Mitochondrial complex III ROS regulate adipocyte differentiation

Abstract

Adipocyte differentiation is characterized by an increase in mitochondrial metabolism. However, it is not known whether the increase in mitochondrial metabolism is essential for differentiation or a byproduct of the differentiation process. Here, we report that primary human mesenchymal stem cells undergoing differentiation into adipocytes display an early increase in mitochondrial metabolism, biogenesis, and reactive oxygen species (ROS) generation. This early increase in mitochondrial metabolism and ROS generation was dependent on mTORC1 signaling. Mitochondrial-targeted antioxidants inhibited adipocyte differentiation, which was rescued by the addition of exogenous hydrogen peroxide. Genetic manipulation of mitochondrial complex III revealed that ROS generated from this complex is required to initiate adipocyte differentiation. These results indicate that mitochondrial metabolism and ROS generation are not simply a consequence of differentiation but are a causal factor in promoting adipocyte differentiation.

Figure 1. Mitochondrial targeted antioxidants diminish adipocyte differentiation

(A) Mitochondrial oxygen consumption rates of human MSCs at day 0 (four hours), 2, and 7 of adipocyte differentiation. Cells were treated with FCCP (10 μM) to obtain maximal oxygen consumption rates. N=4 ± SEM. (B) Intracellular H₂O₂ levels of human MSCs at Day 0 (4 hours) and Day 2 of differentiation. Cells were treated with 500 nM of TPP or MitoCP for 4 hours prior to measurement on day 2. N=4 ± SEM (C) Mitochondrial targeted antioxidants MitoCP and MCTPO attenuate lipid accumulation. Human MSCs were treated with 500nM of TPP control and mitochondrial targeted antioxidants MitoCP or MitoCTPO starting at day 2 of differentiation. Subsequently, optical density (OD) values of Oil Red O were assessed at day 21. N=3± SEM. (D) Mitochondrial targeted antioxidants MitoQ and Mito-Tempol diminish lipid accumulation. Human MSCs were treated starting at day 2 of differentiation with 500 nM of Mito-Tempol C₁₀, Mito-Q, Mito CP or TPP control. Subsequently, at day 21 of differentiation cells were stained with Oil Red O and optical density (OD) values were assessed. N=3 ± SEM. (E) Western blot analysis of PPARγ and C/EBPα protein at days 2, 5, and 7 of differentiation in human MSCs treated with 500 nM of MitoCP or TPP starting at day 2. Day 2 cells were lysed after 4 hours of MitoCP or TPP treatment. (F) Gene expression of PPARγ2 and its target genes at day 7 of differentiation in human MSCs treated with 500 nM MitoCP or TPP starting at day 2. N=3 ± SEM.

Figure 2. Exogenous PPARγ or H₂O₂ rescues adipocyte differentiation in the presence of mitochondrial targeted antioxidants

(A) Human MSCs were infected with AdPPARγ2 or AdNull at day 1 and treated with TPP (500nM) or MitoCP (500nM) +/− 5 μM troglitazone (Tro) starting at day 2. Subsequently, at day 21 of differentiation cells were stained with Oil Red O and optical density (OD) values were assessed. N=3 ± SEM. (B) Gene expression of PPARγ2 and its target genes at day 7 of differentiation in human MSCs infected with AdPPARγ2 or AdNull at day 1 and treated with TPP (500nM) or MitoCP (500nM) +/− 5 μM troglitazone (Tro.) starting at day 2. N=3 ± SEM. (C) Western blot analysis of PPARγ protein at Day 5 of adipocyte differentiation. Human MSCs were infected with AdNull or AdPPARγ2 at day 1 and administered 500 nM of MitoCP or TPP control starting at day 2. (D) Intracellular H₂O₂ levels of human MSCs treated with galactose (0.5mM) with or without galactose oxidase (GAO, 0.015U/ml) in the presence of 500 nM of TPP or MitoCP starting at Day 2. N=3 ± SEM. (E) Human MSCs were treated with galactose (0.5mM) with or without GAO (0.015U/ml) plus 500 nM of TPP or MitoCP starting at day 2. Subsequently, at day 21 of differentiation cells were stained with Oil Red O and optical density (OD) values were assessed. N=3 ± SEM. (F) Human MSCs were treated with galactose (0.5mM) with or without GAO (.015U/ml) plus 500 nM TPP or MitoCP starting at day 2. Gene expression was analyzed at day 7 of differentiation. N=3 ± SEM.

Figure 3. Mitochondrial complex III ROS are required for adipocyte differentiation

(A) Mitochondrial complex III generates ROS through the ubiquinone (Q) cycle. The Q cycle generates superoxide through the donation of an electron from ubisemiquinone (Q^.) to oxygen. Loss of RISP subunit abolishes the generation of Q^. thus superoxide. By contrast, loss of the QPc subunit maintains superoxide production since it functions downstream from the formation of Q^. and superoxide. (B) Western blot showing efficacy of stably transfected shRNA targeted against RISP or QPc in human MSCs. (C) Mitochondrial oxygen consumption rate is decreased in RISP and QPC knockdown MSCs. Cells were treated with FCCP (10μM) to obtain maximal oxygen consumption rates. N=3 ± SEM. D) Intracellular H₂O₂ measured at Day 2 of differentiation in human MSCs infected with RISP or QPc shRNA. N=5 ± SEM. E) Real time PCR analysis of PPARγ2 and its target genes at day 7 of differentiation in human MSCs inflected with scrambled, RISP or QPc shRNA. N=3 ± SEM.

Figure 4. mTORC1 positively regulates ROS production during adipocyte differentiation

(A) Western blot analysis for PPARγ expression and C/EBPα expression of human MSCs treated with LY294002 (20 μM) or rapamycin (10 nM) starting at day 2 of differentiation. Day 2 cells were treated for four hours prior to protein isolation. (B) Mitochondrial oxygen consumption rate was analyzed at day 7 of adipocyte differentiation in human MSCs treated with DMSO, LY-294002 (20 μM) or rapamycin (10nM) at day 2. Cells were treated with FCCP (10 μM) to obtain maximal oxygen consumption rates. N=4 ± SEM. (C) Amplex Red analysis of intracellular H₂O₂ assessed at Day 3 in human MSCs treated with DMSO, rapamycin (10 nM), and LY294002 (20 μM) starting at day 2. N=4 ± SEM. (D) Mitochondrial copy number as assessed by the ratio of mitochondrial gene COXI to nuclear gene PPRC1 at Day 0 (4 hours) and Day 3 of differentiation. Rapamycin (10 nM) was added at starting at day 2. N=6 ± SEM. (E) Western blot showing efficacy of transfected shRNA targeted against raptor in human MSCs. (F) Intracellular H₂O₂ measured at Day 2 of differentiation is decreased in human MSCs infected with raptor shRNA. N=5 ± SEM. (G) mTORC1 dependent increase in mitochondrial complex III ROS is required for PPARγ dependent transcription during adipocyte differentiation.