Hazem Elariny

Predictors of Nonalcoholic Steatohepatitis and Advanced Fibrosis in Morbidly Obese Patients

Background: Nonalcoholic fatty liver disease (NAFLD) is a common form of chronic liver disease in the United States. It is commonly associated with the components of the metabolic syndrome including obesity. From the spectrum of NAFLD, only patients with nonalcoholic steatohepatitis (NASH) have been convincingly shown to have a potential for progression to cirrhosis. We report the prevalence of NAFLD and NASH as well as predictors of NASH and advanced fibrosis in morbidly obese patients. Methods: 212 consecutive patients who underwent bariatric surgery were enrolled in the study. A liver biopsy was performed at the time of the surgery. Causes of chronic liver disease other than NAFLD were excluded by clinical and laboratory evaluation. Results: The prevalence of NAFLD was 93%. Of those with NAFLD, 26% had NASH. 17 patients (9%) had advanced fibrosis (i.e., bridging fibrosis or cirrhosis). Male gender, AST, and type 2 diabetes mellitus were independently associated with NASH. Waistto-hip ratio, AST, and focal hepatocyte necrosis on liver biopsy were independently associated with advanced fibrosis. Interestingly, while AST was associated with NASH and advanced fibrosis, the majority of the patients with either NASH or advanced fibrosis had normal AST. Conclusions: NAFLD and NASH are very common in morbidly obese patients undergoing bariatric surgery. Features associated with the metabolic syndrome and liver cell injury are independently associated with either NASH or advanced fibrosis.

A genomic and proteomic study of the spectrum of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and some of its forms are progressive. This study describes the profiling of hepatic gene expression and serum protein content in patients with different subtypes of NAFLD. Liver biopsy specimens from 98 bariatric surgery patients were classified as normal, steatosis alone, steatosis with nonspecific inflammation, and nonalcoholic steatohepatitis (NASH). Microarray hybridizations were performed in triplicate and the microarray expression levels of a selected group of genes were confirmed using real‐time quantitative reverse‐transcriptase polymerase chain reaction. Serum protein profiles of the same patients were determined by SELDI‐TOF mass spectrometry. Of 98 obese patients, 91 were diagnosed with NAFLD (12 steatosis alone, 52 steatosis with nonspecific inflammation, and 27 NASH), and 7 patients without NAFLD served as obese controls. Each group of NAFLD patients was compared with the obese controls, and 22 genes with more than twofold differences in expression levels were revealed. Proteomics analyses were performed for the same group comparisons and revealed twelve significantly different protein peaks. In conclusion, this genomic/proteomic analysis suggests differential expression of several genes and protein peaks in patients within and across the forms of NAFLD. These findings may help clarify the pathogenesis of NAFLD and identify potential targets for therapeutic intervention. (HEPATOLOGY 2005;42:665–674.)

Gene Expression of Leptin, Resistin, and Adiponectin in the White Adipose Tissue of Obese Patients with Non-Alcoholic Fatty Liver Disease and Insulin Resistance

Background: Adipose tissue is an active endocrine organ that secretes a variety of metabolically important substances including adipokines. These factors affect insulin sensitivity and may represent a link between obesity, insulin resistance, type 2 diabetes (DM), and nonalcoholic fatty liver disease (NAFLD). This study uses real-time polymerase chain reaction (PCR) quantification of mRNAs encoding adiponectin, leptin, and resistin on snap-frozen samples of intra-abdominal adipose tissue of morbidly obese patients undergoing bariatric surgery. Methods: Morbidly obese patients undergoing bariatric surgery were studied. Patients were classified into two groups: Group A (with insulin resistance) (N=11; glucose 149.84 ± 40.56 mg/dL; serum insulin 8.28 ± 3.52 μU/mL), and Group B (without insulin resistance) (N=10; glucose 102.2 ± 8.43 mg/dL; serum insulin 3.431 ± 1.162 μU/mL). Results: Adiponectin mRNA in intra-abdominal adipose tissue and serum adiponectin levels were significantly lower in Group A compared to Group B patients (P<0.016 and P<0.03, respectively). Although serum resistin was higher in Group A than in Group B patients (P<0.005), resistin gene expression was not different between the two groups. Finally, for leptin, neither serum level nor gene expression was different between the two groups. Serum adiponectin level was the only predictor of nonalcoholic steatohepatitis (NASH) in this study (P=0.024). Conclusions: Obese patients with insulin resistance have decreased serum adiponectin and increased serum resistin. Additionally, adiponectin gene expression is also decreased in the adipose tissue of these patients. This low level of adiponectin expression may predispose patients to the progressive form of NAFLD or NASH.

Hepatic gene expression in patients with obesity-related non-alcoholic steatohepatitis.

Abstract: Background: Non‐alcoholic fatty liver disease (NAFLD) is among the most common causes of chronic liver disease. NAFLD includes a spectrum of clinicopathologic syndromes that includes non‐alcoholic steatohepatitis (NASH) that has potential for progression. The pathogenesis of NASH is poorly characterized.

Obesity-related Differential Gene Expression in the Visceral Adipose Tissue

Background:This study investigates the expression patterns in human adipose tissue, and identifies genes that may be involved in the abnormal energy homeostasis. Methods: Subjects were prospectively recruited from morbidly obese patients undergoing bariatric surgery and from non-obese organ donors. Extensive clinical data and visceral fat specimens were obtained from each subject at the time of surgery. A group of 50 obese patients and 9 non-obese controls were selected for further study. Two custom two-color cDNA microarrays were produced with 40,173 human individual cDNA clones. Microarray experiments were performed for each sample, and a selected group of gene expression values were confirmed with real-time RT-PCR. Results: A comparison of gene expression profiles from obese and non-obese patients identified 1,208 genes with statistically significant differential expression between the 2 groups. Most prominent among these genes are multiple glycolysis enzyme encoding genes; others are involved in oxysterol biosynthesis and signaling, or are ATP-binding transporters and solute carriers. Conclusion: Differential gene expression in the adipose tissue of morbidly obese patients includes genes related to lipid and glucose metabolism, membrane transport, and genes promoting the cell cycle. These findings are a first step toward clarifying the molecular pathogenesis of obesity and identifying potential targets for therapeutic intervention.

A systems biology approach to the pathogenesis of obesity-related nonalcoholic fatty liver disease using reverse phase protein microarrays for multiplexed cell signaling analysis†

Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. Omental adipose tissue, a biologically active organ secreting adipokines and cytokines, may play a role in the development of NAFLD. We tested this hypothesis with reverse‐phase protein microarrays (RPA) for multiplexed cell signaling analysis of adipose tissue from patients with NAFLD. Omental adipose tissue was obtained from 99 obese patients. Liver biopsies obtained at the time of surgery were all read by the same hepatopathologist. Adipose tissue was exposed to rapid pressure cycles to extract protein lysates. RPA was used to investigate intracellular signaling. Analysis of 54 different kinase substrates and cell signaling endpoints showed that an insulin signaling pathway is deranged in different locations in NAFLD patients. Furthermore, components of insulin receptor–mediated signaling differentiate most of the conditions on the NAFLD spectrum. For example, PKA (protein kinase A) and AKT/mTOR (protein kinase B/mammalian target of rapamycin) pathway derangement accurately discriminates patients with NASH from those with the non‐progressive forms of NAFLD. PKC (protein kinase C) delta, AKT, and SHC phosphorylation changes occur in patients with simple steatosis. Amounts of the FKHR (forkhead factor Foxo1)phosphorylated at S256 residue were significantly correlated with AST/ALT ratio in all morbidly obese patients. Furthermore, amounts of cleaved caspase 9 and pp90RSK S380 were positively correlated in patients with NASH. Specific insulin pathway signaling events are altered in the adipose tissue of patients with NASH compared with patients with nonprogressive forms of NAFLD. Conclusion: These findings provide evidence for the role of omental fat in the pathogenesis, and potentially, the progression of NAFLD. (HEPATOLOGY 2007;46:166–172.)

Expression of Cytokine Signaling Genes in Morbidly Obese Patients with Non-Alcoholic Steatohepatitis and Hepatic Fibrosis

BackgroundWhite adipose tissue (WAT) from visceral adiposity plays an important role in the pathogenesis of non-alcoholic steatohepatitis (NASH). Development of NASH and its progression to fibrosis is partially due to cytokines and adipokines produced by WAT. The aim of this study was to assess the association of hepatic fibrosis and NASH by evaluating the intrinsic differences in the inflammatory cytokine signaling in the visceral adipose tissue obtained from morbidly obese patients.MethodsWe used targeted microarrays representing human genes involved in the inflammatory and fibrogenic reactions to profile visceral adipose samples of 15 well-matched NASH patients with and without fibrosis. Additionally, visceral adipose samples were subjected to real-time polymerase chain reaction profiling of 84 inflammations related genes.ResultsEight genes (CCL2, CCL4, CCL18, CCR1, IL10RB, IL15RA, and LTB) were differentially expressed in NASH with fibrosis. Additionally, an overlapping but distinct list of the differentially expressed genes were found in NASH with type II diabetes (DM; IL8, BLR1, IL2RA, CD40LG, IL1RN, IL15RA, and CCL4) as compared to NASH without DM.ConclusionsInflammatory cytokines are differentially expressed in the adipose tissue of NASH with fibrosis, as well in NASH with DM. These findings point at the interaction of adipose inflammatory cytokines, DM, hepatic fibrosis in NASH, and its progression to cirrhosis and end-stage liver disease.

Expression of genes for microRNA‐processing enzymes is altered in advanced non‐alcoholic fatty liver disease

Recently, microRNAs (miRNA) have been linked to the pathogenesis of non‐alcoholic fatty liver disease (NAFLD) and its progression to non‐alcoholic steatohepatitis (NASH). First transcribed as pri‐miRNA, these molecules are further processed by a complex of endonuclear and cytosolic RNA binding molecules to form mature miRNAs. The aim of this study is to investigate mechanisms of miRNA regulation in the visceral adipose of obese NAFLD patients via measuring expression of miRNA processing enzymes and pri‐miRNA.

Expression of energy metabolism related genes in the gastric tissue of obese individuals with non-alcoholic fatty liver disease

BackgroundStomach is an integral part of the energy balance regulating circuit. Studies exploring the effects of cross-system changes in the energy homeostasis in stomach tissue are scarce. The proximity of the stomach to liver - the most common secondary target affected by obesity – suggests that these two organs are exposed to each other’s local secretion. Therefore, we aimed at expression profiling of energy metabolism associated genes in the gastric tissue of obese non-alcoholic fatty liver disease (NAFLD) patients.MethodsA total of 24 patients with histologically-proven NAFLD were included. In the gastric tissue, gene expression profiling of 84 energy metabolism associated genes was carried out.ResultsThe accumulation of the fat in the liver parenchyma is accompanied by downregulation of genes encoding for carboxypeptidase E (CPE) and Interleukin 1B (IL1B) in the gastric mucosa of same patient. In patients with high grade hepatic steatosis, Interleukin 1 beta encoding gene with anorexigenic function, IL1B was downregulated. The levels expression of 21 genes, including ADRA2B, CNR1 and LEP were significantly altered in the gastric tissue of NAFLD patients with hepatic inflammation. There were also indications of an increase in the opioid signaling within gastric mucosa that may results in a shift to proinflammatory environment within this organ and contribute to systemic inflammation and the pathogenic processes in hepatic parenchyma.ConclusionsWe have shown differential expression of energy metabolism associated genes in the gastric tissue of obese NAFLD patients. Importantly, these gene expression profiles are associated with changes in the hepatic parenchyma as reflected in increased scores for hepatic steatosis, inflammation, fibrosis and NASH. This study suggests the complex interplay of multiple organs in the pathogenesis of obesity-related complications such as NAFLD and provides further evidence supporting an important role for gastric tissue in promoting obesity-related complications.

Expression of Inflammation-Related Genes Is Altered in Gastric Tissue of Patients with Advanced Stages of NAFLD

Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD.