Exaggerated insulin response to an oral glucose challenge in non-diabetic patients with biopsy evidence of NASH

Balb/c mice for xerograff formation (N =8). Results: In vitro, both PANC-I~xand PANC-lp exhibited similar growth rates. Compared to PANC-lfl (31%), pro-oxidant (peroxynitdte)induced apoptosis was significantly (p
580 Unique Hepatic Gone Expression in NASH - Candidates for a Genetic Basis of Disease RaghavakaimalSreekumar, Russell H. Wiesner, David M. Nagorney, Charles B. Rosen, Michael R. Chariton, Mayo Clin and Fdn, Rochester, MN Introduction: Despite the high prevalence of non-alcoholic steatohepatitis (NASH), little is known aboutthe pathogenesisof this condition in humans.Aim: To better definethe physiological basis of NASH and to identify candidate genes involved in its pathogenesis. Methods: High density synthetic oligonucleotide microarrays (Hu6800 GeneChip,Aflymetdx, CA) were used to define the intrahepatic transcriptional profile of patients with cirrhotic stage NASH (n=4) and compared to: (i) healthy controls with normal liver histology (n=4) and (ii) patients with cirrhosis due to etiologies other than NASH (n=4). Study participants were matched for age, gender and body mass index. Results: When compared to healthy controls, 111 genes were differentially expressedin patients with NASH. Genesthat were differentially expressed in patients with NASH when compared to both control groups are shown below. Thesegenes are consideredto be candidatesfor a genetic basis of NASH.Conclusions: NASH is associatedwith the differential expression of a small but diverse set of genes. While the basis of insulin resistance/sleatosiscannot be elucidated from these experiments, possible causesof inflammationand subsequentfibrosis havebeenidentified.The decreasedexpression of superoxide dismutase is of particular interest as this should be associatedwith increased production of reactive oxygen species and subsequent mutations in mitochondrial DNA. Mitochonddal dysfunction is a hallmark of NASH.

578 Serum Thioredoxin Levels As An Indicator Of Oxidative Stress In Patients With Nonalcoholic Steatohepatitis Toshiaki Nakashima,Yoshio Sumida, Takaharu Yoh, Yuko Kakisaka, Hironod Mitsuyoshi, Takeshi Okanoue, Kyoto Prefectursi Univ of Medicine, Kyoto Japan; Hajima Nakamura, Junji Yodoi, Institute for Virus Research,Kyoto Univ, Kyoto Japan Background/Aim:Oxidativestress could play a crucial role in the pathogenesisof nonalcoholic steatohepatitis (NASH). Thioredoxin (TRX), a thiol-containing protein, is induced by various oxidative stresses, and is therefore a good marker of oxidative stress. We examined the clinical significance of serum TRX levels in patients with HASH. Methods: Serum TRX levels were determinedwith a sandwich enzyme-linkedimmunosorbent assay kit in 18 patients with NASH (11 males and 7 females, median age: 50), in 10 patients with simple fatty liver (FL) (6 and 4, 49) and in17 healthy volunteers (9 and 8, 45). The histological findings of NASH were interpreted and scored according to the classification proposed by Brunt EM et al. (Am J Gastroenterol, 1999). Results: 1) Serum ferdtin concentrations (medians and [ranges], rig/ ml) were significantly higher in NASH patients (218 [24-675]) than in FL patients (134 [17224], p=.049). There were no significant differences in age, gender, body mass index, platelet count, transaminase activities, gamma-glutamyl transpeptidase,blood glucose, total cholesterol, triglycedde, non-esterifiedfatty acid and serum lipidperoxide between NASHand FL patients. 2) Hepatic iron deposits were observed in 10 (66%) of 15 NASH: 5 (33%) with grade 1, 2 (13%) with grade 2, and 3 (20%) with grade 3, whereas only two (22%) of 9 FL had hepatic iron deposits, both with grade 1.3) Serum TRX levels (medians and [ranges], ng/ml) were significantly elevatedin NASH patients (66 [18-77]), as comparedto FL patients (22 [17-30], p =.012) or healthy controls (24 [1-51], p=.0002). Serum TRX levels in NASH patients were significantly correlated with serum ferritin levels (r= 0.417, p = .037), but not with other vadablas. Serum TRX levels in NASH patients were positively correlated with the stage of hepatic fibrosis (r=0.519, p=.032), but not with the grade of inflammation. 4) Removal of hepatic excess iron by phlebotomy significantly decreasedthe serum levels of TRX and ALT (P<.O6) in 6 NASH patients. Conclusions: The pathogenesisof NASH may be associatedwith iron-related oxidativestress, and phlebotomy is indicatedas a suitabletherapy for NASH. Serum TRX levels are useful for discriminating NASH from FL.

Protosseinhibitor/Fatmetabolism 1"4,7 Alpha-l-antltnypsin ,J.3.7 FACLI ,[. 3.7 3-oxoacyI-coAthlolese Energymetabolism ,], 3.7 Cytochrornep450

1"denotesoverexpressedesd $ denotesunderexpressedin NASHvs. bothgroupsof controls. Values indicatefolddifferentin abundanceof RNAin hepaUctissuefrom patientswi~ NASHwhencompared to bothcontrolgroups.

581 A Mouse Model of Long-Chain 3-KetoncyI-CoA Thiolaon Deficiency of Mitochondrial Fatty Acid Beta-OxidaDonis Associated with Low Body Mass, Cold Intolerance and Hepatic Steatesis. Raphael B. Merdman, Univ of CA, San Francisco, San Francisco, CA; Wenning Oin, Washington Univ Sch of Medicine, St. Louis, MO; Arnold W. Strauss, Vanderbilt Univ, Nashville, TN Backround: Long-Chain 3-KetoacyI-CoAThiolase (LKAT) catalysesthe final enzymaticstep in the mitochondrial fatty acid beta-oxidation spiral. Inherited disorders of mitochondrial betaoxidation are associatedwith microvesicular steatosis. LKAT comprises the beta subunit of the tritunctional protein (TFP) complex. Expression of LKAT is necessary for stable TFP formation. TFP deficiency is associatedwith skeletal myopathy, cardiomyoapthyand sudden infant death. Aims: To generate and evaluate a mouse model deficient in LKM, providing insight into mechanisms of regulation of fatty acid oxidation and disease pathogenesis. Methods: We produced a targeted gene deletion of a 5274 base pair segment encompassing 1Kb of the intergenic region and exons 1, 2 and 3 of the beta subunit, and replacementwith a neomycin cassette. Results: LKATdeficient mice were generated.Matings of heterozygotedeleted mice produced the expected ratios of wild type (+/+), heterozygote ( + / - ) and homozygote( - / - ) offspring. Deficiencyof LKATin - / - mice and partial deficiency in +/ - mice was confirmed by Western Blot and Northern Blot analyses. LKAT deficient mice survive in reducednumbers (55%) for at least6 months (p48 hours). The mean core temperature in - / - mice after 1 hour cold room exposure was 72 degrees F compared to 98 degrees F in + / - and +/+ mice (p
579 Exaggerated Insulin Responseto an Oral Glucose Challenge in Non-Diabetic Patients with Biopsy Evidence of NASH Brent A. Neuschwander-Tetri,Craig Sponseller, Bruce R. Bacon, Kent Wehmeier, Elizabeth M. Brunt, Saint Louis Univ Sch of Med, St. Louis, MO BACKGROUND:The pathogenesisof NASH is uncertain, but obesity and type 2 diabetes are major risk factors. Hyperinsulinemiais common in patients with obesity and it may predate a diagnosis of type 2 diabetes.The aim of this study was to characterizethe insulin response to an oral glucose challenge (OGT) in patients with NASH and correlate the insulin levels to clinical parametersof NASH.METHODS:Ten non-diabeticpatientswith NASHreceivedglucose, 75 g orally, after a 10-12 hr fast. Blood glucose and serum insulin were measured at O, 60, and 120 min. Clinical parameterswere obtained within 4 weeks of the OGTthat included liver biopsy, non-contrast CT, and glycated hemoglobin levels. Liver biopsies were graded and staged as described (Brunt, Am J Gastro 94:2467, 1999). RESULTS:All patients had normal Hgb&C levels (range 4.6-6.1%) confirming the absence of overt diabetes. Diabeteswas not identified by OGTalthough 4/10 patients had impaired glucosetolerance. Fasting insulin levels were elevated (> 20 FU/mL) in all patients. Marked hyperinsulinemiawas induced by the oral glucose load and all patients had persistently elevatedserum insulin levels at 2 hr (range: 80-467; normal: 34-58/LU/mL). All had low-density livers by CT imaging (mean liver/spleen density ratio: 0.58, range: 0.25-0.01; normal: 1.00) and the density ratio correlated inversely with the mean post-prandial serum insulin levels (r= -0.74). Liver biopsy grading and staging did not correlate with the magnitude of fasting or post-prandial hypednsulinemia. CONCLUSION: In this group of non-diabetic patients with wall-characterized NASH, postprandial hypednsulinemiawas found in all patients. The degreeof hyperinsulinemiacorrelated with the degreeof hepaticsteatosis.Theseresults provide additional evidencethat hyperinsulinemia is common in patients with NASHalthough a causal role is yet to be established.

582 Hepatocyte Hyperplasia In Obesity-Relatad Fatty Livers Shiqi Yang, Huizhi Lin, Anna Mae Diehl, Johns Hopkins Univ, Baltimore, MD

Glucoseand insulin responseto oral glucosechallenge

Median % above normal

Gluc-O

Gluc-60 Gluc-120 Ins-O

Ins-60

Ins-t20

86 0

167 10

132 90

132 100

123 40

DNAmaintenance/metabolism J~2.5 $uperoxidedismutase1 $ 2.4Aldehydeoxidsse $ 3.2 Catalase Complementactivation 1"3.0 ComponentC3

40 100

BACGROUND:Cancers of the pancreas,colon and liver are increased in obesity. Cancer risk correlates with the severity of insulin resistance, prompting speculation that this hormonal state promotestissue hyperplasia,an early event in tumorigenesis.However,increasedhepatocellular carcinoma (HCC) may simply reflect the increasedprevalenceof cirrhosis in obesity. PURPOSE: To determine if liver hyperplasia occurs in genetically obese, ob/ob mice, a non-cirrhotic model of obesify-related insulin resistance. HYPOTHESIS:Obesity-relatedliver hyperplasia results when hepatocyteproliferation increases relative to apotosis in fatty, noncirrhotic livers. METHODS:Liver mass, hepstocyts proliferation & apotosis, and mechanisms that regulate apotosis [NF-K,8 activity, manganesesuperoxide dismutase (MnSOD) activity, bcl-2-related gene expression,caspese3 activity] and proliferation [mitogen activatedprotein kinase (MAPK) activity, cell cycle gene expression]were compared in 8 wk. old, male oh/oh

glucose:mg/dL;insulin: pU/mLL

A-107