Whole Liver Tissue Research Articles

Key hepatic signatures of human and mouse nonalcoholic steatohepatitis: A transcriptome-proteome data meta-analysis.

BackgroundDespite the global prevalence of nonalcoholic fatty liver disease (NAFLD), its pathophysiology remains unclear. In this study, we established highly confident nonalcoholic steatohepatitis (NASH) gene signatures and evaluated the pathological mechanisms underlying NASH through a systematic meta-analysis of transcriptome and proteome datasets obtained from NASH patients and mouse models.MethodsWe analyzed NASH transcriptome datasets from 539 patients and 99 mice. A whole-liver tissue proteome dataset was used to confirm the protein level dysregulation of NASH signatures significant in both humans and mice.ResultsIn total, 254 human and 1,917 mouse NASH gene signatures were established. Up-regulated genes of 254 human signatures were associated with inflammation, steatosis, apoptosis, and extracellular matrix organization, whereas down-regulated genes were associated with response to metal ions and lipid and amino acid metabolism. When different mouse models were compared against humans, models with high fat and high fructose diet most closely resembled the genetic features of human NAFLD. Cross-species analysis revealed 66 genes that were concordantly dysregulated between human and mouse NASH. Among these, 14 genes were further validated to be dysregulated at the protein level. The resulting 14 genes included some of the well-established NASH associated genes and a promising NASH drug target. Functional enrichment analysis revealed that dysregulation of amino acid metabolism was the most significant hepatic perturbation in both human and mouse NASH.ConclusionsWe established the most comprehensive hepatic gene signatures for NASH in humans and mice to date. To the best of our knowledge, this is the first study to collectively analyze the common signatures between human and mouse NASH on a transcriptome–proteome scale.

Next-generation sequencing of the intrahepatic antibody repertoire delineates a unique B-cell response in HBV-associated acute liver failure.

Hepatitis B virus (HBV) is a major cause of acute liver failure (ALF) worldwide. While liver damage in classic acute hepatitis B is believed to be T-cell mediated, the pathogenesis of HBV-associated ALF remains largely unknown. Access to liver specimens from well-characterized patients with HBV-associated ALF provided us with the opportunity to perform next-generation sequencing (NGS) of the entire VH repertoires of IgM and IgG from the livers of four ALF patients, a control liver donor and a patient with chronic HBV infection. We found that ALF is not associated with expansion of specific B-cell lineages. However, NGS showed that the intrahepatic VH repertoires from ALF patients were characterized by the abundant presence of antibodies in germline configuration in contrast to their marginal prevalence in controls. Moreover, NGS identified a large number of VH genes in germline configuration with identical VDJ sequences in the IgM and IgG repertoires in all four ALF patients, indicating that isotype switch from IgM to IgG had occurred without somatic hypermutation. The results of this study indicate that the presence of intrahepatic antibodies in unmutated germline configuration is a broad phenomenon in the global antibody repertoire generated from total RNA derived from whole-liver tissue that is strongly associated with ALF, suggesting a major role of T cell-independent humoral immunity in the pathogenesis of ALF.

Deficient copper concentrations in dried-defatted hepatic tissue from ob/ob mice: A potential model for study of defective copper regulation in metabolic liver disease

Ob/ob mice provide an animal model for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) in patients with obesity and type-2 diabetes. Low liver copper has been linked to hepatic lipid build-up (steatosis) in animals with systemic copper deficiency caused by low-copper diets. However, hepatic copper status in patients with NAFLD or NASH is uncertain, and a validated animal model useful for the study of hepatic copper regulation in common forms of metabolic liver disease is lacking. Here, we report parallel measurements of essential metal levels in whole-liver tissue and defatted-dried liver tissue from ob/ob and non-obese control mice. Measurements in whole-liver tissue from ob/ob mice at an age when they have developed NAFLD/NASH, provide compelling evidence for factitious lowering of copper and all other essential metals by steatosis, and so cannot be used to study hepatic metal regulation in this model. By marked contrast, metal measurements in defatted-dried liver samples reveal that most essential metals were actually normal and indicate specific lowering of copper in ob/ob mice, consistent with hepatic copper deficiency. Thus ob/ob mice can provide a model useful for the study of copper regulation in NAFLD and NASH, provided levels are measured in defatted-dried liver tissue.

IGF-I Transcript Levels in Whole-Liver Tissue, in Freshly Isolated Hepatocytes, and in Cultured Hepatocytes from Lean and Obese Zucker Rats

Background: The mechanisms underlying the maintenance of normal to high rates of linear growth and plasma insulin-like growth factor I (IGF-I) levels in spite of a low growth hormone secretion in obese children remain unknown. Among the animal models of early-onset obesity, obese Zucker (fa/fa) rats (which are homozygous for an inactivating missense mutation in the leptin receptor) are particularly appropriate, because their linear growth shows this growth hormone independence. Methods: To study the regulation of IGF-I synthesis in this model, we have established primary cultures of hepatocytes derived from 12-week-old Zucker male obese and lean rats. The rat IGF-I gene contains six exons, and alternative splicing generates different mRNAs, one of which (called IGF-1B) has been shown to be decreased by fasting. We report steady state mRNA levels for IGF-I (all transcripts) and for IGF-IB in hepatocytes after 3 days in culture, in freshly isolated hepatocytes, and in whole-liver tissue. RT-PCRs using primers specific for IGF-I or IGF-IB were performed with two different internal competitors for quantification. Results: In primary cultures of hepatocytes, the IGF-IB mRNA was increased by >50-fold (p = 0.01) in cells derived from obese animals as compared with cells from lean animals. However, these transcript levels were not significantly different when measured in freshly isolated hepatocytes or in whole-liver tissue. Conclusions: Increased IGF-IB transcription could be an intrinsic characteristic of cultured hepatocytes harbouring leptin receptors that bear the fa mutation. However, the modulation of this characteristic by cell-cell interactions and by in vivo hormone and metabolic status remains to be studied.

Endothelin antagonism in experimental hepatic fibrosis. Implications for endothelin in the pathogenesis of wound healing.

The liver's response to chronic injury is fibrosis, which is analogous to wound healing in other organs. Hepatic wounding is characterized by the "activation" of resident stellate cells (lipocytes, Ito cells) to myofibroblast-like cells that produce increased amounts of smooth muscle alpha-actin and extracellular matrix. Stellate cells possess abundant endothelin (ET) receptors (ETA/B) and, therefore, are subject to the effects of ET-1 and 3. In this study, we investigated whether ETs contribute to the activation of stellate cells and consequently, fibrogenesis. In cultured stellate cells, ET-1 and sarafotoxin S6C (a potent ETB receptor agonist) stimulated stellate cell activation, as assessed by expression of smooth muscle alpha-actin. Furthermore, the mixed ETA/B receptor antagonist, bosentan, blocked this process. Next, we administered bosentan during the induction of liver injury in two mechanistically distinct forms of hepatic wounding. Bosentan reduced levels of type I collagen and cellular fibronectin mRNAs in whole-liver tissue extracts in both models. In freshly isolated stellate cells from injured livers, bosentan reduced expression of activation markers, including smooth muscle alpha-actin and extracellular matrix mRNAs. We further demonstrate that endothelin antagonism after establishment of fibrosing injury reduced stellate cell activation and matrix production. These data indicate that ET contributes to stellate cell activation and fibrogenesis. Because ET is upregulated in diverse forms of parenchymal injury, we speculate that ET may play an important role in the wound-healing response.

Interactions of glucagon with isolated rat-liver cells fate and subcellular localization of cell-associated hormone

The interaction of glucagon with isolated rat-liver cells was studied, and some results were compared with those obtained in parallel experiments with insulin. Attempts were made, in particular, to examine the functional relationships between receptor-binding and degradation, to assess the subcellular localization of cell-associated glucagon, and to compare the glucagonbinding properties of intact liver cells and whole-liver tissue. Binding determinations at the steady state are compatible with the existence of about 1.3 × 10 −5 sites per cell with a dissociation constant of 1.5 nM. Unlike that of liver membranes, the dissociation of bound glucagon is little affected by guanyl nucleotides. Glucagon in the incubation medium undergoes rapid inactivation and physical changes that suggest cleavage into iodotyrosyl peptides and free iodotyrosines. Most of the degrading activity is unrelated to the receptors as indicated by its low affinity (apparent K m 4 μM), its inhibition by polypeptides chemically unrelated to glucagon, and its partial association (about 50% of the total) with degradative components released extracellularly. In the absence of inhibitors, cell-associated glucagon also undergoes a progressive degradation, with a concomitant release of degraded products in the medium; this process may be, at least in part, receptor-mediated. However, in contrast with insulin, it represents only a minor fraction of the total degrading activity. About 80% of the glucagon, and 90% of the insulin associated to liver cells is recovered in the particulate fraction. On differential centrifugation, this material is almost exclusively recovered in the microsomal fraction; on sucrose density-gradient centrifugation, it is present to the extent of 70% in unidentified membranes of density 1.11–1.17. This pattern of distribution is unaffected by the time of incubation, indicating that if labeled hormone undergoes translocation, the latter is not detectable under the conditions used here. The subcellular distribution of the glucagon and insulinbinding activities is nearly the same whether the binding reaction is performed on intact cells or on isolated fractions, and, at saturation, intact cells bind about the same amount of hormone as does the particulate fraction derived from the cells. Thus, cell disruption does not affect the apparent accessibility of the binding sites. Crude particles and plasma membranes prepared from liver cells show a decrease in their ability to bind insulin and glucagon as compared with the same subcellular fractions obtained from whole liver; this is most pronounced with plasma membranes. Evidence that the collagenase preparation may be involved in these changes is presented.