Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment.

Shortcomings of previously reported preclinical models of nonalcoholic steatohepatitis (NASH) include inadequate methods used to induce disease and assess liver pathology. We have developed a dietary model of NASH displaying features observed clinically and methods for objectively assessing disease progression. Mice fed a diet containing 40% fat (of which ∼18% was trans fat), 22% fructose, and 2% cholesterol developed three stages of nonalcoholic fatty liver disease (steatosis, steatohepatitis with fibrosis, and cirrhosis) as assessed by histological and biochemical methods. Using digital pathology to reconstruct the left lateral and right medial lobes of the liver, we made comparisons between and within lobes to determine the uniformity of collagen deposition, which in turn informed experimental sampling methods for histological, biochemical, and gene expression analyses. Gene expression analyses conducted with animals stratified by disease severity led to the identification of several genes for which expression highly correlated with the histological assessment of fibrosis. Importantly, we have established a biopsy method allowing assessment of disease progression. Mice subjected to liver biopsy recovered well from the procedure compared with sham-operated controls with no apparent effect on liver function. Tissue obtained by biopsy was sufficient for gene and protein expression analyses, providing the opportunity to establish an objective method of assessing liver pathology before subjecting animals to treatment. The improved assessment techniques and the observation that mice fed the high-fat diet exhibit many clinically relevant characteristics of NASH establish a preclinical model for identifying pharmacological interventions with greater likelihood of translating to the clinic.

A primary colonic crypt model enriched in enteroendocrine cells facilitates a peptidomic survey of regulated hormone secretion.

To enable the first physiologically relevant peptidomic survey of gastrointestinal tissue, we have developed a primary mouse colonic crypt model enriched for enteroendocrine L-cells. The cells in this model were phenotypically profiled using PCR-based techniques and showed peptide hormone and secretory and processing marker expression at mRNA levels that were increased relative to the parent tissue. Co-localization of glucagon-like peptide-1 and peptide YY, a characteristic feature of L-cells, was demonstrated by double label immunocytochemistry. The L-cells displayed regulated hormone secretion in response to physiological and pharmacological stimuli as measured by immunoassay. Using a high resolution mass spectrometry-based platform, more than 50 endogenous peptides (<16 kDa), including all known major hormones, were identified a priori. The influence of culture conditions on peptide relative abundance and post-translational modification was characterized. The relative abundance of secreted peptides in the presence/absence of the stimulant forskolin was measured by label-free quantification. All peptides exhibiting a statistically significant increase in relative concentration in the culture media were derived from prohormones, consistent with a cAMP-coupled response. The only peptides that exhibited a statistically significant decrease in secretion on forskolin stimulation were derived from annexin A1 and calcyclin. Biophysical interactions between annexin A1 and calcyclin have been reported very recently and may have functional consequences. This work represents the first step in characterizing physiologically relevant peptidomic secretion of gastrointestinally derived primary cells and will aid in elucidating new endocrine function.

Poly I:CLC induction of the interferon system in mice: an initial study of four detection methods.

Induction of a large number of the components of the interferon (IFN) system (IFN genes, their mRNAs, IFN proteins, IFN receptors, IFN signaling molecules, the IFN response genes, and their effector proteins) has been studied. Less well studied is the comparative induction of these components in vivo. Induction of IFN by double-stranded RNA (dsRNA) treatment mimics certain aspects of viral infection and induces the components of the IFN system. To determine the comparative sensitivity of detection of induction in mice, we initially studied the limiting concentrations of polyribinosinic-polyribocytidylic acid, polylysine complex (poly I:CLC, a synthetic dsRNA preparation), for induction of four representative components of the IFN system: (1) IFN in serum, (2) the IFN response gene mRNA ISG54 in spleen and liver, (3) the IFN-beta mRNA in spleen, and (4) resistance of mice to Banzi viral infection. The results of this initial study showed that resistance to infection was 7-fold more sensitive for detection of the IFN response than was ISG54 mRNA and 70-fold more sensitive than either IFN-beta mRNA or IFN production in serum. In comparison, mouse cells in vitro treated with poly I:CLC were 3-10-fold less sensitive to its antiviral action than is the mouse. The results demonstrate that in the four tests in mice, the most sensitive indicator of poly I:CLC induction of the IFN system was protection against Banzi viral infection, followed by ISG54 mRNA levels, IFN-beta mRNA, and IFN protein levels. It is hypothesized that the highest sensitivity of mouse protection may be due to priming by the initial poly I:CLC-induced IFN of the subsequent Banzi virus-induced IFN, resulting in rapid and high concentrations of IFN at the local site of viral replication. Future studies are needed to study other molecular components of the IFN system to identify those that cause the unanticipated high sensitivity of mice to protection against Banzi virus.