B) PPARγ (one-way ANOVA (p<0.0001), N=6), (C) Cebpα (transcription factor that regulates adipogenesis; one-way ANOVA (p<0.0001), N=6), and (D) Lpl (adipocyte marker; one-way ANOVA (p<0.0001), N=6) mRNA expression, while rosiglitazone increased the expression of all genes in Zenbio 3T3-L1 cells after ten days of differentiation. (E) The PPARγ antagonists GW9662 and T0070907 reduce RSG-induced TG accumulation (one-way ANOVA (p=0.02), N=3), while having no effect on PYRA-induced TG accumulation (one-way ANOVA (p=0.38), N=3). Asterisk denotes statistical significance (p<0.05) for post-hoc comparison (Dunnett’s test) to control. Bars±SEM.

Figure 3. Exposure to pyraclostrobin causes mitochondrial dysfunction

(A) Continuous exposure to pyraclostrobin (PYRA) throughout a ten-day differentiation protocol reduced steady-state ATP levels in Zenbio 3T3-L1 cells (one-way ANOVA (p<0.0001), N=3). Interestingly, 1.0 and 10.0 μM pyraclostrobin had opposing effects (increased (1.0 μM) or decreased (10 μM)) on (B) basal oxygen consumption rate (OCR; one-way ANOVA (p<0.0001), N=15), (C) ATP-linked OCR (a measure of the amount of oxygen consumption directly linked to ATP-production; one-way ANOVA (p<0.0001), N=15), (D) maximal OCR (a measure of the maximum rate at which mitochondria can function; one-way ANOVA (p<0.0001), N=15), (E) spare respiratory capacity (maximal OCR - basal OCR; one-way ANOVA (p<0.0001), N=15), and (F) proton leak (transport of protons across the inner mitochondrial membrane independent of ATP synthase activity; one-way ANOVA (p<0.0001), N=15). Asterisk denotes statistical significance (p<0.05) for post-hoc comparison (Dunnett’s test) to control. Bars±SEM. ¹Maximal and spare respiratory capacity could not be accurately measured for cells treated with 0.1 and 1.0 μM rosiglitazone (RSG), as cells rapidly depleted oxygen from the Seahorse XF^e microchamber resulting in anoxia.

Figure 4. Genes involved in glucose metabolism are down regulated following pyraclostrobin exposure

Continuous exposure to pyraclostrobin (PYRA) over the course of differentiation in Zenbio 3T3-L1 cells, and assessed ten days after induction, had no effect on mRNA expression of (A) Glut-1 (one-way ANOVA (p<0.0001)), while expression of (B) Glut-4 (one-way ANOVA (p<0.0001)), (C) Pkm (muscle isoform; involved in glycolysis; one-way ANOVA (p<0.0001)), (D) Pfkm (muscle isoform; involved in glycolysis; one-way ANOVA (p<0.0001)), and (E) Pfkl (liver isoform; involved in glycolysis; one-way ANOVA (p<0.0001)) were all down regulated. Alternatively, expression of all glucose transport and glycolysis genes were upregulated in response to rosiglitazone (RSG). Asterisk denotes statistical significance (p<0.05) for post-hoc comparison (Dunnett’s test) to control. N=6. Bars±SEM.

Figure 5. Pyraclostrobin increased the extracellular acidification rate

Continuous exposure to pyraclostrobin (PYRA) or rosiglitazone (RSG) increased the extracellular acidification rate (ECAR), an indirect measure of glycolysis, of Zenbio 3T3-L1 cells (one-way ANOVA, p<0.0001) after ten days of differentiation. Asterisk denotes statistical significance (p<0.05) for post-hoc comparison (Dunnett’s test) to control. Bars±SEM. N=15.

Figure 6. Genes involved in fatty acid metabolism are down regulated by pyraclostrobin

Exposure to pyraclostrobin (PYRA) over the course of a ten-day induction of differentiation in Zenbio 3T3-L1 cells decreased expression of (A) mitochondrial Cpt-1b (fatty acid oxidation gene; one-way ANOVA (p<0.0001)), (B) Fasn (fatty acid synthesis gene; one-way ANOVA (p<0.0001)), and (C) Acacα (fatty acid synthesis gene; one-way ANOVA (p<0.0001)) and (D) Acacβ (fatty acid synthesis gene; one-way ANOVA (p<0.0001)). Alternatively, expression of all fatty acid oxidation and synthesis genes were upregulated in response to rosiglitazone (RSG). Asterisk denotes statistical significance (p<0.05) for post-hoc comparison (Dunnett’s test) to control. N=6. Bars±SEM.

Figure 7. CREB plays a role in pyraclostrobin-induced TG accumulation

Inhibiting CREB activity with 5 μM 666-15 reduced rosiglitazone (one-way ANOVA (p=0.048)) and pyraclostrobin (one-way ANOVA (p=0.006))-induced TG accumulation. Data is shown as % control of TG accumulation in pyraclostrobin or rosiglitazone treated cells. Asterisk denotes statistical significance (p<0.05) for post-hoc comparison (Dunnett’s test) to control. N=3. Bars±SEM.

Figure 8. Canonical and proposed non-canonical pathways of triglyceride accumulation

(A) Upon agonist binding, PPARγ, in cooperation with its co-activators (i.e. PGC1α, CREB, Cebpα, RXR), binds to the promoter region of target genes and induces transcriptional changes that facilitate adipocyte differentiation and maturation, TG accumulation, and mitochondrial biogenesis. Mitochondrial retrograde signaling is also depicted, as recent evidence suggests mitochondrial signaling may play a role in adipocyte differentiation (Tormos et al., 2011). (B) Exposure to the electron transport chain inhibitor, antimycin A (AA), causes mitochondrial dysfunction that triggers CREB activation, while inhibiting adipocyte differentiation, PPARγ activation, FAO, and lipogenesis through traditional mechanisms (i.e. through the fatty acid synthase complex). CREB, a transcriptional regulator of lipid and glucose metabolism, promotes increased glucose uptake (via GLUT4) and glycolysis. The glycolytic intermediate, DHAP, can then be converted to G3P and used to fuel the re-esterification of free fatty acids (FFAs) resulting in TG formation and accumulation. Please note, pyruvate and Krebs cycle intermediates can also be used to generate G3P through a process known as glyceroneogenesis; however, radiolabeled glucose experiments suggest a direct glucose-to-TG conversion (Vankoningsloo et al., 2005). Given that pyraclostrobin and AA are both complex III inhibitors that induce similar phenotypes in 3T3-L1 cells, it is plausible that pyraclostrobin is inducing TG accumulation through a similar mechanism. Abbreviations: Cebpα, CCAAT/enhancer binding protein alpha; CREB, cAMP responsive element binding protein; DHAP, dihydroxyacetone phosphate; FAO, fatty acid β-oxidation; G3P, glycerol 3-phosphate; GLUT4, glucose transporter 4; MMP, mitochondrial membrane potential; OXPHOS, oxidative phosphorylation; PPARg, peroxisome proliferator activated nuclear receptor gamma; PCG1α, PPARg coactivator 1-alpha; RXR, retinoid X receptor.