3A) Moreover, quantification of hepatic TAG content by TLC demon

3A). Moreover, quantification of hepatic TAG content by TLC demonstrated reduced TAG accumulation in 24-hour regenerating Balb/CCAV1−/− livers

(Fig. 3C). Taken together, these data demonstrate decreased TAG content and accumulation of LDs in regenerating Balb/CCAV1−/− hepatocytes, supporting our previous results that the absence of CAV1 reduces hepatocyte ability for storage of TAG. Finally, we analyzed hepatic LD accumulation during liver regeneration in JAXCAV1+/+ and JAXCAV1−/− mice. Liver appearance from DAPT molecular weight JAXCAV1−/− mice did not show high levels of steatosis. However, JAXCAV1+/+ also showed great variability in their steatotic appearance (data not shown). Accordingly, western blot analyses showed very variable expression of ADRP protein levels in both JAXCAV1+/+ homogenates and LD fractions (Fig. 3D,E). Thus, these results support the conclusions of Mayoral et al.5 suggesting that there were no significant differences in hepatic LD accumulation between JAXCAV1+/+ and JAXCAV1−/− mice during liver regeneration. To further investigate the importance of CAV1 for the ability of hepatocytes to accumulate TAG and generate LDs, we analyzed two independent physiological Lumacaftor datasheet models of hepatic LD accumulation

in CAV1−/− mice: fasting and maintenance on a high-fat diet. First, we studied hepatic LD accumulation in KCAV1, JAXCAV1, and Balb/CCAV1 mice after 24 hours of fasting (Fig. 4; Supporting Fig. S3). When we compared KCAV1+/+ and KCAV1−/− mice, ADRP and GyK transcript levels, both involved in TAG synthesis, were significantly reduced in KCAV1−/− hepatocytes (Fig. 4A), as were ADRP protein levels in the liver (Fig. 4B,C) and in purified LDs (Fig. 4D) during different periods of fasting. Accordingly, hepatic TAG content and the percentage of the cytosolic area occupied by the LDs were significantly reduced in KCAV1−/− mice (Fig. 4E,F). Similar results were obtained

in liver samples from 24-hour-fasted JAXCAV1+/+ check details and JAXCAV1−/− (Supporting Fig. S3a-c) and from Balb/CCAV1+/+ and Balb/CCAV1−/− mice (Supporting Fig. S3d-f). We next studied the development of steatosis in response to a 12-week high-fat diet (HFD) in mice. Both, KCAV1+/+ and KCAV1−/− mice on the HFD showed increased levels of plasma lipids (TAG, total cholesterol) when compared with mice on a chow diet (Fig. 5A). Moreover, food consumption in KCAV1−/− mice was similar to KCAV1+/+ mice (data not shown). However, KCAV1−/− mice showed a lack of the typical steatotic liver phenotype as judged by several criteria (Fig. 5B). Analysis of ADRP levels in liver homogenates and purified hepatic LD fractions in combination with TLC-liver TAG content quantification and quantitative electron microscopic analysis of liver sections from chow- (data not shown) and HFD-fed mice all showed defective accumulation of TAG in LDs of KCAV1−/− hepatocytes in response to HFD (Fig. 5C-E). Similar results were obtained in liver samples from HFD-fed JAXCAV1+/+ and JAXCAV1−/− (Supporting Fig.

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