Supplementary Material for: Enhanced Liver Regeneration After Partial Hepatectomy in Sterol Regulatory Element-Binding Protein (SREBP)-1c-Null Mice is Associated with Increased Hepatocellular Cholesterol Availability

<b><i>Background/Aims:</i></b> Transient lipid accumulation within hepatocytes preceding the peak proliferative process is a characteristic feature of liver regeneration. However, molecular mediators responsible for this lipid accumulation and their functions are not well defined. Sterol regulatory element-binding proteins-1c (SREBP-1c) are critical transcriptional factors that regulate lipid homeostasis in the liver. We hypothesized that SREBP-1c deficiency induced alterations of lipid metabolism may influence hepatocyte proliferation and liver regeneration. <b><i>Methods:</i></b> 2/3 partial hepatectomy (PH) was performed in wild type C57BL/6J (WT) and <i>Srebp-1c-/-</i> mice. The lipid contents in serum and liver were measured by enzymatic colorimetric methods. Hepatic lipid droplets were detected by Oil Red O staining and immunohistological staining. Hepatic expression of genes involved in lipid metabolism and cellular proliferation was determined by real-time PCR and/or immunoblot. Hepatocyte proliferation and liver regeneration were assessed by BrdU staining and the weight of remanent liver lobes in Srebp-1c-/- mice, respectively. <b><i>Results:</i></b> <i>Srebp-1c-/-</i> mice displayed reduced triglyceride and fatty acids but increased cholesterol in the liver before PH. In response to PH, hepatocellular DNA synthesis was elevated and cell cycle progression was prolonged in <i>Srebp-1c-/-</i> mice, which was associated with enhanced liver regeneration. However, <i>Srebp-1c-/-</i> mice had comparable triglyceride and fatty acid contents and expressions of related genes compared with WT mice during the liver regeneration. In contrast, SREBP-1c-deficiency-induced alteration of cholesterol metabolism was retained during the liver regeneration after PH. <i>Srebp-1c-/-</i> mice exhibited higher cholesterol contents and enhanced expression of SREBP-2 and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR) in the liver than WT mice after PH. Moreover, downregulation of genes involved in cholesterol elimination was observed after PH in <i>Srebp-1c-/-</i> mice. <b><i>Conclusion:</i></b> SREBP-1c deficiency in mice did not interfere with triglyceride and fatty acid metabolism but was associated with significant changes in cholesterol profiles during liver regeneration after PH. These results suggest that increased hepatocellular cholesterol storage and cholesterol availability with the enhanced liver regeneration are identified in <i>Srebp-1c-/-</i> mice. This study also shows that providing requisite cholesterol levels to proliferating hepatocytes and keeping appropriate cholesterol metabolism are required for normal liver regeneration.

**背景与目的**：在肝再生的增殖高峰期之前，肝细胞内出现一过性脂质蓄积是肝再生的典型特征。然而，介导这一脂质蓄积过程的分子介质及其具体功能尚未得到明确阐释。固醇调节元件结合蛋白-1c（SREBP-1c）是调控肝脏脂质稳态的关键转录因子。本研究推测，SREBP-1c缺失所引发的脂质代谢紊乱，可能会对肝细胞增殖与肝再生过程产生影响。
**方法**：对野生型C57BL/6J（WT）小鼠及Srebp-1c基因缺失（Srebp-1c-/-）小鼠施行2/3肝部分切除术（PH）。采用酶比色法检测血清与肝脏组织中的脂质含量；通过油红O染色与免疫组织化学染色检测肝脏脂滴。采用实时定量PCR（real-time PCR）和/或免疫印迹法（immunoblot）检测肝脏脂质代谢与细胞增殖相关基因的表达水平。分别采用溴脱氧尿苷（BrdU）染色法及残余肝叶重量测定，评估Srebp-1c-/-小鼠的肝细胞增殖与肝再生情况。
**结果**：PH术前，Srebp-1c-/-小鼠肝脏内甘油三酯与脂肪酸含量降低，但胆固醇含量升高。接受PH术后，Srebp-1c-/-小鼠的肝细胞DNA合成水平升高，细胞周期进程延长，且伴随肝再生能力增强。在肝再生过程中，Srebp-1c-/-小鼠的甘油三酯、脂肪酸含量及相关基因表达水平与WT小鼠无显著差异。与之相反，SREBP-1c缺失所导致的胆固醇代谢紊乱在PH术后的肝再生过程中持续存在。PH术后，Srebp-1c-/-小鼠肝脏内胆固醇含量显著高于WT小鼠，且SREBP-2与3-羟基-3-甲基戊二酰辅酶A还原酶（HMGCR）的表达水平上调。此外，Srebp-1c-/-小鼠在PH术后，其体内参与胆固醇清除的基因表达出现下调。
**结论**：小鼠体内SREBP-1c缺失并不会干扰甘油三酯与脂肪酸代谢，但会在PH术后的肝再生过程中引发胆固醇谱的显著改变。上述结果表明，Srebp-1c-/-小鼠体内肝细胞胆固醇蓄积量与胆固醇可利用度升高，同时伴随肝再生能力增强。本研究同时证实，为增殖中的肝细胞提供充足的胆固醇水平，并维持正常的胆固醇代谢，是实现正常肝再生的必要条件。