Vikas Chandhoke

Adipokines and cytokines in non‐alcoholic fatty liver disease

Background  Several adipocytokines have been implicated in the pathogenesis non‐alcoholic fatty liver disease (NAFLD).

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 Novel Diagnostic Biomarker Panel for Obesity-related Nonalcoholic Steatohepatitis (NASH)

BackgroundWithin the spectrum of nonalcoholic fatty liver disease (NAFLD), only patients with nonalcoholic steatohepatitis (NASH) show convincing evidence for progression. To date, liver biopsy remains the gold standard for the diagnosis of NASH; however, liver biopsy is expensive and associated with a small risk, emphasizing the urgent need for noninvasive diagnostic biomarkers. Recent findings suggest a role for apoptosis and adipocytokines in the pathogenesis of NASH. The aim of this study was to develop a noninvasive diagnostic biomarker for NASH.MethodsThe study included 101 patients with liver biopsies who were tested with enzyme-linked immunosorbent assay (ELISA)-based assays. Of these, 69 were included in the biomarker development set and 32 were included in the biomarker validation set. Clinical data and serum samples were collected at the time of biopsy. Fasting serum samples were assayed for adiponectin, resistin, insulin, glucose, TNF-alpha, IL-6, IL-8, cytokeratin CK-18 (M65 antigen), and caspase-cleaved CK-18 (M30 antigen).ResultsData analysis revealed that the levels of M30 antigen (cleaved CK-18) predicted histological NASH with 70% sensitivity and 83.7% specificity and area under the curve (AUC) = 0.711, p < 10−4, whereas the predictive value of the levels of intact CK-18 (M65) was higher (63.6% sensitivity and 89.4% specificity and AUC = 0.814, p < 10−4). Histological NASH could be predicted by a combination of Cleaved CK-18, a product of the subtraction of Cleaved CK-18 level from intact CK-18 level, serum adiponectin, and serum resistin with a sensitivity of 95.45% sensitivity, specificity of 70.21%, and AUC of 0.908 (p < 10−4). Blinded validation of this model confirmed its reliability for separating NASH from simple steatosis.ConclusionsFour ELISA-based tests were combined to form a simple diagnostic biomarker for NASH.

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.)

Laser Capture Microdissection and Protein Microarray Analysis of Human Non-small Cell Lung Cancer Differential Epidermal Growth Factor Receptor (EGPR) Phosphorylation Events Associated with Mutated EGFR Compared with Wild Type

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor β suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.

Differential expression of miRNAs in the visceral adipose tissue of patients with non‐alcoholic fatty liver disease

Aliment Pharmacol Ther 2010; 32: 487–497

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.

Secreted Neutral Metalloproteases of Bacillus anthracis as Candidate Pathogenic Factors

To evaluate the pathogenic potential of Bacillus anthracis-secreted proteases distinct from lethal toxin, two neutral zinc metalloproteases were purified to apparent homogeneity from the culture supernatant of a non-virulent delta Ames strain (pXO1–, pXO2–). The first (designated Npr599) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species. The second (designated InhA) is a homolog of the Bacillus thuringiensis immune inhibitor A. These proteases belong to the M4 and M6 families, respectively. Both enzymes digested various substrates, including extracellular matrix proteins, endogenous inhibitors, and coagulation proteins, with some differences in specificity. In addition, InhA accelerated urokinase-mediated plasminogen activation, suggesting that InhA acts as a modulator of plasmin in the host inflammatory system. Relevant to epithelial barrier function, Npr599 and InhA significantly enhanced syndecan-1 shedding from cultured normal murine mammary gland cells without affecting their viability through stimulation of the host cell ectodomain shedding mechanism. In addition, Npr599 and InhA directly cleaved recombinant syndecan-1 fused to glutathione S-transferase. Mass spectrometric analysis suggested that the cleavage sites of Npr599 and InhA are the Asp39–Asp40 and Gly48–Thr49 bonds, respectively. We propose that Npr599 and InhA from B. anthracis are multifunctional pathogenic factors that may contribute to anthrax pathology through direct degradation of host tissues, increases in barrier permeability, and/or modulation of host defenses.

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

BackgroundGiven the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.ResultsCross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes.ConclusionsWe recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.