Cytochrome c oxidase subunits IV (Cox4) and Vb (Cox5b), were up-regulated in SMS1-KO WAT relative to control tissue, as were components of the latter, such as ATP synthase, H+transporting, mitochondrial F1 complex, a subunit 1 (ATP5a1), b polypeptide (ATP5b) and c polypeptide 1 (ATP5c1).DiscussionPreviously, we generated SMS1-KO mice and found that they MedChemExpress ASP-015K exhibit phenotypes suggestive of adipose tissue dysfunction [28]. Here, we confirmed and extended those findings. Histochemical analysis revealed that the adipose cell size was severely reduced in epiWAT of SMS1-KO mice, and epiWAT volume was reduced age-dependently, suggesting that mutant mice exhibit progressive lipodystrophy. No changes were observed in expression of factors required for adipogenesis, suggesting that adipocyte differentiation proceeds normally in WAT cells of knockout mice. However, analysis of sphingolipid composition revealed reduced levels of sphingomyelin species, while ceramide and GM3 species increased in SMS1-KO WAT. Therefore, we conclude that sphingolipid metabolism is perturbed in SMS1-KO WAT cells. Hypertriglyceridemia is reportedly a common feature of 
inherited lipodystrophies [44,45]. We observed increased triglyceride levels in blood plasma of knockout mice, while LPL activity in SMS1-KO liver and WAT was reduced. In vivo analysis confirmed that the primary defect of SMS1-KO mice is in fatty acid Teriparatide web uptake into 22948146 WAT rather than liver. The deficiency may be a common feature among many types of SMS1-KO cells, because SMS1-deficient MEF showed a slight but significant deficiency in fatty acid uptake. We previously showed that increased ceramide species in pancreas islets of SMS1-KO mice perturbed mitochondrial function and increased ROS generation [28]. The present study confirms that WAT of mutant mice is damaged by oxidative stress promoted by increased ROS. Others have reported that increased levels of ceramide species and oxidative stress promote cell deathMitochondrial Function Is Disrupted in SMS1-KO WATTo examine mitochondrial function of SMS1-KO WAT, we first examined ATP level in WAT. We 
found that the ATP content of SMS1-KO WAT was slightly but significantly decreased compared to levels seen in wild-type mice (Fig. 5A). CBB-stain and immunoblot analysis of BN-PAGE indicated that the amounts of respiration complex I and V were reduced in the mitochondria of SMS1-KO WAT, whereas that of complex IV was not (Fig. 5B and 5C). The amount of translocase of the outer membrane (TOM complex) was not changed (Fig. 5C). These results suggest that accumulations of complex I and V are reduced in the mitochondria of SMS1-KO WAT, probably due to their instability. We further examined mitochondrial respiration complex activity in BN-PAGE gel (Fig. 5D). The activity of complex V was reduced in the mitochondria of SMS1-KO WAT, whereas that of complex IV was not. Together, these results suggest that the functions of respiration complexes in the mitochondria of SMS1-KO WAT are disturbed.SMS1 in Adipose Tissue FunctionSMS1 in Adipose Tissue FunctionFigure 4. The oxidative stress response, mitochondrial stress response and mitochondrial biogenesis are activated in SMS1-KO WAT. (A ) WAT of 10-week-old mice was analyzed for mRNA expression levels of genes encoding ROS detoxification enzymes (A), mitochondrial stress-related factors (B), mitochondrial biogenesis-related factors (C) and mitochondrial respiration complex factors (D). Values were normalized to b ctin expression.Cytochrome c oxidase subunits IV (Cox4) and Vb (Cox5b), were up-regulated in SMS1-KO WAT relative to control tissue, as were components of the latter, such as ATP synthase, H+transporting, mitochondrial F1 complex, a subunit 1 (ATP5a1), b polypeptide (ATP5b) and c polypeptide 1 (ATP5c1).DiscussionPreviously, we generated SMS1-KO mice and found that they exhibit phenotypes suggestive of adipose tissue dysfunction [28]. Here, we confirmed and extended those findings. Histochemical analysis revealed that the adipose cell size was severely reduced in epiWAT of SMS1-KO mice, and epiWAT volume was reduced age-dependently, suggesting that mutant mice exhibit progressive lipodystrophy. No changes were observed in expression of factors required for adipogenesis, suggesting that adipocyte differentiation proceeds normally in WAT cells of knockout mice. However, analysis of sphingolipid composition revealed reduced levels of sphingomyelin species, while ceramide and GM3 species increased in SMS1-KO WAT. Therefore, we conclude that sphingolipid metabolism is perturbed in SMS1-KO WAT cells. Hypertriglyceridemia is reportedly a common feature of inherited lipodystrophies [44,45]. We observed increased triglyceride levels in blood plasma of knockout mice, while LPL activity in SMS1-KO liver and WAT was reduced. In vivo analysis confirmed that the primary defect of SMS1-KO mice is in fatty acid uptake into 22948146 WAT rather than liver. The deficiency may be a common feature among many types of SMS1-KO cells, because SMS1-deficient MEF showed a slight but significant deficiency in fatty acid uptake. We previously showed that increased ceramide species in pancreas islets of SMS1-KO mice perturbed mitochondrial function and increased ROS generation [28]. The present study confirms that WAT of mutant mice is damaged by oxidative stress promoted by increased ROS. Others have reported that increased levels of ceramide species and oxidative stress promote cell deathMitochondrial Function Is Disrupted in SMS1-KO WATTo examine mitochondrial function of SMS1-KO WAT, we first examined ATP level in WAT. We found that the ATP content of SMS1-KO WAT was slightly but significantly decreased compared to levels seen in wild-type mice (Fig. 5A). CBB-stain and immunoblot analysis of BN-PAGE indicated that the amounts of respiration complex I and V were reduced in the mitochondria of SMS1-KO WAT, whereas that of complex IV was not (Fig. 5B and 5C). The amount of translocase of the outer membrane (TOM complex) was not changed (Fig. 5C). These results suggest that accumulations of complex I and V are reduced in the mitochondria of SMS1-KO WAT, probably due to their instability. We further examined mitochondrial respiration complex activity in BN-PAGE gel (Fig. 5D). The activity of complex V was reduced in the mitochondria of SMS1-KO WAT, whereas that of complex IV was not. Together, these results suggest that the functions of respiration complexes in the mitochondria of SMS1-KO WAT are disturbed.SMS1 in Adipose Tissue FunctionSMS1 in Adipose Tissue FunctionFigure 4. The oxidative stress response, mitochondrial stress response and mitochondrial biogenesis are activated in SMS1-KO WAT. (A ) WAT of 10-week-old mice was analyzed for mRNA expression levels of genes encoding ROS detoxification enzymes (A), mitochondrial stress-related factors (B), mitochondrial biogenesis-related factors (C) and mitochondrial respiration complex factors (D). Values were normalized to b ctin expression.
