Ecovered aqueous phase was further centrifuged at 10,0006 g to obtain mitochondrial

Ecovered aqueous phase was Epigenetic Reader Domain further centrifuged at 10,0006 g to obtain mitochondrial pellet. The pellet was suspended in the extraction buffer containing 2 digitonin. BN-PAGE analysis was performed by the method as shown previously [35]. When immunoblot 22948146 analysis was performed, proteins in the gel were transferred to PVDF membrane. After 100 methanol treatment, the membrane was washed with water, and then subjected to immunoblot analysis with anti-NDUFA9 antibody (Invitrogen), anti-ATP5A1 antibody (Invitrogen), or anti-Tom40 antibody [34].LPL Activity and Fatty Acid Uptake Are Reduced in WAT of SMS1-KO MiceWe next assessed triglyceride levels in blood plasma. Those concentrations were much higher in SMS1-KO compared to wildtype mice (Fig. 2A), suggesting that metabolic functions of liver and/or WAT are perturbed. LPL plays a critical role in triglyceride homeostasis by catalyzing hydrolysis of triglyceride from plasma lipoproteins [31]. LPL activity in both WAT and liver (Fig. 2B) was significantly reduced in SMS1-KO relative to wild-type mice, although the reduction was more severe in WAT than in liver. We next determined whether fatty acid uptake is altered in WAT and liver of SMS1-KO mice in vivo by assessing radioactivity levels in these tissues after intraperitoneal injection of radiolabeled palmitic acid. After 30 min, wild-type mice showed high radioactivity in both WAT and liver. By contrast, SMS1-KO mice showed relatively lower radioactivity in WAT after 30 min, although radioactivity in liver was comparable to that in wild-type mice. By 48 h, radioactivity in wild-type and SMS1-KO liver was almost completely absent, whereas a larger portion of radioactivity remained in WAT of both genotypes (Fig. 2C). We also undertook assays to evaluate fatty acid uptake in vitro and observed a slight but significant reduction of palmitate uptake in SMS1-deficient MEFs (Fig. 2D). Overall, these results suggest that incorporation of palmitate into WAT rather than liver of SMS1-KO mice is disturbed due to deficient fatty acid uptake function.Measurement of Mitochondrial Respiratory Chain ActivityThe gel slices obtained by BN-PAGE was used to detect mitochondrial respiratory chain activity as described [36]. Complex IV activity (cytochrome oxidase activity) was examined by incubating gel slices in the reaction buffer IV (50 mM sodium phosphate buffer (pH 7.4), 1 mg/ml DAB, 24 units/ml catalase, 1 mg/ml cytochrome c, 0.22 M sucrose). Color development was preserved in fixing buffer (50 methanol, 10 acetic acid), and the gel was stored in 10 acetic acid. Complex V activity (Epigenetic Reader Domain ATPase activity) was assessed by incubating gel slices in the reaction buffer V (35 mM Tris, 270 mM glycine, 14 mM MgSO4, 0.2 Pb(NO3)2, and 8 mM ATP). After overnight incubation, the color-developed gel was washed and stored in water. The remaining gel slice was stained with Coomassie Brilliant blue (CBB).SMS1-KO WAT Is Severely Damaged by Oxidative StressPreviously we observed that islet cells in SMS1-KO mice were chronically damaged by oxidative stress [28]. Therefore, we asked whether oxidative stress also damaged WAT of mutant mice. Immunoblot analysis using anti-2,4-dinitrophenyl (DNP) antibody,Statistical AnalysisData were analyzed using Student’s t-test and reported as means 6 SEM, unless otherwise stated.SMS1 in Adipose Tissue FunctionSMS1 in Adipose Tissue FunctionFigure 1. SMS1-KO mice exhibit a lipodystrophic phenotype. (A) Representative CT images of the lower a.Ecovered aqueous phase was further centrifuged at 10,0006 g to obtain mitochondrial pellet. The pellet was suspended in the extraction buffer containing 2 digitonin. BN-PAGE analysis was performed by the method as shown previously [35]. When immunoblot 22948146 analysis was performed, proteins in the gel were transferred to PVDF membrane. After 100 methanol treatment, the membrane was washed with water, and then subjected to immunoblot analysis with anti-NDUFA9 antibody (Invitrogen), anti-ATP5A1 antibody (Invitrogen), or anti-Tom40 antibody [34].LPL Activity and Fatty Acid Uptake Are Reduced in WAT of SMS1-KO MiceWe next assessed triglyceride levels in blood plasma. Those concentrations were much higher in SMS1-KO compared to wildtype mice (Fig. 2A), suggesting that metabolic functions of liver and/or WAT are perturbed. LPL plays a critical role in triglyceride homeostasis by catalyzing hydrolysis of triglyceride from plasma lipoproteins [31]. LPL activity in both WAT and liver (Fig. 2B) was significantly reduced in SMS1-KO relative to wild-type mice, although the reduction was more severe in WAT than in liver. We next determined whether fatty acid uptake is altered in WAT and liver of SMS1-KO mice in vivo by assessing radioactivity levels in these tissues after intraperitoneal injection of radiolabeled palmitic acid. After 30 min, wild-type mice showed high radioactivity in both WAT and liver. By contrast, SMS1-KO mice showed relatively lower radioactivity in WAT after 30 min, although radioactivity in liver was comparable to that in wild-type mice. By 48 h, radioactivity in wild-type and SMS1-KO liver was almost completely absent, whereas a larger portion of radioactivity remained in WAT of both genotypes (Fig. 2C). We also undertook assays to evaluate fatty acid uptake in vitro and observed a slight but significant reduction of palmitate uptake in SMS1-deficient MEFs (Fig. 2D). Overall, these results suggest that incorporation of palmitate into WAT rather than liver of SMS1-KO mice is disturbed due to deficient fatty acid uptake function.Measurement of Mitochondrial Respiratory Chain ActivityThe gel slices obtained by BN-PAGE was used to detect mitochondrial respiratory chain activity as described [36]. Complex IV activity (cytochrome oxidase activity) was examined by incubating gel slices in the reaction buffer IV (50 mM sodium phosphate buffer (pH 7.4), 1 mg/ml DAB, 24 units/ml catalase, 1 mg/ml cytochrome c, 0.22 M sucrose). Color development was preserved in fixing buffer (50 methanol, 10 acetic acid), and the gel was stored in 10 acetic acid. Complex V activity (ATPase activity) was assessed by incubating gel slices in the reaction buffer V (35 mM Tris, 270 mM glycine, 14 mM MgSO4, 0.2 Pb(NO3)2, and 8 mM ATP). After overnight incubation, the color-developed gel was washed and stored in water. The remaining gel slice was stained with Coomassie Brilliant blue (CBB).SMS1-KO WAT Is Severely Damaged by Oxidative StressPreviously we observed that islet cells in SMS1-KO mice were chronically damaged by oxidative stress [28]. Therefore, we asked whether oxidative stress also damaged WAT of mutant mice. Immunoblot analysis using anti-2,4-dinitrophenyl (DNP) antibody,Statistical AnalysisData were analyzed using Student’s t-test and reported as means 6 SEM, unless otherwise stated.SMS1 in Adipose Tissue FunctionSMS1 in Adipose Tissue FunctionFigure 1. SMS1-KO mice exhibit a lipodystrophic phenotype. (A) Representative CT images of the lower a.