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. 2010 Nov;51(11):3306-15.
doi: 10.1194/jlr.M010256. Epub 2010 Aug 27.

CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance

J Mark Brown  1 , Jenna L Betters, Caleb Lord, Yinyan Ma, Xianlin Han, Kui Yang, Heather M Alger, John Melchior, Janet Sawyer, Ramesh Shah, Martha D Wilson, Xiuli Liu, Mark J Graham, Richard Lee, Rosanne Crooke, Gerald I Shulman, Bingzhong Xue, Hang Shi, Liqing Yu
Affiliations

CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance

J Mark Brown et al. J Lipid Res. 2010 Nov.

Abstract

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80-95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.

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Figures

Fig. 1.
Fig. 1.
ASO-mediated knockdown of CGI-58 prevents HFD-induced obesity. A: CGI-58 mRNA levels in the epididymal adipose tissue of mice treated with control ASO or CGI-58 ASO (CGI-58β). B: Western blot analysis of epididymal adipose tissue in chow-fed mice (exposure time: 15 s). C: Body weight in ad libitum chow-fed or HFD-fed mice (n = 9–10). * P < 0.01 (vs. saline within each time point). D: Gross appearance of HFD-fed mice and their epididymal fat pads, and the total epididymal fat pad weight (g) and the total epididymal fat pad weight expressed as % of body weight (BW) in both chow-fed and HFD-fed mice (n = 9–10). * P < 0.01 (vs. saline or control ASO within each diet).
Fig. 2.
Fig. 2.
ASO-mediated knockdown of CGI-58 causes severe hepatic steatosis. A: Hepatic CGI-58 mRNA expression in mice treated with control ASO or CGI-58 ASO (CGI-58β). B: Western blot analysis of liver homogenates from chow-fed mice (exposure time: 30 s). C: Hepatic TG hydrolase activity (n = 6–7). * P < 0.05 (vs. control ASO within each diet). D: Hepatic NEFA levels (n = 8). * P < 0.05 (vs. control ASO in chow group). ND, not detectable. E: Liver appearance. F: Microscopic examination of liver sections (H and E staining, at 40× magnification). G: Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (n = 6) *P < 0.05 (vs. saline or control ASO within each diet). H: Total hepatic TG levels (n = 8), *P < 0.05 (vs. control ASO within each diet). I–K: Total hepatic contents of diacylglycerol, ceramide, and LCCoA (n = 4–5). * P < 0.05 (vs. control ASO within each diet).
Fig. 3.
Fig. 3.
ASO-mediated knockdown of CGI-58 alters hepatic phospholipid metabolism. A: Total hepatic PL levels (n = 8). * P < 0.05 (vs. control ASO within each diet). B–G: Hepatic contents of total PA, PA species, total phosphatidylcholine (PC), total phosphatidylethanolamine (PE), total phosphatidylserine (PS), and total sphingomyelin (SM) in HFD-fed mice (n = 4). * P < 0.05 (vs. control ASO). H: Total hepatic PG levels in HFD-fed mice (n = 4). * P < 0.01 (vs. control ASO). I: Total hepatic cardiolipin (CL) levels in HFD-fed mice (n = 4).
Fig. 4.
Fig. 4.
CGI-58 regulates plasma lipid metabolism and fasting-induced ketogenesis. At 8 weeks of age, male C57BL/6N mice were either maintained on standard chow (A) or switched to a HFD (B–E) in conjunction with biweekly injections of ASOs for 4 weeks. A: Hepatic secretion rates of TG, CE, and FC determined by recirculating isolated liver perfusion in fed mice (n = 4–5). * P < 0.05. B: Plasma concentrations of TG levels (n = 8). * P < 0.05 (vs. control ASO within each dietary condition). C: Plasma NEFA levels (n = 8). * P < 0.01 (vs. control ASO group within each dietary condition). D: Total plasma cholesterol (TPC) levels (n = 8). E: Plasma β-hydroxybutyrate (β-OH) levels (n = 8). * P < 0.01 (vs. control ASO group within each dietary condition).
Fig. 5.
Fig. 5.
CGI-58 ASO-treated mice are protected against HFD-induced insulin resistance. A: Fasting plasma glucose levels (n = 6). * P < 0.05 (vs. control ASO within each diet). B: Fed and fasting plasma insulin levels in HFD-fed mice (n = 8). * P < 0.05 (vs. control ASO within each dietary condition). C: Glucose tolerance tests (n = 8). * P < 0.01 (vs. control ASO within each time point). D: Plasma insulin levels during glucose tolerance tests (n = 6). * P < 0.01 (vs. control ASO within each time point). E: Insulin tolerance tests (n = 8). * P < 0.01 (vs. control ASO within each time point).
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