Latrunculin A Changes in plasma lipids and glucose handling
Changes in plasma lipids and glucose handling often coincide with changes in liver metabolism. No significant change in liver weight was observed (1.63 ± 0.14 g for WT mice versus 1.38 ± 0.05 g for Prg4 KO mice, respectively; p > 0.05). Gene expression of the rate limiting enzyme in glucose utilization in the liver, glucokinase (Gck), was significantly lower (−30%; p < 0.05) in Prg4 KO mice than in WT mice (Fig. 3A). The glycolysis enzyme pyruvate kinase (PK) showed a similar trend towards lower gene expression levels (−21%; p = 0.06; Fig. 3A). Expression of the insulin-responsive genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxylase (Pepck), both essential in gluconeogenesis, was not changed (Fig. 3A). As glucose intolerance associates with hepatic steatosis [19,20], we subsequently investigated genes involved in hepatic lipid metabolism in these mice. Prg4 KO mice showed significantly lower gene expression levels of the insulin-responsive hepatic lipogenesis genes acetyl-CoA carboxylase alpha (Acc; −21%; p < 0.05) and stearoyl-CoA desaturase 1 (Scd1; −38%; p < 0.001; Fig. 3A) than WT mice. The lipogenesis gene fatty Latrunculin A synthase (Fasn) showed a similar trend towards lower expression (−22%; p = 0.1) in the livers of Prg4 KO mice as compared to WT (Fig. 3A). The lower hepatic lipogenic gene expression coincided with lower hepatic triglyceride levels (−56%, p < 0.001; Fig. 3B). The Prg4 deficiency-associated decrease in lipid accumulation was confirmed histologically by H&E and Oil red O staining. H&E staining showed less macrovesicular lipid vacuoles in livers of Prg4 KO mice after 16 weeks of HFD challenge (Fig. 3C), which was confirmed by less intensive Oil red O staining for neutral lipids (Fig. 3C). These combined findings thus suggest that Prg4 KO mice exhibit a lowered susceptibility for the development of HFD-induced hepatic steatosis as compared to WT mice. Previous studies have shown that Scd1 quantitatively contributes to hepatic triglyceride levels . In line, the Scd1 expression in the liver correlated significantly with the hepatic triglyceride levels (r = 0.73; p < 0.001; Fig. 3D). Furthermore, plasma and hepatic triglyceride levels correlated significantly (r = 0.40; p < 0.05). Notably, expression levels of microsomal triglyceride transfer protein (Mttp) and apolipoprotein b (Apob) were not changed (Fig. 3A), suggesting that the decrease in plasma triglyceride levels were not secondary to alterations in the ability of hepatocytes to generate/secrete very-low-density lipoprotein (VLDL) particles. From these combined findings we anticipate that the effects on hepatic and plasma triglyceride levels are primarily driven by a significant change in the hepatic lipogenesis rate. In further support, relative expression levels of carnitine palmitoyltransferase 1 and 2 were lower (Cpt1: −26%; p < 0.05; Cpt2: −30%; p < 0.01; Fig. 3A), excluding that the decrease in storage of fatty acids within the hepatic triglyceride pool in Prg4 KO mice was due to an increased fatty acid oxidation. Moreover, the relative expression levels of key genes involved in lipoprotein uptake, i.e. the low-density lipoprotein receptor (Ldlr), low-density lipoprotein receptor-related protein 1 (Lrp1), and scavenger receptor BI (Sr-bi), as well as the actual plasma clearance of VLDL-like particle-derived triglycerides labeled with glycerol tri[3H]oleate by the liver was not significantly different between Prg4 KO mice and WT mice (Fig. 3A and E). Previous studies have suggested that the level of endoplasmic reticulum (ER) stress as well as hepatocyte toll-like receptor 4 (Tlr4) activity can influence the lipogenesis rate and associated liver steatosis extent [, , , ]. However, a difference in these parameters also does not seem to underlie the Prg4 deficiency-associated protection against the metabolic disturbances as judged from the similar hepatic expression levels of Tlr4 and the ER stress markers C/EBP homologous protein (Chop) and X-linked inhibitor of apoptosis protein (Xiap) (Fig. 3A).