Idative stress response and a shortened lifespan [22]. SMS2 mutant mice exhibit an attenuated inflammatory response in macrophages [23], decreased atherosclerosis [24] and resistance to high fat dietinduced obesity [25]. Analyses of SMS1 activity undertaken in cultured cells indicate that SMS1 has an important function in lymphoid cell proliferation [20]. Membrane sphingomyelin levels regulated by SMS1 and SMS2 activity are reportedly important for Fas translocation into lipid rafts, which promotes Fas-mediated apoptosis [26]. SMS1 suppression results in enhanced ceramide production and apoptosis after photodamage [27]. To investigate SMS1 function in vivo, we recently generated SMS1 knockout (SMS1-KO) mice and found that SMS1 is required to regulate generation of reactive oxygen species (ROS) and for normalSMS1 in Adipose Tissue Functionmitochondrial function and insulin secretion in pancreatic b-cells [28]. In addition, SMS1-KO mice exhibited loss of epididymal white adipose tissue (WAT) mass [28]. However, the pathological consequences of that loss were not characterized. Here, we analyzed the pathogenesis of lipodystrophic phenotypes observed in SMS1-KO mice. We report that mutant mice exhibit systemic loss of fat tissue mass. Epididymal WAT (epiWAT) mass was MedChemExpress HIV-RT inhibitor 1 Reduced in an age-dependent manner, accompanied by a reduction in adipose cell size. Plasma triglyceride concentrations in mutant mice increased and lipoprotein lipase (LPL) activity and fatty acid uptake activity were reduced in mutant WAT. In vitro analysis using cultured cells also showed reduction of fatty acid uptake by SMS1 depletion. Immunoblot analysis indicated that SMS1-KO WAT proteins were significantly modified by oxidative stress. Mutant mouse WAT showed up-regulation of mRNAs related to apoptosis, redox adjustment, mitochondrial stress response, and mitochondrial biogenesis. Reduced accumulation of mitochondrial respiratory chain complexes and lower ATP content were observed in WAT of SMS1 null mice. Treatment of mutant mice with the antioxidant N-acetyl cysteine (NAC) partially rescued these phenotypes and normalized plasma triglyceride concentrations. These data suggest that SMS1 controls oxidative stress and maintains WAT function.in the tissues was measured by liquid scintillation counting and normalized to total protein concentration.Palmitate Incorporation AssayMouse BIBS39 chemical information embryonic fibroblasts (MEFs) isolated from wild-type and SMS1-deficient embryos were cultured in Dulbecco’s 15755315 modified Eagle’s medium supplemented with 10 fetal calf serum at 37uC in an atmosphere of 5 CO2 and 95 air. For the assay, MEFs were pre-incubated in Krebs-Ringer buffer for 1 h, and then 0.05 mM [3H]palmitic acid bound to fatty acid-free BSA was added. After 10 min, cells were washed three times in the same buffer containing 200 mM phloretin. Cells were then lysed in water containing 0.1 SDS and the incorporated radioactive fatty acids were detected by liquid scintillation counting.Quantitative RT-PCRTotal RNA isolated from WAT was extracted with TRIzol reagent (Invitrogen, Carlsbad, California, USA), and DNasetreated RNA was reverse transcribed with a PrimeScript RT reagent Kit (Takara Bio, Osaka, Japan), following the manufacturer’s protocol. PCR products were analyzed using a Thermal Cycler Dice Real Time system (Takara Bio), and transcript abundance was normalized to that of b-actin mRNA. PCR oligonucleotides and gene abbreviations are listed in Table S1.Materials and Methods Mate.Idative stress response and a shortened lifespan [22]. SMS2 mutant mice exhibit an attenuated inflammatory response in macrophages [23], decreased atherosclerosis [24] and resistance to high fat dietinduced obesity [25]. Analyses of SMS1 activity undertaken in cultured cells indicate that SMS1 has an important function in lymphoid cell proliferation [20]. Membrane sphingomyelin levels regulated by SMS1 and SMS2 activity are reportedly important for Fas translocation into lipid rafts, which promotes Fas-mediated apoptosis [26]. SMS1 suppression results in enhanced ceramide production and apoptosis after photodamage [27]. To investigate SMS1 function in vivo, we recently generated SMS1 knockout (SMS1-KO) mice and found that SMS1 is required to regulate generation of reactive oxygen species (ROS) and for normalSMS1 in Adipose Tissue Functionmitochondrial function and insulin secretion in pancreatic b-cells [28]. In addition, SMS1-KO mice exhibited loss of epididymal white adipose tissue (WAT) mass [28]. However, the pathological consequences of that loss were not characterized. Here, we analyzed the pathogenesis of lipodystrophic phenotypes observed in SMS1-KO mice. We report that mutant mice exhibit systemic loss of fat tissue mass. Epididymal WAT (epiWAT) mass was reduced in an age-dependent manner, accompanied by a reduction in adipose cell size. Plasma triglyceride concentrations in mutant mice increased and lipoprotein lipase (LPL) activity and fatty acid uptake activity were reduced in mutant WAT. In vitro analysis using cultured cells also showed reduction of fatty acid uptake by SMS1 depletion. Immunoblot analysis indicated that SMS1-KO WAT proteins were significantly modified by oxidative stress. Mutant mouse WAT showed up-regulation of mRNAs related to apoptosis, redox adjustment, mitochondrial stress response, and mitochondrial biogenesis. Reduced accumulation of mitochondrial respiratory chain complexes and lower ATP content were observed in WAT of SMS1 null mice. Treatment of mutant mice with the antioxidant N-acetyl cysteine (NAC) partially rescued these phenotypes and normalized plasma triglyceride concentrations. These data suggest that SMS1 controls oxidative stress and maintains WAT function.in the tissues was measured by liquid scintillation counting and normalized to total protein concentration.Palmitate Incorporation AssayMouse embryonic fibroblasts (MEFs) isolated from wild-type and SMS1-deficient embryos were cultured in Dulbecco’s 15755315 modified Eagle’s medium supplemented with 10 fetal calf serum at 37uC in an atmosphere of 5 CO2 and 95 air. For the assay, MEFs were pre-incubated in Krebs-Ringer buffer for 1 h, and then 0.05 mM [3H]palmitic acid bound to fatty acid-free BSA was added. After 10 min, cells were washed three times in the same buffer containing 200 mM phloretin. Cells were then lysed in water containing 0.1 SDS and the incorporated radioactive fatty acids were detected by liquid scintillation counting.Quantitative RT-PCRTotal RNA isolated from WAT was extracted with TRIzol reagent (Invitrogen, Carlsbad, California, USA), and DNasetreated RNA was reverse transcribed with a PrimeScript RT reagent Kit (Takara Bio, Osaka, Japan), following the manufacturer’s protocol. PCR products were analyzed using a Thermal Cycler Dice Real Time system (Takara Bio), and transcript abundance was normalized to that of b-actin mRNA. PCR oligonucleotides and gene abbreviations are listed in Table S1.Materials and Methods Mate.

Idative stress response and a shortened lifespan [22]. SMS2 mutant mice exhibit

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