Cholesterol-fed Prairie Dogs Research Articles

Hepatic and gallbladder acyl-CoA:SN-glycerol-3-phosphate acyltransferase activity in the cholesterol-fed prairie dog: [formula omitted]. Departments of Surgery and Medicine, The Johns Hopkins Medical Institutions, Baltimore, MD

Trois types de photothérapie avec photosensibilisateurs sont actuellement utilisés en dermatologie: la PUVA (psoralène + UVA); la TUV (crude coaltar + UV), la PRT (photoradiation thérapie au dérivé d'hématoporphyrine, HpD); les deux premières ont largement fait la preuve de leur efficacité dans différentes dermatoses, la troisième est au stade expérimental.L'absence de protocole universellement admis, représente l'écueil principal à la TUV. Pour la PUVA, les effets secondaires à long terme, en particulier la carcinogénicité potentielle, sont au centre des préoccupations du clinicien. L'association de rétinoïdes, l'utilisation des pharmacocinétiques pour une meilleure adaptation individuelle du protocole, l'usage de nouveaux psoralènes moins mutagéniques, pourraient représenter des solutions à ce risque potentiel de la PUVA.Enfin, la phototoxicité prolongée, induite par l'injection de l'HpD est l'effet secondaire principal pour le dermatologue de la PRT.Three phototherapeutic regimens with photosensitization are now used in dermatology: PUVA (psoralen + UVA), TUV (crude coaltar + UV), PRT (phototherapy with hematoporphyrin derivative). The efficiency of PUVA and TUV is well known in several dermatoses. PRT is now being tested experimentally.For TUV, the lack of a standardized regimen does not allow a clear-cut evaluation of the therapy. For PUVA, late side-effects, particularly carcinogenicity have to be considered. To improve efficiency and minimize the side-effects of PUVA some procedures, such as association with retinoïds, pharmaco-kinetic studies for individual adaptation of the therapeutic regimen and the use of new less mutagenic psoralens are helpful.The persistent phototoxicity following treatments with hematoporphyrin derivative constitutes the major side-effect observed, for this phototherapy.

Increased in vitro gallbladder mucosal acyl-CoA:sn-glycerol-3-phostphate acyltransferase activity in cholesterol fed prairie dogs: [formula omitted], K Fox-Talbot, A Nakeeb, PA Lipsett, HA Pitt and KD Lillemoe. The Departments of Surgery and Gastroenterology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Increased in vitro gallbladder mucosal acyl-CoA:sn-glycerol-3-phostphate acyltransferase activity in cholesterol fed prairie dogs: [formula omitted], K Fox-Talbot, A Nakeeb, PA Lipsett, HA Pitt and KD Lillemoe. The Departments of Surgery and Gastroenterology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Gallbladder contractility in aspirin- and cholesterol-fed prairie dogs

Background/Aims: Whether aspirin prevents cholesterol gallstone formation is controversial. This study aimed to investigate this issue and determine the depression of gallbladder smooth muscle contractility associated with cholesterol feeding in the prairie dog. Methods: Prairie dogs were divided into four subgroups. Animals were fed control or 1.2% cholesterol diet and treated with placebo or aspirin for 2 weeks. The presence of crystals and stones was determined, and contractile force in response to cholecystokinin octapeptide (CCK-8) of gallbladder muscle strips was measured. Results: Maximal stress of 2.66 ± 0.23 × 104 N/m2 was measured in muscle strips from animals on control diet. Maximal stress was significantly lower in strips from animals on high-cholesterol diet, being 1.49 ± 0.16 × 104 N/m2 with placebo and 1.62 ± 0.23 × 104 N/m2 with aspirin. The difference in maximal stress between aspirin-treated and placebo-treated animals was not significant. Although none of the animals on control diet had crystals or stones, all animals on the high-cholesterol diet, whether receiving placebo or aspirin, had crystals in the bile, and more than 65% had cholesterol stones. Conclusions: Aspirin has no effect on stone formation, nor does it prevent the decrease in contractility despite a profound decrease in endogenous gallbladder prostanoid synthesis.

Cholesterol nucleates rapidly from mixed micelles in the prairie dog

Current theory suggests that the nucleation of cholesterol in human bile requires the aggregation and fusion of cholesterol-enriched phospholipid vesicles. This theory is based on observations which do not exclude the precipitation of cholesterol from mixed micelles. The present study examines the role of mixed micelles and vesicles in the formation of cholesterol monohydrate crystals in the prairie dog. The intermicellar bile salt concentration of prairie dog gallbladder bile was determined using equilibrium dialysis. Model bile equivalent to gallbladder bile from cholesterol-fed prairie dogs was used as dialysant yielding the intermicellar (dialysate) concentration of 9 mM. Cholesterol carriers in gallbladder bile from 11 cholesterol-fed animals were then separated by Sephacryl S200 gel filtration chromatography using eluant buffer containing the intermicellar bile salt concentration. Gel filtration chromatography of fresh bile demonstrated that 100% of cholesterol was carried in the mixed micellar fraction with no vesicles observed in any of the 11 animals. The gallbladder bile nucleation time was 2.0 ± 0.3 days for the cholesterol-fed animals. Gel filtration chromatography immediately after nucleation again revealed a single mixed micellar peak. These data indicate that cholesterol is carried exclusively in and nucleates rapidly from mixed micelles in the cholesterol-fed prairie dog and that cholesterol-phospholipid vesicles are not required in this process.

Increased dietary fat content accelerates cholesterol gallstone formation in the cholesterol-fed prairie dog

Epidemiological studies have provided conflicting information about the relationship between fat consumption and gallstone formation. We studied cholesterol gallstone formation in prairie dogs after 1 wk of the following diets: (group A) a control diet with no added cholesterol and 5% of calories from corn oil, (group B) 1.2% cholesterol with 5% of calories from corn oil or (group C) 1.2% cholesterol with 40% of calories from corn oil. In controls serum cholesterol was 58.9 +/- 4.5 mg/dl, gallbladder bile was unsaturated with cholesterol (cholesterol saturation index = 0.7 +/- 0.1; cholesterol = 3.8 mmol/L) and none of 12 animals formed cholesterol crystals or stones. The low-fat diet supplemented with cholesterol (group B) increased serum and biliary cholesterol concentrations to 292 +/- 76 mg/dl and 7.5 +/- 1.1 mmol/L, respectively (p < 0.05), but cholesterol saturation index was only modestly increased (1.1 +/- 0.1) and in only one of eight animals did cholesterol monohydrate crystals develop. Group C, animals, which received cholesterol plus high levels of corn oil, had higher serum cholesterol levels (457 +/- 66 mg/dl), higher biliary cholesterol concentrations (16.6 +/- 1.3 mmol/L), higher cholesterol saturation indexes (1.7 +/- 0.1) and increased incidence of cholesterol gallstones (5 of 11). The two cholesterol-supplemented diets increased biliary phospholipid concentrations, decreased the ratio of cholic/chenodeoxycholic acid and increased the proportion of biliary lecithins containing linoleic acid, but these abnormalities were greatest in group C, which was given large amounts of corn oil. These findings suggest that cholesterol gallstone formation in the prairie dog is accelerated by increased dietary omega-6 polyunsaturated triglycerides.

Increased dietary fat content accelarates cholesterol gallstone formation in the cholesterol-fed prairie dog

Increased dietary fat content accelarates cholesterol gallstone formation in the cholesterol-fed prairie dog

Gallbladder mucosal blood flow increases during early cholesterol gallstone formation

Gallbladder absorption increases during early cholesterol gallstone formation and is influenced by the intraluminal presence of lithogenic bile. The effect of lithogenic bile on gallbladder mucosal blood flow is unknown. The current study tested the hypothesis that the presence of lithogenic gallbladder and hepatic bile enhances gallbladder mucosal blood flow in cholesterol-fed (0.4%) prairie dogs, as determined by hydrogen gas clearance. Gallbladder mucosal blood flow in control animals was 35.57 ± 3.9 mL · min−1 sd 100 g−1. In contrast, basal gallbladder mucosal blood flow in cholesterol-fed animals was significantly (P < 0.01) increased to 64.94 ± 8.7 mL · min−1 · 100 g−1. In crossover studies, the addition of lithogenic gallbladder bile to control animals (n = 6) resulted in a significant (P < 0.025) 26% increase in gallbladder mucosal blood flow, whereas the addition of nonlithogenic gallbladder bile into gallbladders of cholesterol-fed prairie dogs resulted in a significant (P < 0.025) 58% decrease in gallbladder mucosal blood flow. Similarly, hepatic bile crossover studies showed that the addition of lithogenic hepatic bile to control gallbladders significantly increased (P < 0.025) gallbladder blood flow by 30%, whereas instillation of nonlithogenic hepatic bile in gallbladders of cholesterol-fed animals significantly (P < 0.025) decreased gallbladder mucosal blood flow by 29%. These results suggest that alterations in gallbladder mucosal blood flow, influenced by the presence and absence of lithogenic bile, may play a role in cholesterol gallstone formation.

Inhibition of prostaglandin synthesis fails to prevent gallbladder mucin hypersecretion in the cholesterol-fed prairie dog

Gallstone formation in the cholesterol-fed prairie dog is preceded by an increase in mucin secretion by the gallbladder epithelium, and mucin hypersecretion is believed to promote cholesterol gallstone formation by accelerating the nucleation of cholesterol monohydrate crystals. Some studies have suggested that gallbladder mucin hypersecretion is mediated by increases in gallbladder prostaglandin synthesis, but other observations are difficult to reconcile with this view. An organ culture technique was used to measure mucin secretion in normal prairie dog gallbladder in response to exogenous prostaglandins and agents that increased or decreased endogenous prostaglandin production. Incubation with indomethacin produced a concentration-dependent inhibition of endogenous prostaglandin synthesis with virtually complete inhibition at 10−5 mol/L indomethacin. However, indomethacin had no effect on gallbladder mucin secretion at concentrations as high as 10−5 mol/L, and significant inhibition of mucin secretion was only found at 10−4 mol/L indomethacin, a concentration that also produced a significant increase in lactate dehydrogenase release from cultured explants. Incubation of gallbladder explants with the calcium ionophore A23187 significantly stimulated endogenous prostaglandin synthesis in a concentration-dependent manner, increasing synthesis of prostaglandins E and F to as much as 278% ± 20% and 335% ± 21% of basal values, respectively; however, the same concentrations of A23187 did not stimulate mucin secretion. Incubation of gallbladder explants in the presence of exogenous prostaglandin E2 or prostaglandin F2a in concentrations as high as 10−6 mol/L also did not stimulate mucin secretion. Prairie dogs fed a lithogenic 1.2% cholesterol diet showed a significant increase in gallbladder mucin secretion after 1 week (117.5 ± 10.2% of control, P < 0.05), and 4 of 5 had formed cholesterol monohydrate crystals after 3 weeks. Long-term treatment with indomethacin, 1.2 mg · kg−1 · day−1, failed to inhibit gallbladder mucin hypersecretion (129.2 ± 10.7% of control after 1 week) or cholesterol monohydrate crystal formation (3/5) in cholesterol-fed prairie dogs. Furthermore, incubation of explants with 10−5 mol/L indomethacin failed to prevent in vitro mucin hypersecretion in cholesterol-fed animals. These findings suggest that prostaglandins do not regulate gallbladder mucin secretion in the prairie dog, and it is unlikely that increases in gallbladder prostaglandin synthesis are responsible for mediating gallbladder mucin hypersecretion during cholelithiasis in the prairie dog.

Effect of bile diversion and sphincterotomy on gallbladder muscle contractility and gallstone formation

Feeding prairie dogs a diet rich in cholesterol induces gallstone formation that is preceded by a sustained decrease in gallbladder smooth muscle contractility. Sphincterotomy is known to prevent gallstone formation in cholesterol-fed prairie dogs. Experiments were designed to determine whether the effect of Sphincterotomy is a consequence of hepatic bile diversion, and whether bile diversion prevents the altered contractility. Following sham operation, surgical biliary enteric bypass, or sphincterotomy, prairie dogs were fed a high-cholesterol or a regular diet. Gallbladder muscle contractility and the presence of crystals and stones were determined. In sham-operated animals, the cholesterol diet induced a decrease in gallbladder muscle contractility and caused the formation of cholesterol gallstones. In animals with bile diversion and sphincterotomy, the effects of cholesterol feeding were reduced or prevented. Thus, these procedures may prevent stone formation by preventing a reduction in gallbladder contractility. Contractility was depressed in animals with bile diversion fed a regular diet, compared with animals with a sham operation fed a regular diet. The mechanism for this depression may differ from that induced by the cholesterol diet. Diversion, and perhaps sphincterotomy, impairs gallbladder filling. Thus, gallbladder muscle is not stretched and does not contract against a load. This could result in a “disuse atrophy.” If the results from our study apply to humans, sphincterotomy may reduce stone formation by preventing the effects of lithogenic bile on gallbladder muscle contractility and by enhancing the ability of the muscle to empty the lithogenic bile.

Effects of dietary fish oil on biliary phospholipids and prostaglandin synthesis in the cholesterol‐fed pairie dog

Cholesterol gallstone formation in the prairie dog is accompanied by an increase in the percentage of biliary phospholipids containing arachidonic acid, and an increase in gallbladder prostaglandin (PG) synthesis, but the pathogenetic significance of these changes is unclear. Dietary supplementation with eicosapentaenoic acid (EPA), an omega-3 fatty acid which is commonly found in fish oil, decreases prostaglandin synthesis in some tissues by replacing arachidonic acid, and by competitively inhibiting prostaglandin synthesis. We studied the effect of dietary fish oil on gallbladder PG synthesis, and the relative abundance of various molecular species of phosphatidylcholines and phosphatidylethanolamines in bile and gallbladder epithelium in the cholesterol-fed prairie dog. Prairie dogs were maintained for 4 weeks on one of four diets: i) control, ii) cholesterol-supplemented (0.34%), iii) menhaden oil (50 g/kg chow), or iv) cholesterol plus menhaden oil. Supplementation with menhaden oil resulted in a replacement of arachidonic and linoleic acids with EPA and docosahexaenoic acids in the phospholipids of bile and gallbladder mucosa. In cholesterol-fed animals, supplementation with menhaden oil prevented increased gallbladder PG synthesis. Menhaden oil also reduced the incidence of cholesterol monohydrate crystals among cholesterol-fed animals (9/20 with cholesterol plus menhaden oil vs 21/22 with cholesterol alone), but the improvement could not clearly be attributed to decreased PG synthesis since supplementation with menhaden oil also increased the total phospholipid concentration in bile, and decreased the degree of cholesterol saturation. These results demonstrate that dietary supplementation with omega-3 fatty acids significantly influences biliary phospholipids, and decreases the incidence of cholesterol monohydrate crystal formation in this animal model.

Gallbladder absorption increases during early cholesterol gallstone formation

The hypothesis that the presence of cholelithogenic bile during the early stages of cholesterol gallstone formation promotes gallbladder absorption of water and electrolytes was tested in a prairie dog gallstone model. An increase in gallbladder transport of water and sodium was observed in cholesterol-fed prairie dogs at a time when cholesterol crystals were present, but before gallstone formation. These data suggest that in the presence of cholesterol-saturated bile, in vivo gallbladder absorption is increased during the early stages of cholesterol gallstone formation. The resulting increase in the solute concentration may promote nucleation and, therefore, be an important etiologic factor in cholesterol gallstone formation.

Role of gallbladder mucin in pathophysiology of gallstones.

A critical step in the formation of cholesterol gallstones in nucleation (i.e., the formation of cholesterol monohydrate crystals from supersaturated bile). The rate of nucleation of cholesterol depends upon a critical balance between pronucleating and antinucleating factors in bile. Mucin, a high molecular weight glycoprotein secreted by the gallbladder and biliary duct epithelium, is a pronucleating agent in experimental and human gallstone disease. Gallbladder mucin shares with other epithelial mucins the ability to bind lipids and bile pigment. The hydrophobic binding sites in the polypeptide core of mucin may provide a favorable environment for nucleation of cholesterol monohydrate from supersaturated bile. In nearly all animal models of cholelithiasis, mucin hypersecretion is prominent. The stimulus for gallbladder mucin hypersecretion appears to be a component of lithogenic bile. Prostaglandins regulate mucin release in gallbladder epithelium in vitro and probably in vivo. In the cholesterol-fed prairie dog, blockage of mucin release with aspirin inhibits gallstone formation. These findings suggest that inhibition of mucin release may prevent cholesterol stone formation during high-risk periods or after dissolution therapy with bile salts.

Impaired Gallbladder Emptying Before Gallstone Formation in the Prairie Dog

Several human and experimental observations suggest that gallbladder stasis is an important link between the hepatic secretion of cholesterol saturated bile and the formation of cholesterol gallstones. In the cholesterol-fed prairie dog model, gallbladder stasis occurs before gallstone formation. In this study we sought to determine the specific defects in extrahepatic biliary physiology responsible for gallbladder stasis in this model. Adult male prairie dogs were fed either a trace cholesterol or a 0.4% cholesterol-enriched diet. In acute terminal experiments, gallbladder contents were examined for cholesterol crystals and gallstones, and gallbladder function was determined at rest and in response to intravenous cholecystokinin-octapeptide. The following alterations in gallbladder function developed concurrently with biliary cholesterol crystallization, but before gallstone formation: (a) decreased gallbladder emptying, (b) increased intragallbladder pressure in response to cholecystokinin-octapeptide, (c) increased cystic duct closing pressure, and (d) increased resistance to outflow through the cystic duct.

Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones.

Because mucin glycoproteins may be important in the pathophysiology of gallstones, we studied the relationship among biliary lipids, gallbladder mucin secretion, and gallstone formation in cholesterol-fed prairie dogs. Organ culture studies of gallbladder explants revealed that the incorporation of [(3)H]glucosamine into tissue and secretory gallbladder glycoproteins was significantly increased at 3, 5, 8, and 14 d of feeding. Peak secretion of labeled mucin occurred at 5 d, when total tissue and secreted glycoprotein production was fivefold greater than control. Gel filtration of the secreted glycoprotein on Sepharose 4B indicated that the majority of radioactivity was present in a macromolecule of > 1 million molecular weight. The increased secretion of gallbladder mucin was organ specific, in that [(3)H]glucosamine incorporation into glycoproteins of stomach and colon was unaffected by cholesterol feeding. Similarly, the incorporation of [(3)H]mannose into gallbladder membrane glycoproteins was not altered by cholesterol feeding. The rate of glycoprotein synthesis and secretion returned to normal upon withdrawal of the cholesterol diet, and ligation of the cystic duct before cholesterol feeding prevented gallbladder mucin hypersecretion. Both results indicate that the stimulus to mucin secretion was a constituent of bile. Gallbladder bile after 5 d contained cholesterol in micelles, liquid crystals, and crystals, whereas hepatic bile remained a single micellar phase throughout cholesterol feeding. For this reason the cholesterol-saturation indices of gallbladder bile were compared in both homogenized and centrifuged samples. The micellar phase of gallbladder bile was appreciably less saturated than homogenized bile at 5 and 8 d, which reflects the continuous nucleation of cholesterol in the gallbladder. Purified human gallbladder mucin gels were shown to induce nucleation of lecithin-cholesterol liquid crystals from supersaturated hepatic bile. These in turn gave rise to cholesterol monohydrate crystals within 18 h. Control supersaturated hepatic bile could not be nucleated by the addition of other proteins, and was stable for days upon standing. These results suggest that the increase in cholesterol content of bile in cholesterolfed prairie dogs stimulates gallbladder mucus hypersecretion, and that gallbladder mucus gel is a nucleating agent for biliary cholesterol.

Chronic cholelithiasis and decreased bile salt pool size: Cause or effect?

The hypothesis that the decreased bile salt pool size observed in humans with chronic cholelithiasis is a result rather than a cause of gallstones was tested in a prairie dog gallstone model. A decrease in bile salt pool size was observed in cholesterol-fed prairie dogs only after the chronic presence of stones had induced physiologic, anatomic and microscopic changes typical of human chronic cholecystitis. These data suggest that the decreased bile salt pool size associated with chronic cholelithiasis is a result rather than a cause of gallstones and that the link between the hepatic secretion of cholelithogenic bile and gallstones is altered gallbladder function.