Apolipoprotein A-V is present in bile and its secretion increases with lipid absorption in Sprague-Dawley rats

Abstract

Apolipoprotein (apo) A-V is a protein synthesized only in the liver that dramatically modulates plasma triglyceride levels. Recent studies suggest a novel role for hepatic apoA-V in regulating the absorption of dietary triglycerides, but its mode of action on the gut remains unknown. The aim of this study was to test for apoA-V in bile and to determine whether its secretion is regulated by dietary lipids. After an overnight recovery, adult male Sprague-Dawley bile fistula rats indeed secreted apoA-V into bile at a constant rate under fasting conditions. An intraduodenal bolus of intralipid (n = 12) increased the biliary secretion of apoA-V but not of other apolipoproteins, such as A-I, A-IV, B, and E. The lipid-induced increase of biliary apoA-V was abolished under conditions of poor lymphatic lipid transport, suggesting that the stimulation is regulated by the magnitude of lipids associated with chylomicrons transported into lymph. We also studied the secretion of apoA-V into bile immediately following bile duct cannulation. Biliary apoA-V increased over time (∼6-fold increase at hour 16, n = 8) but the secretions of other apolipoproteins remained constant. Replenishing luminal phosphatidylcholine and taurocholate (n = 9) only enhanced apoA-V secretion in bile, suggesting that the increase was not due to depletion of phospholipids or bile salts. This is the first study to demonstrate that apoA-V is secreted into bile, introducing a potential route of delivery of hepatic apoA-V to the gut lumen. Our study also reveals the uniqueness of apoA-V secretion into bile that is regulated by mechanisms different from other apolipoproteins.

Keywords: bile fistula; bile salts; chylomicron; fat absorption; negative feedback regulation.

Fig. 1.

Under fasting conditions, Apolipoprotein (apo) A-V is present in bile and secreted at a constant rate. A: ApoA-V output into bile with a representative Western blot shown (20 second outputs of bile loaded into each well). P, diluted plasma (1:5) as a comparative marker for apoA-V. B: bile flow rate. After a 24-h recovery from surgery, bile fistula rats were given a continuous intraduodenal infusion of saline. ApoA-V was measured in bile by Western blot and quantified by densitometry. Output was determined by concentration × bile flow. Values are means ± SE.

Fig. 2.

Supplementing bile fistula rats with a continuous intraduodenal infusion of phosphatidylcholine (PL) and taurocholate (NaTC) restored lymphatic triglyceride (TG) transport to the same level as bile intact rats. Bile intact or bile fistula rats were given an intraduodenal bolus of lipids, respectively. Another cohort of bile fistula rats was given an intraduodenal supplement of phospholipid-taurocholate showed the same lymphatic triglyceride output as bile-intact rats. Without the phospholipid-taurocholate supplemental infusion, bile fistula rats transported triglycerides poorly into lymph. All values are means ± SE. **P < 0.01, ***P < 0.001 is significance comparing Lipid, Bile fistula vs. Lipid, Bile-intact.

Fig. 3.

Dietary lipids stimulated the secretion of apoA-V into bile and required transport of lipids into lymph. A: biliary apoA-V output in rats given an intraduodenal bolus of dietary lipids or saline (with PL+NaTC supplemental infusion), **P < 0.01. B: biliary outputs of other apolipoproteins in response to dietary lipids, *P < 0.05, **P < 0.01 ApoA-I with lipid treatment vs. with saline treatment. C: bile flow between the 2 groups. D: biliary apoA-V output in rats given dietary lipid but no PL+NaTC supplementation (a condition of poor lymphatic triglyceride transport). Apolipoprotein concentration in bile was determined by Western blot and quantified by densitometry. Output was determined by concentration × bile flow. Experimental protocols described in materials and methods. All values are means ± SE.

Fig. 4.

Effects of dietary lipids on the biliary outputs of cholesterol (A), bile acids (B), and phospholipids (C). Dietary lipids significantly affected total biliary phospholipid output (P < 0.05 for treatment factor, 2-way ANOVA), but there was no statistical significance between groups at individual time points by multiple comparisons posttest analysis. Biliary concentrations were measured by chemical assays described in materials and methods. Output was determined by concentration × bile flow. All values are means ± SE. Ch, cholesterol; BS, bile salts; PL, phospholipids.

Fig. 5.

Biliary output of apoA-V in bile increased dramatically after bile fistula surgery while the outputs of other apolipoproteins remained constant. A: biliary apoA-V output with representative Western blot (20-s output of bile loaded into each well). B: biliary outputs of apoA-I, apoA-IV, apoB, and apoE. Apolipoprotein concentration was determined by Western blot and quantified using densitometry. Output was determined by concentration × bile flow. All values are means ± SE. **P < 0.01 hour 16 vs. hour 1.

Fig. 6.

After bile fistula surgery, bile flow and biliary lipid and bile salt outputs decrease. Bile flow rate (A) and biliary outputs of cholesterol (B), bile acids (C), and phospholipids (D). Bile fistula rats were given a continuous intraduodenal infusion of 5% glucose-saline immediately after surgery. Biliary concentrations of lipids were measured by chemical assays. Output is determined by concentration × bile flow. All values are means ± SE.

Fig. 7.

Biliary apoA-V output remained increased after bile fistula surgery even after phospholipid-taurocholate treatment. A: biliary apoA-V output. B: bile flow. Immediately after bile fistula surgery, rats were given either a continuous intraduodenal infusion of 5% glucose-saline or a physiological concentration of phospholipid-taurocholate for 27 h. Biliary apoA-V concentration was determined by Western blot and quantified by densitometry. Output was determined by concentration × bile flow. All values are means ± SE. **P < 0.01, ***P < 0.001