Staffan Sahlin

ACAT2 Is Localized to Hepatocytes and Is the Major Cholesterol-Esterifying Enzyme in Human Liver

Background—Two acyl-coenzyme A:cholesterol acyltransferase (ACAT) genes, ACAT1 and ACAT2, have been identified that encode 2 proteins responsible for intracellular cholesterol esterification. Methods and Results—In this study, immunohistology was used to establish their cellular localization in human liver biopsies. ACAT2 protein expression was confined to hepatocytes, whereas ACAT1 protein was found in Kupffer cells only. Studies with a highly specific ACAT2 inhibitor, pyripyropene A, in microsomal activity assays demonstrated that ACAT2 activity was highly variable among individual human liver samples, whereas ACAT1 activity was more similar in all specimens. ACAT2 provided the major cholesterol-esterifying activity in 3 of 4 human liver samples examined. Conclusions—The data suggest that in diseases in which dysregulation of cholesterol metabolism occurs, such as hypercholesterolemia and atherosclerosis, ACAT2 should be considered a target for prevention and treatment.

Changes in gallbladder bile composition and crystal detection time in morbidly obese subjects after bariatric surgery

The aim of the present study was to elucidate the mechanisms of development of cholesterol crystals and gallstones during weight reduction in obese subjects. Twenty‐five morbidly obese, gallstone‐free subjects underwent vertical‐banded gastroplasty. Gallbladder bile was collected at the time of the operation via needle aspiration and 1.1–7.3 months after the operation via ultrasound‐guided transhepatic puncture of the gallbladder. The mean weight loss was 17 kg. Two patients developed gallstones and 10 patients displayed cholesterol crystals in their bile. In patients with a follow‐up time of less than 2 months (n = 13), cholesterol saturation increased from 90% to 114% but tended to decrease in the patients with a follow‐up time of more than 2 months. The extraction of the concanavalin‐A–binding fraction from gallbladder bile obtained after weight reduction in 7 patients prolonged crystallization detection time from 6 to 10 days. The hexosamine concentration, a marker for mucin, was increased by about 100% in bile obtained in 6 of 7 patients after weight reduction. In conclusion, the results indicate that crystallization‐promoting compounds (mucin) are of great importance in the development of cholesterol crystals and gallstones in obese subjects during weight reduction, probably because of defective gallbladder emptying. (HEPATOLOGY 2005.)

Biliary lipid composition in patients with cholesterol and pigment gallstones and gallstone-free subjects: deoxycholic acid does not contribute to formation of cholesterol gallstones.

Four main disturbances have been attributed to cholesterol gallstone disease: hypersecretion of cholesterol from the liver with cholesterol supersaturation in bile; disturbed motility with defective absorption and secretion by the gallbladder; increased crystallisation of cholesterol in the gallbladder bile; and slow intestinal transit with increased amount of deoxycholic acid in the bile acid pool. We aimed to evaluate the biliary lipid composition in a large series of gallstone patients, with emphasis on the amount of deoxycholic acid and with respect to number of stones, compared to gallstone free subjects.

Effects of high-dose statin on the human hepatic expression of genes involved in carbohydrate and triglyceride metabolism.

Abstract.  Pramfalk C, Parini P, Gustafsson U, Sahlin S, Eriksson M (Department of Laboratory Medicine, Division of Clinical Chemistry; Department of Biosciences and Nutrition, Centre for Nutrition and Toxicology, Molecular Nutrition Unit, NOVUM; Department of Surgery, Karolinska Institutet at Danderyd Hospital, Danderyd; Department of Endocrinology, Metabolism Unit, Metabolism and Diabetes, and Department of Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, S‐141 86 Stockholm, Sweden). Effects of high‐dose statin on the human hepatic expression of genes involved in carbohydrate and triglyceride metabolism. J Intern Med 2010; 269: 333–339.

Cholesterol synthesis inhibition elicits an integrated molecular response in human livers including decreased ACAT2.

Objective—The purpose of this study was to identify how different degrees of cholesterol synthesis inhibition affect human hepatic cholesterol metabolism. Methods and Results—Thirty-seven normocholesterolemic gallstone patients randomized to treatment with placebo, 20 mg/d fluvastatin, or 80 mg/d atorvastatin for 4 weeks were studied. Based on serum lathosterol determinations, cholesterol synthesis was reduced by 42% and 70% in the 2 groups receiving statins. VLDL cholesterol was reduced by 20% and 55%. During gallstone surgery, a liver biopsy was obtained and hepatic protein and mRNA expression of rate-limiting steps in cholesterol metabolism were assayed and related to serum lipoproteins. A marked induction of LDL receptors and 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase was positively related to the degree of cholesterol synthesis inhibition (ChSI). The activity, protein, and mRNA for ACAT2 were all reduced during ChSI, as was apoE mRNA. The lowering of HDL cholesterol in response to high ChSI could not be explained by altered expression of the HDL receptor CLA-1, ABCA1, or apoA-I. Conclusions—Statin treatment reduces ACAT2 activity in human liver and this effect, in combination with a reduced Apo E expression, may contribute to the favorable lowering of VLDL cholesterol seen in addition to the LDL lowering during statin treatment.

Effects of combined treatment with pravastatin and ursodeoxycholic acid on hepatic cholesterol metabolism

Background Treatment with ursodeoxycholic acid and also, to some degree, statins reduces cholesterol saturation of bile. The present study aimed [ 1 ] to study the effects of combined treatment with ursodeoxycholic acid and pravastatin on hepatic cholesterol metabolism and [ 2 ] to evaluate if the addition of pravastatin to ursodeoxycholic acid treatment has beneficial effects on the lipid composition of gallbladder bile in gallstone patients.

Studies on the mechanism of accumulation of cholesterol in the gallbladder mucosa. Evidence that sterol 27-hydroxylase is not a pathogenetic factor

BACKGROUND/AIMS Cholesterolosis is characterized by accumulation of esterified cholesterol in human gallbladder mucosa. The present study aimed at investigating possible pathogenetic factors for cholesterolosis. The hypothesis was tested that a reduced sterol 27-hydroxylase or an increased amount of ACAT-1 enzyme may be of importance. METHODS Gall bladder mucosa and bile were obtained from patients with cholesterol gallstones undergoing cholecystectomy (30 with and 43 without cholesterolosis). RESULTS In cholesterolosis, the gall bladder mucosa was characterized by a several-fold increase in esterified cholesterol and normal content of free cholesterol. The amount of ACAT-1 protein, measured by immunoblotting, was similar in patients with and without cholesterolosis. The level of 27-hydroxycholesterol in gallbladder mucosa was elevated sevenfold as compared with cholesterol in patients with cholesterolosis. Most (87%) of this oxysterol was esterified and the accumulation is most probably secondary to the higher total amount of cholesterol in the cells. Patients with cholesterolosis had normal levels of both sterol 27-hydroxylase mRNA (real time polymerase chain reaction) and protein (immunoblotting). The enzymatic activity of the sterol 27-hydroxylase in gallbladder mucosa was normal or increased in cholesterolosis. CONCLUSIONS The pathogenesis of cholesterolosis may be multifactorial, but is not caused by reduced efflux of cholesterol due to a defect sterol 27-hydroxylase mechanism.

Occurrence of cholesterol monohydrate crystals in gallbladder and hepatic bile in man: influence of bile acid treatment

Abstract The occurrence of cholesterol monohydrate crystals was examined and related to the degree of cholesterol saturation in gallbladder bile and hepatic bile of gallstone (GS) patients (n= 34), gallstone‐free (GSF) subjects (n= 33) and GS patients treated with chenodeoxycholic acid (CDCA (n= 7) or ursodeoxycholic acid (UDCA) (n= 11) for 3 weeks prior to cholecystectomy. Twenty‐five untreated GS patients (74%) and four UDCA‐treated patients (40%) displayed cholesterol crystals in the gallbladder bile. Only two GSF subjects (6%) and none of the CDCA‐treated patients had crystals. Half of the patients with crystals in the gallbladder bile had crystals also in the hepatic bile. Cholesterol saturation of the gallbladder bile was higher in GS (142 ± 15%, mean ± SEM) than in GSF patients (74 ± 5%). Saturation was also higher in GS patients with crystals (157 ± 20%) than in those without crystals (99 ± 12%). Gallblader bile was unsaturated in all CDCA‐ and UDCA‐treated patients. The results underline the importance of the degree of cholesterol saturation for the formation of cholesterol crystals. The data also give further support to the concept that the mechanism for inducing gallstone dissolution is different for CDCA and UDCA.

DNA Ploidy and S-Phase Fraction in Carcinoma of the Gallbladder Related to Histopathology, Number of Gallstones and Survival 1

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T‐category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S‐phase values in triploid tumours (p=0.05). S‐phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T‐category and tumour grade were independent prognostic factors. The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S‐phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.

Lysosomal enzyme activities in gallbladder mucosa of gallstone-free subjects and patients with gallstones

BACKGROUND/AIMS Gallstone patients have a reduced cellular lysosome content in the gallbladder mucosa cells compared with gallstone-free subjects. The purpose of the study was to further evaluate the possible role of lysosomes in the pathogenesis of cholesterol gallstone formation in humans. METHODS Lysosomal enzyme activities were assayed in gallbladder mucosa and for comparison in liver specimens of 19 gallstone-free subjects and 24 gallstone patients undergoing cholecystectomy. RESULTS Gallstone patients had 25-50% lower activities of the lysosomal proteases cathepsin B, D and L in their gallbladder mucosa compared with gallstone-free subjects. The activity of acid phosphatase also tended to be decreased in gallstone patients. The liver lysosomal enzyme activities were not significantly different between the two groups. CONCLUSIONS The results show that gallstone patients have diminished lysosomal enzyme activities in the gallbladder mucosa, a finding which may be related to decreased intracellular degradation of proteins and/or mucin in the mucosal cells. This may lead to a higher concentration of mucin in gallbladder bile and thus an increased risk of precipitation of cholesterol crystals and gallstone formation.