Lack of endogenous cholecystokinin promotes cholelithogenesis in mice

Abstract

Background: Celiac disease is an autoimmune enteropathy caused by a permanent intolerance to dietary gluten in genetically predisposed individuals. Cholecystokinin (CCK) release from the proximal small intestine and gallbladder emptying in response to a fatty meal are greatly reduced in celiac patients before they start the gluten-free diet, showing a genetic predisposition to gallstones.

Methods: To elucidate the complex pathophysiological mechanisms determining the biliary characteristic of celiac disease, we investigated the effect of the absence of endogenous CCK on cholesterol crystallization and gallstone formation in mice fed a lithogenic diet for 28 days.

Key results: Fasting gallbladder volumes were increased and the response of gallbladder emptying to the high-fat diet was impaired in CCK knockout (KO) mice compared to wild-type mice. Because of the absence of CCK, small intestinal transit time was prolonged and intestinal cholesterol absorption was increased. During 28 days of feeding, elevated biliary cholesterol concentrations and gallbladder stasis promoted the growth and agglomeration of solid cholesterol crystals into microlithiasis and stones. Thus, cholesterol crystallization and gallstone formation were accelerated in CCK KO mice. In contrast, daily intraperitoneal administration of CCK-8 reduced gallstone formation in CCK KO mice even on the lithogenic diet.

Conclusions & inferences: The lack of endogenous CCK enhances susceptibility to gallstones by impairing gallbladder contractile function and small intestinal motility function. These findings show that celiac disease is an important risk factor for gallstone formation and the gallbladder motility function should be routinely examined by ultrasonography and gallbladder stasis should be prevented in celiac patients.

Keywords: bile salt; celiac disease; cholesterol absorption; cholesterol crystallization; gallbladder motility; lithogenic bile.

Figure 1

(A) Representative photographs of mucin gel, liquid crystals, cholesterol monohydrate crystals, and gallstones observed in CCK WT and KO mice during lithogenic diet feeding for 28 days. All magnifications are ×400, except for the left top panel, which is ×800, by polarizing light microscopy. (B) It should be emphasized that the growth and agglomeration of solid cholesterol crystals, as well as the development of cholesterol gallstones are dramatically accelerated in CCK KO mice. Thus, gallstone prevalence is significantly higher in CCK KO mice compared with wild-type mice.

Figure 2

Bile flow is comparable between CCK WT and KO mice. Hepatic output of biliary cholesterol and phospholipids, but not bile salts during the first hour of interruption of the enterohepatic circulation is significantly higher in CCK KO mice compared to that in CCK WT mice fed the lithogenic diet for 28 days.

Figure 3

(A) The relative lipid composition of pooled gallbladder bile from CCK KO and WT mice at each time point before (day 0, on chow diet) and at 14 and 28 days on the lithogenic diet are plotted on a condensed phase diagram. The one-phase micellar zone at bottom is enclosed by a solid curved line. Above the micellar zone, two solid lines divide the two-phase zones from a central three-phase zone. Based upon the solid and liquid crystallization sequences present in bile, the left two phase and central three-phase regions are divided by dashed lines into region A to E. With passage of time, relative lipid composition of gallbladder bile shift upward and to the right in both CCK KO and WT mice. Only relative lipid composition of bile in CCK KO mice pass through region B and enter region C. In contrast, relative lipid composition of bile in CCK WT mice enter region C directly from the micellar zone. Black symbols represent CCK KO mice and white symbols for CCK WT mice. (B) The CSI values of pooled gallbladder bile reach supersaturation at 14 days on the lithogenic diet in both CCK WT and KO mice. Notably, the CSI values are markedly higher in CCK KO mice than in WT mice.

Figure 4

Gallbladder emptying in response to duodenal infusion of corn oil or exogenously administered IV CCK-8 (as indicated by the arrows) is significant in CCK WT mice. BY contrast, gallbladder contractile function in response to duodenal infusion of corn oil is impaired in CCK KO mice fed the lithogenic diet for 14 days. Although exogenously administered CCK-8 stimulates gallbladder emptying in both CCK WT and KO mice, the percentage of gallbladder emptying in CCK WT mice is significantly greater than that in CCK KO mice.

Figure 5

(A) Small intestinal transit times in CCK WT and KO mice fed the lithogenic diet for 14 days as determined by the distribution of radioactivity along the small intestine at 30 minutes after intraduodenal infusion of [³H]sitostanol dissolved in medium-chain triglyceride. Each bar is the mean percentage of radioactivity in each segment. Segments 1 and 20 represent evenly divided segments from the most proximal to the distal part of the small intestine. Arrows indicate the geometric center that is significantly shorter in CCK KO mice, showing significantly slower small intestinal transit times compared with CCK WT mice. (B) CCK KO mice display significantly higher intestinal cholesterol absorption efficiency compared with CCK WT mice, as measured by the fecal dual-isotope ratio method.

Figure 6

In the lithogenic state, gallstone prevalence is significantly reduced in CCK KO mice treated with CCK-8 twice per day for 28 days compared to that in CCK KO mice administered with 0.9% NaCl as a control.

Figure 7

Effect of CCK on mRNA levels of gallbladder mucin genes, as well as the genes involved in lipid metabolism and transporters in (A) the gallbladder, (B) liver, and (C) small intestine in CCK WT and KO mice fed the lithogenic diet for 28 days. See text for further details and for abbreviations.