Coordinate regulation of gallbladder motor function in the gut-liver axis


  • Piero Portincasa,

    Corresponding author
    1. Department of Internal Medicine and Public Medicine, Clinica Medica “A. Murri”, University of Bari Medical School, Bari, Italy
    • Section of Internal Medicine, Department of Internal and Public Medicine, University Medical School, Piazza Giulio Cesare 11, Policlinico, 70124 Bari, Italy
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    • fax +39 80-5478232

  • Agostino Di Ciaula,

    1. Department of Internal Medicine and Public Medicine, Clinica Medica “A. Murri”, University of Bari Medical School, Bari, Italy
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  • Helen H. Wang,

    1. Department of Medicine, Liver Center and Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School and Harvard Digestive Diseases Center, Boston, MA
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  • Giuseppe Palasciano,

    1. Department of Internal Medicine and Public Medicine, Clinica Medica “A. Murri”, University of Bari Medical School, Bari, Italy
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  • Karel J. van Erpecum,

    1. Department of Gastroenterology, University Medical Center Utrecht, Utrecht, The Netherlands
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  • Antonio Moschetta,

    1. Department of Internal Medicine and Public Medicine, Clinica Medica “A. Murri”, University of Bari Medical School, Bari, Italy
    2. Department of Translational Pharmacology, Consorzio Mario Negri Sud, Santa Maria Imbaro (CH), Italy
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  • David Q.-H. Wang

    1. Department of Medicine, Liver Center and Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School and Harvard Digestive Diseases Center, Boston, MA
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  • Potential conflict of interest: Nothing to report.


Gallstones are one of the most common digestive diseases with an estimated prevalence of 10%-15% in adults living in the western world, where cholesterol-enriched gallstones represent 75%-80% of all gallstones. In cholesterol gallstone disease, the gallbladder becomes the target organ of a complex metabolic disease. Indeed, a fine coordinated hepatobiliary and gastrointestinal function, including gallbladder motility in the fasting and postprandial state, is of crucial importance to prevent crystallization and precipitation of excess cholesterol in gallbladder bile. Also, gallbladder itself plays a physiopathological role in biliary lipid absorption. Here, we present a comprehensive view on the regulation of gallbladder motor function by focusing on recent discoveries in animal and human studies, and we discuss the role of the gallbladder in the pathogenesis of gallstone formation. (HEPATOLOGY 2008;47:2112–2126.)

Gallstones are one the most common gastrointestinal diseases that require hospitalization in the western world,1 and this is a true health burden2–4 with enormous costs estimated at 6.5 billion dollars each year in United States.4, 5 Because life expectancy and the incidence rate of obesity are both rising worldwide,6 the incidence rate of gallstones may increase, often in the context of the “metabolic syndrome”.7 The estimated prevalence of gallstones is 10%-15% in the general population of developed countries. In the United States, approximately 20 million to 25 million adults have gallstones. The burden of the disease is highest (60%-70%) in American Indians followed by Hispanics of mixed Indian origin. In contrast, the rate is lowest (less than 5%) in Asian and African populations.5 The prevalence is intermediate (10%-15%) in Caucasians in developed countries. In Europe, the Multicenter Italian Study on Cholelithiasis (MICOL) ultrasonographic survey, which was performed across Italy including 29,000 subjects aged 30-69 years, reported an overall prevalence rate of 18.8% and 9.5% in women and men, respectively.8

Major risk factors for the development of gallstones have been better identified over the past years and include age, female sex, some genetic polymorphisms within a background of a complex genetic disorder, pregnancy, obesity and rapid weight loss on low caloric diets or following bariatric surgery, delayed small intestine transit time, liver cirrhosis, hemolytic anemia, increased serum triglycerides, metabolic syndrome, terminal ileal resection, reduced physical activity, and some medications (for example, estrogens and oral contraceptives, octreotide, clofibrate, and ceftriaxone).1, 5, 9, 10

The gallbladder is deemed as the end-organ of gallstone disease, and a defective gallbladder motility has been strongly linked to the formation of gallstones and particularly of cholesterol-enriched stones (approximately 75%-80% of all gallstones in Western countries).11, 12 Abnormal gallbladder motility has been documented in humans in both the fasting and postprandial state.13, 14 Additionally, an abnormal gallbladder motor function has also been reported in subjects at high risk of gallstone formation (total parenteral nutrition, very low calorie dieting, treatment with somatostatin analogs). As a consequence, the pathophysiology of gallbladder motility is seen with growing interest, where a complex interplay exists between factors involving the content of the gallbladder lumen, the intrinsic ability of the gallbladder smooth muscle to contract, and extrinsic neurohormonal factors. Recently, major new developments have been obtained concerning the molecular mechanisms driving gallbladder dynamics. Of note, the identification of fibroblast growth factor 19 (FGF19) as the hormonal signal responsible for gallbladder distension represents a formidable step forward in the understanding of gallbladder modulation in the context of the gut-liver axis.15 The exploitation of these innovative theoretical attainments is just beginning, with potential implications for future pharmacological approaches.

This review will offer the most recent insights into the mechanisms implicated in the regulation of gallbladder motor function and in the pathogenesis of gallbladder dysmotility by focusing on both basic and clinical aspects. Features concerning the clinical setting, in vitro studies, and the role of luminal, parietal, and neurohormonal factors will be highlighted.

Physiology and Pathophysiology of Gallbladder Contraction

In the interdigestive phase, bile is stored and concentrated in the gallbladder before being actively expelled in the duodenum in response to food intake. In the intestine, bile plays a key role in lipid digestion and absorption. Because of the importance of timely bile delivery in the intestine for the physiology of the digestive system, both interprandial and postprandial gallbladder motility are regulated by complex mechanisms involving the interplay of neural, humoral, and paracrine factors.16, 17 These mechanisms participate in the multiorgan regulatory function operating between intestine and liver, which defines the gut-liver axis. Several substances and receptors18 mediating either contraction or relaxation of the gallbladder are involved in this complex scenario; a list is given in Table 1.

Table 1. Principal Substances Involved in Humoral and Paracrine Control of the Gallbladder Smooth Muscle Contraction/Relaxation
SubstanceReceptor(s)/ channelsMechanism(s)/ NotesReference(s)
  1. Abbreviations: B, bradykinin; BRP, bombesin-related peptides; CGRP, calcitonin gene-related peptide; ERG1, ether-a-go-go–related gene 1; ET-1, endothelin-1; FGFR3, fibroblast growth factor receptor 3; GRP, gastrin-releasing peptide; H, histamine; LT, leukotriens; NMB, NMB-preferring receptor; NT, neurotensin; PACAP, pituitary adenylate cyclase-activating peptide; PHI, peptide histidine isoleucine; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; VIP, vasoactive intestinal peptide.

AcetylcholineMuscarinic M3 >M2, M4, M1Parasympathetic vagal pathways13, 40, 41
CholecystokininCCK-1Direct effect on muscle. Also via presynaptic facilitory effect on ganglionic transmission (increased release of acetylcholine from vagal terminals in GB ganglia)13, 23, 156–160
Endothelins (ET1, 2, 3)ETA, ETB, mobilization of extracellular and intracellular Ca2+Vasoconstrictor peptides, produced by several tissues, including GB epithelial cells132, 137, 161–164
ERG1Ether-a-go-go–related gene 1 (ERG1) protein K(+) channelsHuman, mouse, and guinea pig GB smooth muscle. Excitation-contraction coupling165
EstrogenEstrogen receptor α that induces signal-transduction decoupling of the CCK-1 receptor-G-protein pathwayDirect effect on muscle.166
GRP, Substance K, Substance P, ET-1, BRP, PACAPGRP, NK2, (NK3) SP, ET-1, NMB, PACAP-1NANC neuroendocrine and paracrine transmission117, 136
Histamine, bradykinin, prostaglandins, LTC4, LTD4H1, B2, (B1), LTD4Mediators released by inflammatory cells118–122
MotilinMotilide receptors (?)Hormone, intestinal release during fasting167, 168
Nitric oxide (NO+ or peroxynitrite redox species)Activation of LT metabolism and extracellular Ca2+Extracellular Ca2+ entry via L-type Ca2+ channels133
SerotoninSerotonin receptors (?)Maybe involved in severe chronic cholecystitis120
Adrenaline, Noradrenalineβ-adrenergicStimulation of the sympathetic nervous system169
Bile saltsCCK receptor + cholinergic nervesPossibly dependent on bile salt hydrophobic/ hydrophilic properties112
CGRP, PACAP, VIP, NT, PHICGRP, PACAP-2, VIP2, NT, PHINANC neuroendocrine and paracrine transmission133, 136
Fibroblast Growth Factor 15/19FGFR3Increase gallbladder cAMP concentrations15
HistamineH2Release via inflammatory cells117, 119
Nitric oxide (NO* form)Sensitive to guanylyl cyclase inhibitor. Not altered by KCl channel blockersConcentration-dependent effect133, 170–172
ProgesteroneTyrosine kinase and PKA/cAMP activitySeveral pathways involved173
PTH and PTHrPcAMP mediatedConcentration-dependent effect138

In patients developing cholesterol gallstones, a disrupted interdigestive gallbladder emptying is present due to a poor integration with the intestinal migrating motor complexes.14 Also, in the postprandial phase, increased gallbladder volume and delayed emptying have been documented in about one-third of patients with cholesterol stones.13, 19–22 Although the presence of cholesterol-supersaturated bile is considered the primum movens of cholesterol gallstone disease (see below), alterations of gallbladder motility, which often anticipate the formation of cholesterol gallstones, favors the formation and growth of stones by multiple, interconnected mechanisms. Impaired gallbladder emptying prolongs the residence time of concentrated gallbladder bile, with the result that more time is available for nucleation and crystallization of cholesterol crystals from supersaturated hepatic bile, and less cholesterol crystals can be ejected into the duodenum.

Among humoral factors, the most relevant impact for gallbladder contraction and relaxation is currently attributed to cholecystokinin and the recently identified FGF19, respectively.


Cholecystokinin (CCK) is one of the most important circulating gastrointestinal hormones involved in gallbladder motility.23 In healthy subjects, the ingestion of a standard fat-containing meal induces gallbladder emptying up to 80% of the fasting gallbladder volume by stimulating CCK secretion from the duodenal I cells. CCK influences gallbladder muscular tone mainly in the postprandial state,24 principally through specific CCK-1 receptors,25 by a complex mechanism leading to smooth muscle contraction and relaxation.26, 27In vitro evidence suggests that changes of CCK-receptor number/expression and of processes governing signal transduction play a key role in the determination of the motility dysfunction observed in vivo.28, 29 In a recent integrated in vivo and in vitro study, it was shown that the amount of gallbladder CCK receptor is lower in patients with gallstones who have poor gallbladder contraction compared with that in both healthy subjects or a subgroup of patients with gallstones who have well-contracting gallbladders. Also, gallbladder ejection fraction has been positively correlated with the density of CCK receptors on gallbladder smooth muscle cells.30 Furthermore, it has been demonstrated that, whether on chow or a lithogenic diet, mice with deletion of CCK-1 receptor (Cck-1r) present larger gallbladder volumes (predisposing to bile stasis), significant retardation of small intestinal transit times (resulting in increased cholesterol absorption), and increased biliary cholesterol secretion rates.31 This scenario facilitates nucleation, growth, and agglomeration of cholesterol monohydrate crystals. Indeed, these sequences of events result in a significantly higher prevalence of cholesterol gallstones in the CCK-1 receptor knockout mice. Interestingly, it has been found that some polymorphisms in the CCK-1R gene are associated with gallstone formation in humans.32 Interestingly, a recent observation suggests that density of CCK-1 receptors on gallbladder smooth muscle cells is significantly decreased in patients with the combination of gallstones and diabetes mellitus compared to those with only gallstones,33 thus underlining the possibility that cholesterol gallstone disease is an associated condition of the metabolic syndrome.7

Fibroblast Growth Factor 19.

It has been recently shown that in response to bile acid, the distal small intestine secretes FGF15 in mice (human ortholog FGF19), a hormone required for gallbladder filling.15 The role of FGF19 in the endocrine regulation of the gut-liver axis was first identified in the regulation of hepatic bile acid synthesis in response to intestinal bile acid concentrations.34 After being actively absorbed in the distal ileum, bile acids activate the nuclear receptor farnesoid X receptor (FXR),35 which in turn promotes the expression of FGF19. FGF19 is a circulating hormone, secreted in the portal circulation and active in the liver, where it binds to FGF receptor 4 (FGFR4), and ignites a metabolic cascade which results in inhibited bile acid synthesis. The effect is mediated by the atypical nuclear receptor small heterodimer partner (SHP), which inhibits gene expression of 7α-hydroxylase (CYP7α), the rate-limiting enzyme for bile acid synthesis.36, 37

Compelling new evidence now extends the physiological role of FGF19 in the gut-liver axis to include endocrine control of gallbladder motility. FGF15 knockout mice display a gallbladder completely devoid of bile, in the absence of any impairment of gallbladder histology, bile flow, and interprandial or postprandial CCK release. In parallel, administration of recombinant FGF15 or FGF19 doubles gallbladder volume of wild-type mice, and restores gallbladder volume in FGF15 knockout mice at levels just as high as that of the wild-type. Although the mechanism for this effect is not completely solved, the relaxation induced by FGF15/FGF19 is likely dependent on increased production of cyclic adenosine monophosphate (cAMP), which antagonizes the effect of CCK. The ability of FGF15/FGF19 to actively mediate gallbladder relaxation is conserved in FGFR4 knockout mice. Apparently, this receptor does not mediate the effects of FGF15/FGF19 on the gallbladder.15 Gene expression profiling demonstrates high levels of FGFR3 in the gallbladder, thus suggesting this receptor as the top candidate to mediate the effects of FGF19.15 Furthermore, the identification of different isoforms of FGFRs in additional anatomical districts of the enterohepatic system (that is, bile duct and the sphincter of Oddi) holds the promise for new exciting discoveries on the role of FGF19 in the endocrine regulation of digestive physiology. Also, we believe that the tissue specificity for different receptors of the same hormone might be of future pharmacological relevance.

These studies demonstrate that gallbladder filling is actively regulated by an endocrine pathway and suggest a postprandial timing mechanism that controls gallbladder motility. After the ingestion of a meal, CCK secretion would induce gallbladder contraction and provide the adequate concentration of duodenal amphipathic bile acids, which directly contribute to solubilization and absorption of cholesterol, fat, and fat-soluble vitamins. Once bile acids arrive at the terminal ileum, they regulate the FXR-induced production and the secretion of FGF15 (or the human ortholog FGF19), which now signals back to the gallbladder in order to guide its refilling and prepare the target organ for the next meal. This intriguing scenario is depicted in Fig. 1. Future studies are needed to clarify if this regulatory network would also act as the ileal brake of the digestion, possibly ending postprandial pancreatic secretion and regulating gastric emptying.

Figure 1.

A dynamic scenario for the regulation of gallbladder motility. In the fasting state, motilin secretion at the end of phase II of the migrating motor complexes14 induces a weak but significant gallbladder contraction. After the ingestion of a meal, CCK secretion would induce gallbladder contraction and provide the adequate concentration of duodenal amphipathic bile acids, which directly contribute to solubilization, digestion, and absorption of cholesterol, fat, and liposoluble vitamins. Once bile acids arrive at the terminal ileum, they regulate the FXR-induced production and the secretion of FGF15 in mice (or the human ortholog FGF19), which now signals back to the gallbladder in order to guide its refilling and prepare the target organ for the next meal. The inset represents the gallbladder mucosa and smooth muscle with a set of proteins that play a key role in lipid absorption and smooth muscle contractility (see text for details). Adapted and modified from Choi et al.15

Neuronal Control.

Apart from humoral factors, gallbladder motility is under control of neuronal stimuli. The gallbladder is innervated by both sympathetic and parasympathetic (vagal) terminations reaching the smooth muscle and interconnecting with nonadrenergic nerve cells. Ganglia, connected with both sympathetic and parasympathetic terminations, are distributed throughout the three layers of the gallbladder tissues (subserosal, myenteric, and mucosal plexuses) and provide intrinsic innervation.38 Ganglia represent the site of complex modulatory interactions influencing muscle and epithelial cell function.39 CCK may also be active presynaptically within ganglia to increase acetylcholine release from vagal terminals.39 During fasting and in the early postprandial phase, gallbladder motility is also under the control of neural cholinergic pathways mediated by muscarinic receptors,40, 41 which, if stimulated, invariably involve myosin light-chain phosphorylation.42 Because neural pathways also govern gallbladder motility, the presence of autonomic neuropathy might affect gallbladder motor function in several conditions and be a further factor predisposing an individual to gallstone formation. In diabetic patients, a link between autonomic neuropathy and impaired gallbladder motility has been suggested.43–51 Patients with chronic functional constipation have a subclinical form of autonomic neuropathy,52 and gastrointestinal motility is defective at various levels, including at the gallbladder.53 A pathophysiological link between autonomic neuropathy and gallbladder motility has also been observed in patients with chronic alcohol abuse54 or chronic liver diseases.55, 56 It has been proposed that autonomic neuropathy contributes to (pigment) gallstone formation in patients with advanced liver cirrhosis (Child class C), who indeed show a high prevalence of both gallstone disease and autonomic neuropathy.57 Also, in a recent study from our group, 86% of adult patients with β-thalassemia major had abnormal tests of sympathetic and parasympathetic system and, in these subjects, positive tests for autonomic neuropathy tended to correlate with abnormal gallbladder motility.58

Conditions Associated with Impaired Gallbladder Motility.

Impaired gallbladder motor function has been also documented as a complication in a number of diseases, as depicted in Table 2.12 Such conditions include patients who develop pigment gallstones59, 60 as a consequence of chronic hemolysis, that is, sickle hemoglobinopathy,61 and adults with liver cirrhosis62–64 and β-thalassemia major.58 Patients with nonhemolytic black pigment stones may also exhibit some degree of gallbladder motility defects, although these are less, compared with patients with cholesterol stones (Fig. 2).17 Other conditions can be found as part of the so-called “metabolic syndrome”,7 inflammatory bowel diseases or celiac disease, spinal cord injury, total parenteral nutrition, and gastrectomy.

Table 2. Conditions Potentially Associated with Impaired Gallbladder Motility
ConditionPrincipal mechanism(s)Reference(s)
  • Abbreviation: CCK, cholecystokinin.

  • *

    Can be components of the metabolic syndrome.7, 216

5-Hydroxytryptamine - inhibitorsInhibition of 5-hydroxytryptamine reuptake174
Acute hepatitis ADelayed gastric emptying, viraemia175
Celiac diseaseDecreased release of endogenous CCK176, 177
Chronic pancreatitisIncreased fasting and residual gallbladder volumes; decreased release of endogenous CCK178, 179
Crohn's diseaseIncreased endogenous CCK release, decreased fasting gallbladder volume180–183
Diabetes mellitus*Autonomic neuropathy, gallbladder stasis43–51, 184, 185
Down syndromeDecreased fasting and emptying gallbladder volumes186
Growth hormone deficiencyPartial decrease of endogenous CCK release187
Hypertriglyceridaemia*Impaired gall bladder motility due to decreased sensitivity to CCK188
Irritable bowel syndromeDecreased gallbladder emptying; impaired response to endogenous CCK189, 190
Insulin resistance*Decreased gallbladder emptying191
Liver cirrhosisDecreased gallbladder emptying: lack of coordination with gastric emptying63, 64, 192–194
Obesity*/rapid weight lossEnlarged fasting / residual gallbladder volume; decreased postprandial emptying195–197
Octreotide therapy (e.g. acromegaly)Inhibition of endogenous CCK release; gallbladder stasis198–200
Oral bile acid therapyIncreased fasting and residual gallbladder volume149, 201, 202
PregnancyProgesterone-induced gallbladder stasis203–206
Primary sclerosing cholangitisIncreased fasting and residual gallbladder volume207
SomatostatinomaInhibition of endogenous CCK release; gallbladder stasis9, 208
Spinal cord injuryGallbladder stasis209, 210
β-thalassemia majorDecreased gallbladder emptying and autonomic neuropathy58, 211
Total parenteral nutritionGallbladder stasis (lack of enteral nutrition, decreased release of endogenous CCK)5, 212, 213
Total/partial gastric resectionVagotomy, increased fasting gallbladder volume and decreased gallbladder emptying214, 215
Figure 2.

Fasting and postprandial gallbladder volumes in patients with cholesterol and pigment gallstones compared to healthy controls. Patients with cholesterol gallstones showed significantly larger fasting volume and postprandial volumes than controls and patients with pigment stones at each time point during the 2-hour study period (*). Patients with pigment gallstones showed similar fasting volume but significantly larger postprandial volumes than controls from 20-60 minutes after meal ingestion (* and †) (analysis of variance followed by Fishers's LSD multiple comparison test, 0.01 < P < 0.05). Adapted from Portincasa et al.17

Certain medications can worsen gallbladder motility and increase the predisposition to gallstone formation.12 In acromegalic patients, the use of the long-acting octreotide formulation resulted in the increase of fasting gallbladder volume, severe inhibition of postprandial cholecystokinin release, and gallbladder emptying (all changes associated with gallbladder stasis and high risk of gallstone formation).65 In these patients, the motility defects of the gallbladder and intestine during octreotide therapy are associated with increased cholesterol saturation in bile;66 potential prophylactic therapies for gallstones might therefore include prokinetics and/or hydrophilic ursodeoxycholic acid.


ABC, ATP-dependent binding cassette transporters; AE, anion exchanger; ASBT, apical sodium-dependent bile acid transporter; B, bradykinin; BRP, bombesin-related peptides; CCK, cholecystokinin; CFTR, cystic fibrosis transmembrane conductance regulator; CGRP, calcitonin gene–related peptide; CMC, critical micellar concentration; cPLA2, cytosolic phospholipase A2; CYP7α, cholesterol 7α-hydroxylase; cAMP, cyclic adenosine monophosphate; ERG1, ether-a-go-go–related gene 1; ET-1, endothelin-1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FXR, farnesoid X receptor; GRP, gastrin-releasing peptide; H, histamine; HDL, high-density lipoprotein; LT, leukotriene; LXR, liver X receptor; MAPK, mitogen-activating protein kinase; MDR, multidrug resistance; MICOL, Multicenter Italian Study on Cholelithiasis; MUC, mucin gene; NMB, NMB-preferring receptor; NPC1L1, Niemann-Pick C1-like 1; NT, neurotensin; OATP, organic anion transporting polypeptide; PACAP, pituitary adenylate cyclase-activating peptide; PGE2, prostaglandin E2; PHI, peptide histidine isoleucine; PKC, protein kinase C; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; SHP, small heterodimer partner; SRBI, scavenger receptor class B type I; VIP, vasoactive intestinal peptide.

Bile Composition and Gallbladder Motility

At present, gallbladder motility defects observed in patients with gallstones are mainly interpreted as secondary events related to cholesterol supersaturation. Nucleation of solid cholesterol monohydrate crystals from supersaturated bile leads to the precipitation of the sterol, absorption by the gallbladder wall,67 and ensuing incorporation into the smooth muscle sarcolemma. Excessive incorporation of cholesterol inside gallbladder smooth muscle cells would then trigger biochemical alterations, resulting in impaired motility of the organ (see below). In addition, at the moment, there is no direct evidence that the impaired gallbladder motor function observed in cholelithiasis associated with metabolic disorders, as diabetes, obesity, or insulin resistance, is the primary event; rather, also in these conditions, gallbladder dyskinesia is likely the result of systemic impaired lipid metabolism and altered biliary lipid composition. The pathophysiological scenario could be that of a vicious cycle: an impairment of biliary lipid composition causes cholesterol supersaturation and precipitation. Following epithelial absorption, cholesterol is incorporated into the sarcolemma of gallbladder smooth muscle cells, causing contraction defects and gallbladder relaxation. The impairment of gallbladder motor function delays gallbladder emptying, and this alteration increases contact time of precipitated cholesterol with the mucosa, leading to additional cholesterol absorption and more extensive muscular damage.68, 69

Bile contains also plant sterols (campesterol, sitosterol) and oxysterols that may play a role in the pathogenesis of chronic inflammation of the gallbladder. Complex relationships exist between bile compositions and gallbladder motor function, because different patterns of postprandial gallbladder emptying can regulate bile compositions.21 Conversely, luminal biliary lipids and/or proteins can influence gallbladder smooth muscle contractility.27 Because of the importance of biliary lipid composition for the pathogenesis of gallbladder motor dysfunction and gallstone disease, the biochemical and biophysical events driving lipid physiology in the gallbladder have been the object of intense research.

Molecular Mechanisms Controlling Lipid Absorption in the Gallbladder Epithelia.

In addition to its active role in absorbing water, the gallbladder wall is also capable of absorbing significant amounts of cholesterol from bile. In healthy subjects, this mechanism might keep biliary cholesterol saturation within physiological ranges by absorption of excess cholesterol.67 Interestingly, the absorptive capacity of gallbladder mucosa is not limited to cholesterol, but also includes bile acids and phospholipids.67, 70In vitro experiments performed in isolated, intra-arterially perfused gallbladder suggest that the human gallbladder displays preferential absorption for cholesterol and phospholipids, whereas bile acid absorption capacity is significantly smaller. In addition, in vitro evidence suggests that the gallbladder epithelium is also capable of the active efflux of cholesterol through both the apical and the basolateral membranes.71 At a molecular level, the identification of the proteins involved in the processes of cholesterol absorption and secretion has taken advantage of the progresses made regarding the mechanisms responsible for cholesterol transport in liver and intestine. Indeed, gallbladder epithelia and enterocytes of the proximal gut express similar molecular mechanisms regulating cholesterol absorption. A coordinated interplay between different lipid transport proteins might exist, because the net cholesterol absorption in the gallbladder, as in the gut, probably depends on the interaction between scavenger receptor class B type I (SR-BI), Niemann-Pick C1-like 1 (NPC1L1), ABCG5-ABCG8, and ABCA1 activities. Two of these proteins are potential candidates for mediating active cholesterol absorption: SR-BI and NPC1L1.

In 1998, SR-BI was suggested as the protein responsible for intestinal cholesterol absorption.72 SR-BI had been isolated 5 years earlier73 and later identified as the high-affinity receptor for circulating high-density lipoprotein (HDL),74, 75 and a high-affinity cholesterol binding protein on intestinal brush-border membrane vesicles.75 However, the importance of SR-BI for intestinal cholesterol absorption has been challenged by the finding of unaltered intestinal cholesterol absorption in SR-BI knockout mice.76 Also, an accelerated lipid absorption has been observed in mice overexpressing SR-BI.77 SR-BI is present in gallbladder epithelial cells, where it localizes on the apical membrane and may participate in the process of cholesterol absorption, because its expression has been found to be reduced in conditions of cholesterol hypersecretion.78, 79 Nevertheless, the translational value of these findings is still controversial, because gallstone formation and gallbladder wall cholesterol content do not correlate with SR-BI expression levels in experimental models of diet-induced cholelithiasis.79

Identified and characterized in 2000,80 NPC1L1 was later proposed as the protein mediating intestinal cholesterol absorption at the brush-border membrane of the enterocyte.81 Although NPC1L1 is expressed only in the intestine and gallbladder in mice, NPC1L1 is present at high levels in liver and intestine in humans.81 The recent generation of transgenic mice with selective overexpression of NPC1L1 in the liver has proven the role that this protein plays in restraining excessive biliary cholesterol secretion, by actively reabsorbing cholesterol at the hepatocyte canalicular membrane.82 Given the species differences between humans and mice, it is fascinating to speculate that NPC1L1 in mouse gallbladder fulfills the same role as human NPC1L1 in the liver. The involvement of NPC1L1 in gallstone disease has not yet been established, but theoretically its loss of function would favor gallstone formation by increasing the amount of biliary cholesterol. Ezetimibe is a drug that reduces intestinal cholesterol absorption in hypercholesterolemic patients, probably via inhibition of NPC1L1.83 In the near future, the impact of ezetimibe therapy upon hepatic and gallbladder handling of cholesterol and on gallstone disease needs to be evaluated.

Regarding the process of cholesterol efflux, the role of ATP-dependent binding cassette (ABC) transporters appears critical; these transport proteins are located both on plasma and intracellular membranes, and are responsible for active, energy-dependent transport of molecules across biological membranes. The heterodimer ABCG5/ABCG8 is responsible for cholesterol secretion into bile at the canalicular side of the hepatocyte, and also for apical secretion of cholesterol in the intestinal lumen at the brush border membrane of the enterocyte.84, 85 By simultaneously enhancing biliary secretion and restraining intestinal absorption, ABCG5/ABCG8 play a critical role in enhancing fecal cholesterol disposal. Mutations of either ABCG5 or ABCG8 result in sitosterolemia, a genetic syndrome characterized by excessive accumulation of cholesterol and phytosterols in the body.86 ABCG5/ABCG8 are expressed in human gallbladder epithelial cells, where they could act to limit excessive cholesterol absorption.87In vitro evidence generated in canine gallbladder epithelial cells suggests that ABCG5/ABCG8 are localized intracellularly in basal conditions, but are shuttled on the apical membrane in conditions of cholesterol overload.87 The translocation is triggered by activation of the oxysterol liver X receptors (LXRα and LXRβ), which are nuclear receptors activated by increased intracellular levels of oxysterols and responsible for the upregulation of genes that limit cholesterol overload, including cholesterol effluxers ABCG5/ABCG8.88 ABCA1 is yet another ABC transporter involved in cholesterol trafficking. In the circulation, ABCA1 mediates the transfer of intracellular cholesterol and phospholipids to circulating HDL.89 ABCA1 is mutated in Tangier disease, a clinical condition characterized by nearly absent plasma levels of HDL and increased cardiovascular mortality.90–92 Although confocal imaging proves the basolateral localization of ABCA1 in gallbladder epithelial cells, the role of ABCA1 in gallbladder needs further investigation.71

Both human and mouse gallbladder epithelia also expressed megalin, which is a protein able to mediate the endocytosis of numerous ligands, including HDL/apolipoprotein A-I (apoA-I). Bile acids and a lithogenic diet are able to strongly increase the expression of megalin, thus supporting a pathophysiological role of megalin in gallstone pathogenesis.93 Lastly, several other transporters are present in the epithelial cells of the gallbladder, including the bile acid transporters apical sodium-dependent bile acid transporter (ASBT)94 and organic anion transporting polypeptide (OATP-A),95 cystic fibrosis transmembrane conductance regulator (CFTR),96 multidrug resistance 1 (MDR1), and anion exchanger 2 (AE2).97 The eventual contribution of these proteins in the development of gallstone disease is, at the present time, purely speculative.

Intraluminal Cholesterol.

Defective lipid absorption in the gallbladder has been reported in patients with cholesterol gallstones.70 However, the cholesterol-absorbing capacity of the gallbladder wall might induce excess membrane accumulation of cholesterol in the muscular layer. In fact, gallbladder muscle from patients with cholesterol stones displays an increased mole ratio of membrane cholesterol/phospholipid and decreased membrane fluidity. The increased cholesterol content in gallbladder smooth muscles results in either defective contractility69, 98, 99 and/or relaxation.100, 101 Interestingly, similar to what is observed in arterial myocytes during atherogenesis,102 proliferative modifications of smooth muscle cells have also been reported in gallbladder smooth muscle exposed to excess cholesterol,13 depicting a form of gallbladder hypertrophic leiomyopathy. The presence of bile supersaturated with cholesterol is therefore a condition predisposing to impaired receptor–G protein activation and reduced gallbladder contractility.69, 103–106 Isolated gallbladder smooth muscle cells from patients with cholesterol gallstone show greater dysfunction in response to CCK than smooth muscle cells from gallbladders of patients with pigment stones.13 Also, excess cholesterol could contribute to reduced gallbladder contractility by affecting calcium channel activity, whereas potassium-channels and chloride-channels are not affected.107 These abnormalities may be corrected by removing the excess cholesterol from the plasma membranes, at least in the in vitro model of isolated gallbladder smooth muscle cells.69 Of note, a recent study on patients with gallstones who were treated with ursodeoxycholic acid before cholecystectomy suggests that, after bile acid administration, an improvement of gallbladder muscle contractility is associated with a decreased cholesterol content in the plasma membranes of muscle cells.98

Intraluminal Bile Acids.

Bile acids are able to relax the smooth muscle. Increased biliary/serum concentration of more hydrophobic bile salts (for example, deoxycholate) might lead to impaired gallbladder smooth muscle contractility,108 probably by an effect on intramural neurons.109

In an animal model (guinea pig), incubation of isolated gallbladder smooth muscle cells with a hydrophobic taurochenodeoxycholic acid causes muscle cell dysfunction by inducing formation of H2O2 (activation of NADPH and xanthine oxidase). This leads to lipid peroxidation and activated cytosolic phospholipase A2 (cPLA2) to increase prostaglandin E2 (PGE2) production and, ultimately, to increased free-radical scavengers through protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) pathway.110 In contrast, the more hydrophilic ursodeoxycholate seems to prevent the deoxycholate-dependent impairment of smooth muscle contractility, as demonstrated by in vitro studies of gallbladder smooth muscle strips in an animal model110 and from patients with gallstones.108, 111 The relaxing mechanism of bile acids might also involve their ability to enhance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells, as demonstrated by patch-clamp studies.112

Intraluminal Mucin.

Intraluminal mucin also plays an important role in cholesterol gallstone formation and, as demonstrated by a recent animal study, the increased gallbladder epithelial mucin encoded by mucin gene 1 (MUC1) strongly influences cholelithogenesis by impairing gallbladder motility in mice transgenic for the human MUC1 gene, through an increased cholesterol absorption by the gallbladder wall,113 a lithogenic mechanism completely different from the gel-forming mucins. Thus, it is well possible that inhibiting the secretion and accumulation of not only the gel-forming mucins but also the epithelial mucins in the gallbladder may prevent the formation of cholesterol gallstones. Indeed, decreased MUC1 mucin in the gallbladders of mice with disrupted Muc1 gene reduces susceptibility to cholesterol gallstone formation.114

In addition, expression levels of the gallbladder Muc5ac, a gel-forming mucin gene, are significantly reduced in Muc1/ mice challenged to the lithogenic diet. Consequently, cholesterol crystallization and gallstone formation are drastically retarded. This finding suggests that there may be gene-gene interactions between Muc1 and Muc5ac, which might influence mucin secretion and accumulation in the gallbladder. Elevated concentration of mucin115 and its marker hexosamine116 have been measured in bile of patients with cholesterol crystals and stone, thus opening the speculative hypothesis that changes in bile viscosity secondary to soluble mucin content in bile, could also influence gallbladder emptying. Overall, these results suggest that the epithelial mucin genes may influence gallbladder mucin accumulation by regulating expression and function of the gel-forming mucin genes. A picture of this experimental condition in mice is shown in Fig. 3.

Figure 3.

Because of gallbladder emptying, bile flow rates and biliary bile salt outputs are increased sharply in response to exogenously administered CCK-8 (as shown by the arrows) in mice (A) on chow or (B) fed the lithogenic diet. We observed that gallbladder contractile function is totally impaired in MUC1.Tg mice and partially in wild-type mice, in the lithogenic state. Modified from Wang et al.113

Gallbladder Wall Inflammation.

Substances involved in inflammation processes can strongly influence gallbladder contractility.117–122 Increased proportion of arachidonyl-phosphatidylcholine species (PLA2 activity, PGE2), as a marker of gallbladder mucosal inflammation and mucin concentrations, are found in the animal model fed a lithogenic diet.123 Mice susceptible to gallstone formation show, after the lithogenic diet, an altered gallbladder histology associated with impaired motility and reduced concentrating function.124, 125 Prostaglandins are produced during the inflammatory process in the gallbladder and they induce relaxation of gallstone-containing gallbladders; these mediators might therefore be a determinant of the impaired gallbladder motility.126

Studies on human gallbladder tissues have shown that CCK-induced smooth muscle contraction via the CCK-1 receptor pathway is modulated by prostaglandins in the healthy state. This modulation disappears in gallstone-containing gallbladders, but the excessive serotonin release in advanced cholecystitis normalizes the CCK-induced contraction.120 Several animal studies demonstrate a strong interplay between factors such as inflammation, oxidative stress, and cytotoxic bile salts, and impaired CCK effect during cholesterol gallstone formation.110, 123, 127, 128 In humans, the administration of ursodeoxycholic acid (a more hydrophilic and less cytotoxic bile acid) has been demonstrated to play a role in decreasing chronic gallbladder inflammation in patients with cholesterol gallstones, with better in vitro contractility to CCK and other mediators, such as acetylcholine and KCl.111, 129 The mechanism of action of ursodeoxycholic acid might include decreased biliary cholesterol saturation, decreased incorporation of cholesterol molecules into smooth muscle plasma membrane, and decreased inflammatory mediators such as arachidonyl-phosphatidylcholine species and PLA2,130 and markers of oxidative stress.131

Studies of Gallbladder Motor Function

Gallbladder Contractility In Vitro.

The ability of the gallbladder smooth muscle to contract in the absence of extrinsic neuroendocrine control and/or effects of bile composition/intraluminal content can be effectively studied in vitro with isolated smooth muscle cells and smooth muscle strips obtained from animals and patients following cholecystectomy. The whole gallbladder has also been studied in animal models.27 Similarly to what is seen in vivo, the contractile function of gallbladder smooth muscle cells of cholesterol gallstone patients is abnormal in vitro, as shown by several tensiometric studies.13, 25, 42, 69, 104, 106, 121, 123, 132–144 Gallbladders containing cholesterol stones show a defective muscular contractility13, 28, 68, 69, 99, 120, 126, 145 and/or relaxation.100, 101 This latter phenomenon seems to parallel the decreased gallbladder refilling observed in vivo which, in turn, can increase the tendency toward cholesterol crystal precipitation and aggregation into macroscopic stones.146 The potassium K(ATP) channels seem to play an important role for gallbladder smooth muscle relaxation.134

Gallbladder Motility in a Clinical Setting.

Real-time abdominal ultrasonography provides key information about the morphology and content of the gallbladder including the features of the wall (thickness) and of the content (that is, anechoic bile, sludge, solitary or multiple stones, polyps, neoplasms). This method is noninvasive, highly accurate, and widely available.1, 147 The study of gallbladder motor function, however, requires repeated measurements of the gallbladder volume at different time points after meal ingestion, as markers of fasting gallbladder volume, postprandial gallbladder emptying and refilling ability, and, indirectly, of cystic duct patency.147–149 Biliary scintigraphy, although providing information on gallbladder emptying, refilling, and cystic duct patency, does not allow the study of gallbladder morphology and requires use of radiation.19, 146, 150 Afterward, a negative feedback exists between intraduodenal bile and CCK release.151 The impaired gallbladder emptying which may anticipate the formation of cholesterol gallstones prolongs the residence time of concentrated gallbladder bile, provides more time for nucleation and crystallization of cholesterol crystals from supersaturated bile, and lessens the amount of nucleated cholesterol crystals that can be ejected into the duodenum. Microscopic cholesterol crystals are evolved through aggregation and growth into macroscopic stones with assistance of mucin gels. A subgroup of patients with cholesterol gallstones have defective gallbladder emptying and refilling146 and increased fasting gallbladder volume.13, 14, 20, 21 In the fasting state, the fluctuations of gallbladder volume is under the subtle control of the fasting gastrointestinal hormone motilin.152

Finally, from a clinical point of view, the evaluation of gallbladder motility is also important in the decision-making for patients symptomatic for gallstones. In a recent study, an efficient gallbladder motility in patients with gallstones represented a risk factor for the development of biliary pain.153 By contrast, oral ursodeoxycholic acid increases gallbladder residual volume and reduces the percentage of gallbladder emptying, without affecting the rate of biliary symptoms in the highly symptomatic patient.154, 155

Conclusions and Future Perspectives

The gallbladder plays an essential dynamic role if one thinks that liver bile—a solute highly enriched in cholesterol and lipids—is accumulated, mixed, concentrated in the gallbladder, and secreted intraduodenally during fasting and in the postprandial status. The results of integrated in vivo and in vitro studies in humans and in animal models depict a complex scenario in which subtle mechanisms govern gallbladder motility. Mechanisms include hormonal and extrinsic neuroendocrine factors, bile composition, biliary cholesterol saturation, and intrinsic gallbladder smooth muscle contractile properties with receptorial/postreceptorial features. These mechanisms are directly coordinated via regulatory mechanisms finely tuned in the gut-liver axis. The gallbladder motility abnormalities observed in patients with gallstones are considered mainly secondary rather than primary events. Although some well-known metabolic risk conditions for gallstone disease such as diabetes, obesity, and insulin resistance may be associated with impaired gallbladder motility, there is no direct evidence that in a subgroup of individuals with these conditions impaired gallbladder motility is the main factor involved in gallstone formation. Longitudinal clinical studies are needed to address this intriguing issue. Understanding the pathogenesis of gallbladder motor-dysfunction leading to gallstone formation could be fostered by wide integrated in vivo and in vitro studies of gallbladder function, in particular at a molecular and genetic level. These studies would eventually provide a better strategy for the treatment and prevention of cholesterol gallstones.