Publications by authors named "Sum P Lee"

45 Publications

Pcsk9 Deletion Promotes Murine Nonalcoholic Steatohepatitis and Hepatic Carcinogenesis: Role of Cholesterol.

Hepatol Commun 2021 Nov 24. Epub 2021 Nov 24.

Division of Gastroenterology, Department of Medicine, Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA.

Proprotein convertase subtilisin/kexin type 9 (Pcsk9) binds to hepatic low-density lipoprotein receptor (LDLR) and induces its internalization and degradation. Pcsk9 inhibition increases LDLR expression by hepatocytes, which causes increased uptake of circulating LDL, thereby reducing plasma LDL-cholesterol. However, by increasing the uptake of LDL by the liver, Pcsk9 inhibition increases the exposure of the liver to cholesterol, which may result in higher risk of steatohepatitis and ever carcinogenesis. We compared Pcsk9-/- knockout (KO) mice and appropriate wild-type (WT) controls of the same strain assigned to a high-fat (15%, wt/wt) diet for 9 months supplemented with 0.25%, 0.5%, or 0.75% dietary cholesterol. Pcsk9 KO mice on a high-fat, high-cholesterol diet exhibited higher levels of hepatic free cholesterol loading and hepatic cholesterol crystallization than their WT counterparts. Pcsk9 KO mice developed crown-like structures of macrophages surrounding cholesterol crystal-containing lipid droplets and hepatocytes, exhibited higher levels of apoptosis, and developed significantly more hepatic inflammation and fibrosis consistent with fibrosing steatohepatitis, including 5-fold and 11-fold more fibrosis at 0.5% and 0.75% dietary cholesterol, respectively. When injected with diethylnitrosamine, a hepatic carcinogen, early-in-life Pcsk9 KO mice were more likely to develop liver cancer than WT mice. Conclusion: Pcsk9 KO mice on high-cholesterol diets developed increased hepatic free cholesterol and cholesterol crystals and fibrosing steatohepatitis with a higher predisposition to liver cancer compared with WT mice. Future studies should evaluate whether patients on long-term treatment with anti-PSCK9 monoclonal antibodies are at increased risk of hepatic steatosis, steatohepatitis or liver cancer, while accounting for concurrent use of statins.
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http://dx.doi.org/10.1002/hep4.1858DOI Listing
November 2021

Cholesterol Crystals in Hepatocyte Lipid Droplets Are Strongly Associated With Human Nonalcoholic Steatohepatitis.

Hepatol Commun 2019 Jun 1;3(6):776-791. Epub 2019 Apr 1.

Division of Gastroenterology, Department of Medicine Veterans Affairs Puget Sound Health Care System Seattle WA.

It is unclear what drives the development of fibrosing nonalcoholic steatohepatitis (NASH). We aimed to determine whether cholesterol crystallization within hepatocyte lipid droplets (LDs) distinguishes patients with fibrosing NASH from patients with isolated hepatic steatosis and to study pathways leading to cholesterol accumulation in hepatocyte LDs Patients with fibrosing NASH (n = 16) were compared to patients with isolated steatosis (n = 14). Almost all patients with fibrosing NASH had free cholesterol staining by filipin (16/16) and cholesterol crystals (15/16) in hepatocyte LDs, mostly in association with the LD membrane, compared to only 3/14 with cholesterol crystals and 3/14 with faint filipin staining in patients with isolated steatosis ( < 0.05). We were unable to identify significant differences in the expression of genes in liver tissue related to cholesterol homeostasis or LD proteins between patients with fibrosing NASH and isolated steatosis. Human hepatoma cell line (HepG2) cells were supplemented with low-density lipoprotein (LDL)-cholesterol and oleic acid to develop large LDs, similar to those observed in patients with NASH. Fluorescent markers were used to track the uptake and intracellular trafficking of LDL-cholesterol. LDL-cholesterol was taken up by HepG2 cells and transported through the endosomal-lysosomal compartment directly to LDs, suggesting direct contact sites between late endosomes and LDs. Exposure of HepG2 cells to LDL-cholesterol resulted in a high concentration of cholesterol and cholesterol crystallization in LDs. Excess cholesterol is stored in the liver primarily within hepatocyte LDs where it can crystallize. Our findings are best explained by direct transport of cholesterol from late endosomes/lysosomes to LDs in hepatocytes. We found a strong association between the presence of LD cholesterol crystals and the development of fibrosing NASH in humans, suggesting a causal relationship.
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http://dx.doi.org/10.1002/hep4.1348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545865PMC
June 2019

Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.

J Lipid Res 2017 06 12;58(6):1067-1079. Epub 2017 Apr 12.

Division of Gastroenterology, Department of Medicine, Veterans Affairs Puget Sound Health Care System, Seattle, WA.

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of , and interleukin 1beta () mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.
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http://dx.doi.org/10.1194/jlr.M072454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5456359PMC
June 2017

Gallstones: new insights into an old story.

F1000Res 2016 26;5. Epub 2016 Jul 26.

Division of Gastroenterology, University of Washington, Seattle, WA, USA.

Gallstones, particularly cholesterol gallstones, are common in Western populations and may cause symptoms such as biliary colic or complications such as acute cholecystitis or gallstone pancreatitis. Recent studies have allowed for a better understanding of the risk of symptoms or complications in patients with gallstones. In addition, newer data suggest an association of gallstones with overall mortality, cardiovascular disease, gastrointestinal cancers, and non-alcoholic fatty liver disease. Knowledge of appropriate indications and timing of cholecystectomy, particularly for mild biliary pancreatitis, has gradually accumulated. Lastly, there are exciting possibilities for novel agents to treat or prevent cholesterol stone disease. This review covers new advances in our understanding of the natural history, clinical associations, and management of gallstone disease.
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http://dx.doi.org/10.12688/f1000research.8874.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962289PMC
August 2016

The changing faces of cholangitis.

F1000Res 2016 17;5. Epub 2016 Jun 17.

Department of Medicine, Division of Gastroenterology , University of Washington School of Medicine, Seattle, WA, USA.

A variety of diseases are included under the umbrella term 'cholangitis', including hepatobiliary diseases with an autoimmune pathogenesis (such as primary sclerosing cholangitis, primary biliary cholangitis, and IgG4-associated sclerosing cholangitis) and disease processes associated with intraductal stones and infectious etiologies (such as ascending bacterial cholangitis, recurrent pyogenic cholangitis, and liver fluke-associated cholangitis). Recent advances in the pathophysiologic bases of these disorders, particularly with respect to the autoimmune variety, are allowing improved diagnosis and prognostication as well as providing the opportunity to refine and re-imagine treatment modalities. The aim of this review is to highlight selected advances in cholangitis research that point to novel insights into the pathophysiology, diagnosis, and treatment of this diverse array of disorders.
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http://dx.doi.org/10.12688/f1000research.8745.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916985PMC
June 2016

Physical activity, maternal metabolic measures, and the incidence of gallbladder sludge or stones during pregnancy: a randomized trial.

Am J Perinatol 2014 Jan 1;31(1):39-48. Epub 2013 Mar 1.

Department of Epidemiology, University of Washington, Seattle, Washington.

Objective: To evaluate the effect of a physical activity intervention upon the incidence of gallbladder sludge or stones during pregnancy.

Study Design: Pregnant women without gallstones were randomized to an intervention to increase moderate to vigorous physical activity or control. Intervention group women received motivational materials and small-group instruction to increase physical activity. Gallbladder ultrasound and blood draws were obtained at entry, 18 weeks' gestation, and 36 weeks' gestation.

Results: In all, 591 were randomized to the intervention and 605 women to control groups. Women in the intervention group reported modestly higher levels of physical activity compared with control women, and fewer women in the intervention group reported no physical activity during pregnancy. The incidence of gallbladder sludge or stones was similar in intervention and control groups at 18 weeks (4.8% versus 5.4%; relative risk 0.89; 95% confidence interval 0.53, 1.47) and 36 weeks (4.3% versus 3.3%; relative risk 1.31; 95% confidence interval 0.70, 2.54). Fasting glucose, lipid, insulin, leptin, and adiponectin levels were similar in the two groups, as was insulin sensitivity and the incidence of gestational diabetes.

Conclusion: An intervention to increase moderate to vigorous physical activity did not decrease the incidence of gallbladder sludge or stones during pregnancy and did not result in improvement in maternal metabolic measures.
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http://dx.doi.org/10.1055/s-0033-1334455DOI Listing
January 2014

Liver fluke-induced hepatic oxysterols stimulate DNA damage and apoptosis in cultured human cholangiocytes.

Mutat Res 2012 Mar 21;731(1-2):48-57. Epub 2011 Oct 21.

Department of Biochemistry, Khon Kaen University, Khon Kaen, Thailand.

Oxysterols are cholesterol oxidation products that are generated by enzymatic reactions through cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols have been identified in bile in the setting of chronic inflammation, suggesting that biliary epithelial cells are chronically exposed to these compounds in certain clinical settings. We hypothesized that biliary oxysterols resulting from liver fluke infection participate in cholangiocarcinogenesis. Using gas chromatography/mass spectrometry, we identified oxysterols in livers from hamsters infected with Opisthorchis viverrini that develop cholangiocarcinoma. Five oxysterols were found: 7-keto-cholesta-3,5-diene (7KD), 3-keto-cholest-4-ene (3K4), 3-keto-cholest-7-ene (3K7), 3-keto-cholesta-4,6-diene (3KD), and cholestan-3β,5α,6β-triol (Triol). Triol and 3K4 were found at significantly higher levels in the livers of hamsters with O. viverrini-induced cholangiocarcinoma. We therefore investigated the effects of Triol and 3K4 on induction of cholangiocarcinogenesis using an in vitro human cholangiocyte culture model. Triol- and 3K4-treated cells underwent apoptosis. Western blot analysis showed significantly increased levels of Bax and decreased levels of Bcl-2 in these cells. Increased cytochrome c release from mitochondria was found following treatment with Triol and 3K4. Triol and 3K4 also induced formation of the DNA adducts 1,N(6)-etheno-2'-deoxyadenosine, 3,N(4)-etheno-2'-deoxycytidine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in cholangiocytes. The data suggest that Triol and 3K4 cause DNA damage via oxidative stress. Chronic liver fluke infection increases production of the oxysterols Triol and 3K4 in the setting of chronic inflammation in the biliary system. These oxysterols induce apoptosis and DNA damage in cholangiocytes. Insufficient and impaired DNA repair of such mutated cells may enhance clonal expansion and further drive the change in cellular phenotype from normal to malignant.
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http://dx.doi.org/10.1016/j.mrfmmm.2011.10.009DOI Listing
March 2012

Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis.

Gastroenterology 2011 Oct 23;141(4):1393-403, 1403.e1-5. Epub 2011 Jun 23.

Liver Research Group, ANU Medical School at The Canberra Hospital, Garran, ACT, Australia.

Background & Aims: Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH.

Methods: Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice.

Results: Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis.

Conclusions: In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.
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http://dx.doi.org/10.1053/j.gastro.2011.06.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186822PMC
October 2011

Effects of lipopolysaccharide on platelet-derived growth factor isoform and receptor expression in cultured rat common bile duct fibroblasts and cholangiocytes.

J Gastroenterol Hepatol 2009 Jul;24(7):1218-25

Division of Gastroenterology, Department of Medicine, University of Washington and Veterans Affairs Puget Sound Health Care System, Seattle Division, Seattle, Washington 98195, USA.

Background And Aim: Little is known about the role of platelet-derived growth factor (PDGF) in biliary fibrosis in the setting of bacterial colonization of the biliary tree. We therefore sought to investigate whether exposure to bacterial lipopolysaccharide (LPS) alters PDGF isoform and receptor expression in cultured rat common bile duct fibroblasts (CBDF) and normal rat cholangiocytes (NRC).

Methods: Collagen content in cells and media was assessed by colorimetric assay and gel electrophoresis. mRNA levels of PDGF-A and -B, and PDGF-Receptors (PDGF-R) alpha and beta were measured by relative quantitative real-time PCR. Protein levels of PDGF-AA, AB and BB were measured by ELISA, and PDGF-Ralpha and PDGF-Rbeta by Western blot.

Results: In CBDF, LPS increased total soluble collagen synthesis and secretion. PDGF-Ralpha and beta mRNA and protein were also increased by LPS treatment in CBDF. Lipopolysaccharide treatment elicited an increase in PDGF-A and -B mRNA levels in CBDF. In NRC, levels of PDGF-AmRNAincreased in a dose-dependent fashion following LPS treatment, whereas PDGF-B mRNA showed no response. PDGF-AA secretion was higher by CBDF than by NRC. PDGF-BB levels were also higher in CBDF than in NRC. While PDGF-BB levels did not respond to LPS treatment in CBDF, there was a dosedependent response of this isoform to LPS in NRC. Intracellular and secreted PDGF-AB increased with LPS treatment in NRC.

Conclusions: These results support a model in which chronic bacterial colonization of the biliary tree induces fibrosis through PDGF-dependent mechanisms.
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http://dx.doi.org/10.1111/j.1440-1746.2008.05729.xDOI Listing
July 2009

Association between dietary nutrient composition and the incidence of cirrhosis or liver cancer in the United States population.

Hepatology 2009 Jul;50(1):175-84

Division of Gastroenterology, Department of Medicine, Veterans Affairs Puget Sound Health Care System, Seattle, WA 98108, USA. fax:

Unlabelled: Little is known about the impact of dietary factors on the progression of liver disease. Our aim was to determine whether dietary intake was associated with the risk of cirrhosis-related or liver cancer-related death or hospitalization in the U.S. population. Participants included 9221 persons aged 25-74 years without evidence of cirrhosis at entry into the study or during the first 5 years of follow-up, who were subsequently followed for a mean of 13.3 years as part of the first National Health and Nutrition Examination Survey. Dietary intake was ascertained at baseline using a 24-hour dietary recall questionnaire. During follow-up, 123 of 9221 participants had a diagnosis of cirrhosis (n = 118) or liver cancer (n = 5) in hospitalization records or death certificates, including 36 who were diagnosed only on the basis of death certificates. Participants who reported a diet high in protein were at a higher risk of hospitalization or death due to cirrhosis or liver cancer (P = 0.001), whereas those who reported a diet high in carbohydrates were at a lower risk (P = 0.003), after adjusting for potential confounders (daily consumption of protein, carbohydrate, fat, tea or coffee, and alcohol, gender, race, age, educational attainment, U.S. geographical region, diabetes, body mass index, and subscapular-to-triceps skinfold ratio). Although total fat consumption was not significantly associated with the risk of cirrhosis or liver cancer, cholesterol consumption was associated with higher risk (P = 0.007), whereas serum cholesterol level was not associated with risk of cirrhosis or liver cancer.

Conclusion: Diet may be an important and potentially modifiable determinant of liver disease.
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http://dx.doi.org/10.1002/hep.22941DOI Listing
July 2009

Peripheral amyloid-beta levels regulate amyloid-beta clearance from the central nervous system.

J Alzheimers Dis 2009 ;16(2):325-9

Geriatric Research, Education, and Clinical Center, Seattle, WA, USA.

Amyloid-beta (Abeta) is cleared from the brain by both proteolytic digestion and transport across the blood-brain-barrier into the peripheral circulatory system. To investigate the role peripheral Abeta levels play in regulating Abeta brain clearance, we measured the clearance of [125I]-Abeta(1-40) injected into the brains of liver-ligated rats that allowed peripheral Abeta levels to be maintained at elevated levels for approximately one hour with/without a single peripheral bolus of unlabeled Abeta(1-40). We found that elevating peripheral Abetalevels significantly decreased [125I]-Abeta(1-40) brain clearance, thus supporting the hypothesis that peripheral Abeta levels regulate Abeta clearance from the central nervous system.
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http://dx.doi.org/10.3233/JAD-2009-0964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3066174PMC
June 2009

Gallbladder epithelial cells that engraft in mouse liver can differentiate into hepatocyte-like cells.

Am J Pathol 2009 Mar 13;174(3):842-53. Epub 2009 Feb 13.

Division of Gastroenterology, Department of Medicine, University of Washington, Puget Sound Veterans Affairs Health Care System, Seattle, WA 98195, USA.

We tested the hypothesis that well-differentiated gallbladder epithelial cells (GBECs) are capable of engrafting and surviving in murine liver and acquire phenotypic characteristics of hepatocytes. GBECs isolated from transgenic mice that constitutively express green fluorescent protein (GFP) were either cultured before transplantation or transplanted immediately following isolation. Recipient mice with severe-combined immunodeficiency underwent retrorsine treatment and either partial hepatectomy before transplantation or carbon tetrachloride treatment following transplantation. From 1 to 4 months following transplantation, the livers of recipient mice contained discrete colonies of GFP(+) cells. Most GFP(+) cells surrounded vesicles, were epithelial cell-like in morphology, and expressed the biliary epithelial markers cytokeratin 19 and carbonic anhydrase IV. Subpopulations of GFP(+) cells resembled hepatocytes morphologically and expressed the hepatocyte-specific markers connexin-32 and hepatic nuclear factor-4alpha, but not cytokeratin 19 or carbonic anhydrase IV. At 4 months, cells in GFP(+) colonies were not actively proliferating as determined by proliferating cell nuclear antigen expression. Thus, GBECs are capable of engrafting and surviving in damaged mouse livers, and some can differentiate into cells with hepatocyte-like features. These findings suggest that environmental cues in the recipient liver are sufficient to allow a subpopulation of donor GBECs to differentiate into hepatocyte-like cells in the absence of exogenous transcriptional reprogramming. GBECs might be used as donor cells in a cell transplantation approach for the treatment of liver disease.
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http://dx.doi.org/10.2353/ajpath.2009.080262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665745PMC
March 2009

Serotonin metabolism is dysregulated in cholangiocarcinoma, which has implications for tumor growth.

Cancer Res 2008 Nov;68(22):9184-93

Department of Medicine, Texas A&M Health Science Center, College of Medicine, Scott & White Hospital, Temple, TX 76504, USA.

Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We present novel data indicating that the metabolism of serotonin is dysregulated in cholangiocarcinoma cell lines, compared with normal cholangiocytes, and tissue and bile from cholangiocarcinoma patients. Specifically, there was an increased expression of tryptophan hydroxylase 1 and a suppression of monoamine oxidase A expression (enzymes responsible for the synthesis and degradation of serotonin, respectively) in cholangiocarcinoma. This resulted in an increased secretion of serotonin from cholangiocarcinoma and increased serotonin in the bile from cholangiocarcinoma patients. Increased local serotonin release may have implications on cholangiocarcinoma cell growth. Serotonin administration increased cholangiocarcinoma cell growth in vitro, whereas inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. The data presented here represent the first evidence that serotonin metabolism is dysregulated in cholangiocarcinoma and that modulation of serotonin synthesis may represent an alternative target for the development of therapeutic strategies.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-2133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2593938PMC
November 2008

Physical chemistry of intestinal absorption of biliary cholesterol in mice.

Hepatology 2008 Jul;48(1):177-85

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

Unlabelled: Although many putative sterol transporters influencing cholesterol absorption and physical-chemical factors affecting dietary cholesterol absorption have been extensively investigated, it is still unclear how biliary cholesterol contributes to the regulation of intestinal cholesterol absorption. We studied whether the gallbladder can modulate the microaggregates of cholesterol carriers, which may in turn influence the intestinal absorption of biliary cholesterol. Supersaturated, crystallized, or micellar model biles were delivered via a duodenal catheter to conscious, freely moving C57L mice daily for 2 days. Intestinal uptake and absorption of biliary cholesterol and its fecal excretion, as well as expression levels of intestinal sterol transporters, were analyzed. Cholesterol uptake and absorption by the enterocyte were dramatically reduced in mice treated with crystallized biles compared with supersaturated biles. This correlated with the higher cumulative radioactivity of cholesterol recovered in the feces at 24 hours. Such findings were absent with the added reference compound sitostanol. After removing cholesterol crystals from crystallized biles, micellar biles showed essentially identical effects on intestinal absorption but with lower fecal cholesterol excretion compared with the original samples containing crystals. Expression levels of the jejunal Abcg5 (ATP-binding cassette transporter G5) and Abcg8, but not Npc1l1 (Niemann-Pick C1 like 1), were significantly increased by supersaturated biles compared with crystallized biles.

Conclusion: Different physical forms of biliary cholesterol dramatically determine intestinal uptake and absorption of cholesterol. Solid plate-like cholesterol monohydrate crystals in bile are probably not absorbed and are totally excreted in feces from the body. The gallbladder may have a role in regulating cholesterol homeostasis by modulating the physical forms of biliary cholesterol.
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http://dx.doi.org/10.1002/hep.22286DOI Listing
July 2008

Hepatic free fatty acids accumulate in experimental steatohepatitis: role of adaptive pathways.

J Hepatol 2008 Apr 28;48(4):638-47. Epub 2008 Jan 28.

Department of Gastroenterology and Hepatology, ANU Medical School, Australian National University at The Canberra Hospital, ACT, Australia.

Background/aims: We determined the effects of dietary lipid composition on steatohepatitis development with particular attention to the nature of lipid molecules that accumulate in the liver and pathways of hepatic triglyceride synthesis.

Methods: Mice were fed methionine and choline deficient (MCD) diets supplemented with 20% fat as lard (saturated) or olive oil (monounsaturated), for 3 weeks.

Results: Irrespective of dietary lipid composition, MCD-fed mice developed steatosis, ballooning degeneration and lobular inflammation. MCD-feeding increased hepatic free fatty acid (FFA) levels 2-3-fold, as well as total triglyceride levels. Hepatic FFA composition was characterized by increased ratio of monounsaturated: saturated FFA. There were reduced nuclear levels of the lipogenic transcription factor sterol regulatory element binding protein-1 in MCD-fed mice, but no consistent reduction in fatty acid synthesis genes (acetyl-CoA carboxylase and fatty acid synthase). Consistent with pathways of hepatic triglyceride synthesis, expression of diacylglycerol acyltransferase-1 and -2 was increased, as were delta-5- and delta-6- fatty acid desaturase mRNA levels.

Conclusions: In this nutritional model of steatohepatitis, accumulation of FFA occurs despite substantial suppression of lipogenesis and induction of triglyceride synthesis genes. Accumulation of FFA supports a lipotoxicity mechanism for liver injury in this form of fatty liver disease.
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http://dx.doi.org/10.1016/j.jhep.2007.12.011DOI Listing
April 2008

Insulin resistance and incident gallbladder disease in pregnancy.

Clin Gastroenterol Hepatol 2008 Jan 11;6(1):76-81. Epub 2007 Dec 11.

Department of Medicine, University of Washington, Seattle, Washington 98195, USA.

Background & Aims: Insulin resistance is associated with prevalent gallstones, but its effect on initial gallstone formation is not well-understood.

Methods: We conducted a nested case-control study to examine whether insulin resistance is a risk factor for initial gallbladder sludge and stone formation during pregnancy. Cases were 205 women with new gallbladder sludge and stones during pregnancy and the early postpartum. Controls were 443 randomly selected women without sludge or stones during pregnancy. Gallbladder ultrasounds were obtained during each trimester and at 4-6 weeks post partum. Fasting serum glucose, lipids, and insulin were measured at 26-28 weeks gestation. Insulin resistance was measured by the homeostasis model. Logistic regression was used to identify independent risk factors for gallstone formation.

Results: Insulin resistance was significantly greater in cases than in controls on univariate analysis (P < .001). Pre-pregnancy body mass index was strongly associated with gallstone formation on univariate analysis (P < .001), but this association was diminished after adjusting for insulin resistance (P = .01). On multivariate analysis, insulin resistance was significantly associated with gallstone formation (P = .004), even after adjustment for pre-pregnancy body mass index and other confounding factors including high-density lipoprotein cholesterol and physical activity. This association was strongest in women with pre-pregnancy body mass index <30 kg/m(2).

Conclusions: Insulin resistance is a risk factor for incident gallbladder sludge and stones during pregnancy, even after adjustment for body mass index. Insulin resistance might represent a causal link between obesity and overweight and gallstones.
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http://dx.doi.org/10.1016/j.cgh.2007.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693050PMC
January 2008

Murine gallbladder epithelial cells can differentiate into hepatocyte-like cells in vitro.

Am J Physiol Gastrointest Liver Physiol 2007 Nov 23;293(5):G944-55. Epub 2007 Aug 23.

Division of Gastroenterology, University of Washington, Seattle, WA 98195, USA.

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific beta-galactosidase (beta-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no beta-Gal activity. beta-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4alpha, aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, alpha-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease.
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http://dx.doi.org/10.1152/ajpgi.00263.2006DOI Listing
November 2007

Gastric fluid bile concentrations and risk of Barrett's esophagus.

Interact Cardiovasc Thorac Surg 2007 Jun 8;6(3):304-7. Epub 2007 Feb 8.

Department of Surgery, University of Washington, Seattle, WA, USA.

Patients with Barrett's esophagus are at high risk of progression to adenocarcinoma. A growing, but conflicting body of evidence implicates bile reflux as a contributor to Barrett's esophagus. To investigate whether duodenogastric reflux was associated with an increased risk of Barrett's esophagus, a case-control study of incident Barrett's esophagus was performed. Cases (n=72) were identified by new histologically-confirmed diagnosis of specialized intestinal metaplasia (indicative of Barrett's esophagus) following upper endoscopy for refractory gastroesophageal reflux between October 1997 and September 2000. Cases were compared to gastroesophageal reflux patients without specialized intestinal metaplasia (controls; n=72). There was no difference in total bile acid concentrations between cases and controls. Risk of Barrett's esophagus did not significantly vary with increasing concentrations of total or free bile acids, respectively (OR 0.35 (95% CI 0.12, 1.02) and 0.60 (95% CI 0.22, 1.66)). Low gastric fluid pH (toxic range 3-5), was associated with a non-significant increase in the risk of Barrett's esophagus. In conclusion, no significant association between Barrett's esophagus and total or free bile acids in gastric refluxate was found. Patients with low gastric fluid pH (3-5) may represent a subset of patients at high risk of developing Barrett's esophagus.
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http://dx.doi.org/10.1510/icvts.2006.143370DOI Listing
June 2007

Paclitaxel interrupts TGF-beta1 signaling between gallbladder epithelial cells and myofibroblasts.

J Surg Res 2007 Aug 14;141(2):183-91. Epub 2007 Jun 14.

Division of Gastroenterology, Department of Medicine, University of Washington, Seattle, Washington. 98195, USA.

Background: The cellular and molecular mechanisms of fibrogenesis in the extrahepatic biliary epithelium are not known. Transforming growth factor-beta1 (TGF-beta1) is a cytokine implicated in signaling pathways that mediate collagen formation. An observation that paclitaxel (PT), applied topically into the rat common bile duct, inhibited stricture formation led us to hypothesize that PT's effects might be due to interruption of TGF-beta1 signaling between biliary epithelial cells and subepithelial myofibroblasts.

Materials And Methods: We tested this hypothesis using an in vitro cell-culture model in which murine gallbladder epithelial cells (GBEC) are cultured separately or cocultured with human gallbladder myofibroblasts (GBMF).

Results: Exposure to Escherichia coli lipopolysaccharide (LPS) enhanced TGF-beta1 mRNA expression and stimulated TGF-beta1 protein secretion into both apical and basolateral compartments in GBEC. This effect was more prominent with basolateral secretion and was also more pronounced in the coculture system. In GBMF, collagen I mRNA expression and protein secretion were stimulated by treatment with LPS or TGF-beta1. GBMF also expressed TGF-beta1 mRNA, whose levels were enhanced by exposure to either LPS or exogenous TGF-beta1. PT inhibited LPS-induced TGF-beta1 mRNA expression and protein secretion in GBEC in both culture systems. Tumor necrosis factor-alpha mRNA expression and protein secretion were not affected by PT in GBEC, demonstrating that the effects were specific for TGF-beta1. PT also inhibited LPS- and TGF-beta1-induced collagen I mRNA expression and protein secretion in GBMF.

Conclusions: These findings support a model in which GBEC communicate with subepithelial GBMF via TGF-beta1, leading to collagen deposition and fibrosis, and in which GBMF possess autocrine mechanisms involving TGF-beta1 that could regulate collagen production. PT inhibits these fibrogenic pathways.
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http://dx.doi.org/10.1016/j.jss.2006.12.558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571727PMC
August 2007

Incidence and predictors of hepatocellular carcinoma in patients with cirrhosis.

Clin Gastroenterol Hepatol 2007 Aug 16;5(8):938-45, 945.e1-4. Epub 2007 May 16.

Division of Gastroenterology, Department of Medicine, Veterans Affairs Puget Sound Health Care System, University of Washington, Seattle, Washington 98108, USA.

Background & Aims: Independent predictors of hepatocellular carcinoma in patients with cirrhosis are not well established.

Methods: We created a cohort of 2126 patients (41% with hepatitis C virus [HCV] infection) who sought care from all Veterans Affairs health care centers in the northwest United States from 1994 to 2005 and who had a diagnosis of cirrhosis recorded in inpatient or outpatient medical records.

Results: During a mean follow-up period of 3.6 years, 100 patients were diagnosed with hepatocellular carcinoma (incidence, 1.3 per 100 patient-years). Important predictors of hepatocellular carcinoma in multivariate models included HCV infection (adjusted hazard ratio [ahr], 3.0; 95% confidence interval [CI], 1.7-5.3); hepatitis B virus (HBV) surface antigen (ahr, 3.3; 95% CI, 1.4-7.7); HBV core antibody (ahr, 1.7; 95% CI, 1.1-2.8); obesity (ahr, 2.5; 95% CI, 1.3-4.9), and overweight (ahr, 2.8; 95% CI, 1.5-5.4) relative to patients with a body mass index of < 25 kg/m2, diabetes (ahr, 1.5; 95% CI, 0.9-2.5), and low platelet count (relative to patients with a platelet count of > 266 thousands/microL, the ahr was 2.1 [95% CI, 0.8-5.6] in patients with a platelet count of 180-266 thousands/microL, 3.3 [95% CI, 1.3-8.0] in patients with a platelet count of 111-179 thousands/microL, and the ahr was 4.7 [95% CI, 2.0-11.4] in patients with a platelet count of < or = 110 thousands/microL).

Conclusions: We identified 6 important predictors of hepatocellular carcinoma in multivariate models (including relatively novel predictors such as increased body mass index, HBV core antibody, and low platelet count), which suggest a means of predicting the risk of hepatocellular carcinoma in patients with cirrhosis and optimizing surveillance strategies.
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http://dx.doi.org/10.1016/j.cgh.2007.02.039DOI Listing
August 2007

Gene expression patterns in pancreatic tumors, cells and tissues.

PLoS One 2007 Mar 28;2(3):e323. Epub 2007 Mar 28.

Department of Medicine, Stanford University Medical Center, Stanford, California, United States of America.

Background: Cancers of the pancreas originate from both the endocrine and exocrine elements of the organ, and represent a major cause of cancer-related death. This study provides a comprehensive assessment of gene expression for pancreatic tumors, the normal pancreas, and nonneoplastic pancreatic disease.

Methods/results: DNA microarrays were used to assess the gene expression for surgically derived pancreatic adenocarcinomas, islet cell tumors, and mesenchymal tumors. The addition of normal pancreata, isolated islets, isolated pancreatic ducts, and pancreatic adenocarcinoma cell lines enhanced subsequent analysis by increasing the diversity in gene expression profiles obtained. Exocrine, endocrine, and mesenchymal tumors displayed unique gene expression profiles. Similarities in gene expression support the pancreatic duct as the origin of adenocarcinomas. In addition, genes highly expressed in other cancers and associated with specific signal transduction pathways were also found in pancreatic tumors.

Conclusion: The scope of the present work was enhanced by the inclusion of publicly available datasets that encompass a wide spectrum of human tissues and enabled the identification of candidate genes that may serve diagnostic and therapeutic goals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000323PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1824711PMC
March 2007

Absence of CFTR is associated with pleiotropic effects on mucins in mouse gallbladder epithelial cells.

Am J Physiol Gastrointest Liver Physiol 2006 Dec 6;291(6):G1148-54. Epub 2006 Jul 6.

Division of Gastroenterology, Department of Medicine, University of Washington, Seattle, WA 98195, USA.

Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr((-/-)) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr((-/-)) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr((-/-)) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr((-/-)) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr((-/-)) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr((-/-)) cells. Cftr((-/-)) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr((-/-)) cells compared with wild-type cells. Secreted mucins from Cftr((-/-)) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.
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http://dx.doi.org/10.1152/ajpgi.00547.2005DOI Listing
December 2006

Hypertonic saline for cystic fibrosis.

N Engl J Med 2006 Apr;354(17):1848-51; author reply 1848-51

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April 2006

Elevated serum alanine aminotransferase activity and calculated risk of coronary heart disease in the United States.

Hepatology 2006 May;43(5):1145-51

Research Enhancement Award Program, Department of Medicine, Veterans Affairs Puget Sound Health Care System, Seattle, WA 98108, USA.

In the United States, elevated serum alanine aminotransferase (ALT) activity in the absence of viral hepatitis or excessive alcohol consumption is most commonly attributed to nonalcoholic fatty liver disease (NAFLD). NAFLD is related to predictors of coronary heart disease (CHD) such as insulin resistance and central obesity. We examined the association between elevated serum ALT activity and the 10-year risk of CHD as estimated using the Framingham risk score (FRS). We performed a cross-sectional analysis comparing participants in the Third National Health and Nutrition Examination Survey with normal and elevated ALT activity (>43 IU/L), examining the mean levels of FRS. Among participants without viral hepatitis or excessive alcohol consumption, those with elevated ALT activity (n=267) had a higher FRS than those with normal ALT activity (n=7259), both among men (mean difference in FRS 0.25, 95% CI 0.07-0.4; hazard ratio for CHD 1.28, 95% CI 1.07-1.5) and women (mean difference in FRS 0.76, 95% CI 0.4-1.1; hazard ratio for CHD 2.14, 95% CI 1.5-3.0). The ALT threshold for increased risk of CHD was higher in men (>43 IU/L) than in women (>30 IU/L). Elevated ALT activity was not associated with higher FRS among nonobese participants with viral hepatitis or excessive alcohol consumption. In condusion, individuals with elevated serum ALT activity in the absence of viral hepatitis or excessive alcohol consumption, most of whom have NAFLD, have an increased calculated risk of CHD. This association is more prominent in women.
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http://dx.doi.org/10.1002/hep.21171DOI Listing
May 2006

The production of oxysterols in bile by activated human leukocytes.

Biochem Biophys Res Commun 2006 May 9;343(2):467-9. Epub 2006 Mar 9.

Division of Gastroenterology, VA Medical Center and Department of Medicine, University of Washington, Seattle, WA 98108, USA.

Oxysterols are naturally occurring intermediates in the conversion of cholesterol to bile acids, the major route for elimination of cholesterol. Additionally, they are important signaling agents, particularly in control of cholesterol synthesis; however, some species also are cytotoxic and carcinogenic. Oxysterols in plasma, contained in oxidized low-density lipoprotein, are strongly correlated with atherosclerosis. Oxysterols are found in infected human bile and the oxysterol content in gallstones correlates with bacterial DNA in the stones. Here we demonstrate that human leukocytes, activated by the presence of bacterial lipopolysaccharide, are able to oxidize cholesterol to a variety of oxysterols, including species known to be carcinogenic.
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http://dx.doi.org/10.1016/j.bbrc.2006.02.165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628770PMC
May 2006

The prevalence and predictors of elevated serum aminotransferase activity in the United States in 1999-2002.

Am J Gastroenterol 2006 Jan;101(1):76-82

Research Enhancement Award Program, Department of Medicine, Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108, USA.

Objectives: The presence of elevated serum aminotransferase activity is a sign of possible underlying liver disease. We aimed to describe the prevalence and associations of elevated serum aminotransferase activity in a recent, nationally representative U.S. survey.

Methods: We described the prevalence and predictors of elevated alanine aminotransferase (ALT >43 IU/L) or elevated aspartate aminotransferase (AST >40 IU/L) activity among 6,823 participants of the National Health and Nutrition Examination Survey (NHANES) conducted between 1999 and 2002. We compared our findings to the results already published based on the NHANES conducted between 1988 and 1994.

Results: In NHANES 1999-2002, the prevalences of elevated ALT, AST, or either ALT or AST were 8.9%, 4.9%, and 9.8%, respectively, in the entire population and 7.3%, 3.6%, and 8.1%, respectively, after excluding participants who tested positive for hepatitis C virus (HCV) antibody or reported excessive alcohol consumption. Strong predictors of elevated ALT activity included increasing waist circumference and body mass index, alcohol consumption, male sex, Mexican American ethnicity, decreasing age, and presence of HCV antibody. In NHANES 1988-1994, which employed a different assay methodology, the prevalences of elevated aminotransferases were approximately half of the prevalences we describe in NHANES 1999-2002, but the predictors of elevated ALT activity were similar.

Conclusions: The current prevalence of elevated ALT activity in the United States (8.9%) is more than double that of previously available estimates. This prevalence is very high (7.3%) even among persons without viral hepatitis C or excessive alcohol consumption and is strongly associated with risk factors for nonalcoholic fatty liver disease.
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http://dx.doi.org/10.1111/j.1572-0241.2005.00341.xDOI Listing
January 2006

Biliary sludge is formed by modification of hepatic bile by the gallbladder mucosa.

Clin Gastroenterol Hepatol 2005 Jul;3(7):672-8

Department of Medicine, Veterans Affairs Medical Center, Seattle, Washington, USA.

Background & Aims: We studied 22 patients with symptomatic microlithiasis to determine whether a contributory role of the gallbladder in the early stage of cholesterol gallstone formation exists. We compared the merits of different methods (ultrasonography and microscopy) and sources (hepatic or gallbladder) of bile samples for diagnosing microlithiasis.

Methods: Paired hepatic and gallbladder bile samples were studied with polarizing microscopy. Nucleation time, bile salts, phospholipid, cholesterol, cholesterol saturation index (CSI), bilirubin, total protein, albumin and mucin concentration were measured. All patients had abdominal ultrasound examination.

Results: With polarizing microscopy as the standard, ultrasonography was positive in 13 patients (59%) and negative in 9 (41%). All gallbladder bile samples were positive for microlithiasis by microscopy. Only one hepatic bile sample was positive (P < .0001). There was a disproportional enrichment of total protein, albumin, and mucin (P < .05) in the gallbladder bile and a conversion of bilirubin diglucuronide to monoglucuronide (P < .01). Gallbladder samples had lower CSI but a faster nucleation time (P < .001), which correlates inversely with CSI, total protein, and mucin concentration.

Conclusion: Biochemical composition and physical chemical behavior of hepatic bile are modified during residence in the gallbladder, contributing to sludge formation. Gallbladder bile has a lower calculated CSI, higher deconjugation of bilirubin, protein and mucin concentration and crystals were present. Hepatic bile samples are inappropriate for microscopic detection of microlithiasis.
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http://dx.doi.org/10.1016/s1542-3565(05)00369-1DOI Listing
July 2005

Interactions between sphingomyelin and cholesterol in low density lipoproteins and model membranes.

J Colloid Interface Sci 2006 Jan 12;293(1):203-12. Epub 2005 Jul 12.

Chemical Engineering Department, Drexel University, Philadelphia, PA 19104, USA.

This work examines three related, but previously unexplored, aspects of membrane biophysics and colloid science in the context of atherosclerosis. First, we show that sphingomyelinase (SMase)-induced aggregation of low density lipoproteins (LDLs), coupled with LDL exposure to cholesterol esterase (CEase), results in nucleation of cholesterol crystals, long considered the hallmark of atherosclerosis. In particular, this study reveals that the order of enzyme addition does not effect the propensity of LDL to nucleate cholesterol crystals, raising the possibility that nucleation can proceed from either the intra- or extracellular space. Second, we demonstrate that ceramide-rich aggregates of LDL release cholesterol to neighboring vesicles far more rapidly, and to a greater extent, than does native LDL. A likely explanation for this observation is displacement of cholesterol from SM-Chol rafts by "raft-loving" ceramide. Third, we demonstrate that a time-independent Förster resonance energy transfer (FRET) assay, based on dehydroergosterol and dansylated lecithin and used previously to study cholesterol nanodomains, can be used to measure raft sizes (on the order of 10 nm) in model membrane systems. Taken together, these observations point to the possibility of an extracellular nucleation mechanism and underscore the important role that biological colloids play in human disease.
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http://dx.doi.org/10.1016/j.jcis.2005.06.043DOI Listing
January 2006

Dietary carbohydrates and gallstones: is there a link?

Gastroenterology 2005 Jul;129(1):373-5

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http://dx.doi.org/10.1053/j.gastro.2005.05.042DOI Listing
July 2005

Combined interaction of phospholipase C and apolipoprotein A-I with small unilamellar lecithin-cholesterol vesicles: influence of apolipoprotein A-I concentration and vesicle composition.

Biochemistry 2005 May;44(19):7294-304

Department of Chemical and Biological Engineering, Drexel University, Philadelphia, Pennsylvania 19104, USA.

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small ( approximately 5-15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400-800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0-60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 microg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Forster resonance energy transfer; structures are confirmed by cryo TEM.
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http://dx.doi.org/10.1021/bi047317mDOI Listing
May 2005
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