Absence of perilipin 2 prevents hepatic steatosis, glucose intolerance and ceramide accumulation in alcohol-fed mice

Abstract

Background: Perilipin 2 (Plin2) is a lipid droplet protein that has roles in both lipid and glucose homeostasis. An increase in Plin2 in liver is associated with the development of steatosis, glucose intolerance, and ceramide accumulation in alcoholic liver disease. We investigated the role of Plin2 on energy balance and glucose and lipid homeostasis in wildtype and Plin2 knockout (Plin2KO) mice chronically fed a Lieber-DeCarli liquid ethanol or control diet for six weeks.

Methods: We performed in vivo measurements of energy intake and expenditure; body composition; and glucose tolerance. After sacrifice, liver was dissected for histology and lipid analysis.

Results: We found that neither genotype nor diet had a significant effect on final weight, body composition, or energy intake between WT and Plin2KO mice fed alcohol or control diets. Additionally, alcohol feeding did not affect oxygen consumption or carbon dioxide production in Plin2KO mice. We performed glucose tolerance testing and observed that alcohol feeding failed to impair glucose tolerance in Plin2KO mice. Most notably, absence of Plin2 prevented hepatic steatosis and ceramide accumulation in alcohol-fed mice. These changes were related to downregulation of genes involved in lipogenesis and triglyceride synthesis.

Conclusions: Plin2KO mice chronically fed alcohol are protected from hepatic steatosis, glucose intolerance, and hepatic ceramide accumulation, suggesting a critical pathogenic role of Plin2 in experimental alcoholic liver disease.

Figure 1. Confirmation of plin2ko mice.

A) Genotyping of wildtype +/+ and Plin2KO (−/−) mice using tail DNA. Bands correspond to 485 BP KO PCR product and 406 bp WT PCR product. B) Immunoblots of Plin2 and GAPDH from liver lysates of wildtype and Plin2KO mice. Each lane represents liver lysate from an individual mouse. Plin2 = Perilipin 2 GAPDH =  glycerol-3-phosphate dehydrogenase housekeeping protein.

Figure 2. Measurement of glucose tolerance in wildtype and Plin2KO (KO) mice fed control liquid or ethanol diet.

Glucose tolerance test after A) 4 weeks and B) 6 weeks of feeding. # P = 0.001 WTCtrl vs WTEtoh; ##P = 0.03 WTCtrl vs KOEtoh; ###P<0.05 WTCtrl vs WTEtoh; C) Serum insulin levels at 6 weeks. *P<0.05. N = 5 mice/group. P<0.05 is considered significant.

Figure 3. Liver histology of wild-type and Plin2KO mice fed control liquid or ethanol diet for 6 weeks.

Representative hematoxylin and eosin-stained liver sections. 40x magnification. WT = wildtype KO = Plin2KO Ctrl = control Etoh = ethanol

Figure 4. Serum and liver chemistries of wildtype and Plin2KO mice fed control or ethanol diets.

Graphs depict results of serum and liver assays on wildtype (WT) and Plin2KO (KO) mice after six weeks of feeding a control (Ctrl) or ethanol (Etoh) diet. N = 5 mice/group. Data reported are mean ±SEM. *p = 0.01 **p = 0.02 ***p = 0.04 ****p = 0.002. p<0.05 is considered significant.

Figure 5. Liver mRNA of wildtype and Plin2KO mice fed control or ethanol diets.

Hepatic mRNA expression relative to 36B4 (phosphoriboprotein) using liver lysates of wildtype (WT) and Plin2KO (KO) mice fed six weeks of control (Ctrl) or ethanol (Etoh) diets. N = 5 mice/group. DGAT2 =  diacylglycerol O-acyltransferase 2; CPT1 =  Carnitine palmitoyltransferase I; PPARα =  peroxisome proliferator-activated receptor alpha; SREBP1 = Sterol regulatory element-binding protein 1; Plin3 =  Perilipin 3. Data reported are means ±SEM. *p = 0.02 **p = 0.003. p<0.05 is considered significant.

Figure 6. Hepatic ceramide measurements in WT and Plin2KO mice fed control or ethanol diets.

A) Distribution of short (C2-C5), medium (C6-C12), long (C13-C21) and very long chain (>21) hepatic ceramides as measured by mass spectrometric analysis in WT and Plin2KO (KO) mice fed control (Ctrl) or ethanol (Etoh) diets for six weeks B) Comparison of predominant ceramide species in WT and KO mice fed control or ethanol diets. N = 5 mice/group. Data reported are mean ±SEM. *p<0.05.