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Knockdown of aquaporin-8 induces mitochondrial dysfunction in 3T3-L1 cells.

ABSTRACT: Background:Aquaporin-8 (AQP8), a member of the aquaporin water channel family, is expressed in various tissue and cells, including liver, testis, and pancreas. AQP8 appears to have functions on the plasma membrane and/or on the mitochondrial inner membrane. Mitochondrial AQP8 with permeability for water, H2O2 and NH3 has been expected to have important role in various cells, but its information is limited to a few tissues and cells including liver and kidney. In the present study, we found that AQP8 was expressed in the mitochondria in mouse adipose tissues and 3T3-L1 preadipocytes, and investigated its role by suppressing its gene expression. Methods:AQP8-knocked down (shAQP8) cells were established using a vector expressing short hairpin RNA. Cellular localization of AQP8 was examined by western blotting and immunocytochemistry. Mitochondrial function was assessed by measuring mitochondrial membrane potential, oxygen consumption and ATP level measurements. Results:In 3T3-L1 cells, AQP8 was expressed in the mitochondria. In shAQP8 cells, mRNA and protein levels of AQP8 were decreased by about 75%. The shAQP8 showed reduced activities of complex IV and ATP synthase; it is probable that the impaired mitochondrial water handling in shAQP8 caused suppression of the electron transport and ADP phosphorylation through inhibition of the two steps which yield water. The reduced activities of the last two steps of oxidative phosphorylation in shAQP8 cause low routine and maximum capacity of respiration and mitochondrial hyperpolarization. Conclusion:Mitochondrial AQP8 contributes to mitochondrial respiratory function probably through maintenance of water homeostasis. General significance:The AQP8-knocked down cells we established provides a model system for the studies on the relationships between water homeostasis and mitochondrial function.

SUBMITTER: Ikaga R

PROVIDER: S-EPMC5668916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 (PPAR?), the canonical pathway mediating adipogenesis. Furthermore, cells failed to express many markers of adipogenesis (PPAR?, lpl, CEBP?), while co-exposure to pyraclostrobin and two different PPAR? antagonists (GW9662, T0070907) failed to mitigate TG accumulation, suggesting TG accumulation occurred through a PPAR?-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, Acac?, Acac?) further suggested a disruption of metabolism. Finally, inhibition of cAMP responsive element binding protein (CREB), a PPAR? coactivator, partially mitigated pyraclostrobin-induced TG accumulation, suggesting TG accumulation is occurring through a CREB-driven mechanism. In contrast, rosiglitazone, a known PPAR? agonist, induced TG accumulation in a PPAR?-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.

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Knockdown of aquaporin-8 induces mitochondrial dysfunction in 3T3-L1 cells.

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