RenaSci

Non-alcoholic Steatohepatitis (NASH)

Hepatic Steatosis and Liver Function

 

Non-alcoholic fatty liver disease (NAFLD) is the excessive accumulation of lipid in the liver. Simple hepatic steatosis is a relatively benign condition and usually reversible. However, if lipids continue to accumulate and the liver becomes inflamed it can progress into non-alcoholic steatohepatitis (NASH). NASH is characterised by lobular inflammation and hepatocellular ballooning and is often associated with fibrosis (collagen deposition).  It can progress to cirrhosis, hepatocellular carcinoma and end-stage liver failure. Risk factors for NASH include obesity and diabetes. 

 

We offer a range of assays for evaluating the effects of novel drugs on liver lipids, liver function and liver pathology. 

 

Liver lipids


Total liver fat can be determined by chemical analysis (Soxhlet extraction) and assays can be performed on liver extracts to determine liver triglycerides, liver cholesterol, liver NEFA and liver glycogen levels. There is a good correlation between total liver fat determined by Soxhlet extraction and total liver triglycerides determined by enzyme assay.

Correlation between Total Liver Fat and Total Liver Triglycerides

We have used these assays to show that our dietary-induced obese mice and rats develop hepatic steatosis. Antiobesity agents such as the GLP-1 receptor agonist, liraglutide, significantly reduce liver lipids in dietary-induced obese mice.

 

Liver enzymes


Liver function can be assessed by measuring plasma levels of the liver enzymes, alanine aminotransferase ALT); aspartate aminotransferase (AST) and alkaline phosphatase (ALP), throughout and/or at the end of studies.

 

Liver pathology


At the end of studies, liver lobes can be collected and processed using standard histological staining procedures eg :-

 

     •     Haematoxylin and Eosin (H&E) staining to examine cell structures

     •     Oil Red O (ORO) staining for lipids and neutral triglycerides 

     •     Sirius Red (SR) staining for collagen deposition (fibrosis)


Liver sections can be sent for assessment by a fully-qualified pathologist (PathCelerate) using a light microscope and established graded scoring systems :-

 

     •    Steatosis scores (based on H&E staining; graded 0-3)

     •    Hepatocellular ballooning scores (based on H&E staining; graded 0-2)

     •    Lobular inflammation scores (based on H&E staining; graded 0-3)

 

The steatosis, hepatocellular ballooning and lobular inflammation scores are added together to form a non-alcoholic fatty liver disease (NAFLD) activity score (NAS; graded 0-8). .

 

Lipid deposition (lipid-postive vacuolation) can also be scored (based on ORO staining; graded 0-4).


The degree of fibrosis can be assessed by collagen deposition scores, which are based on SR staining and graded 0-4.  

 

Total collagen content in liver tissue can also be quantified as a direct measure of liver fibrosis. Total collagen content is determined using commercially available kits which measure levels of the amino acid, hydroxyproline, following acid hydrolysis of the tissue.

 

We have examined liver histology in dietary-induced obese mice maintained on high fat diet. An accumulation of hepatocellular-associated lipid was observed in vehicle-treated control mice in the absence of notable inflammation or fibrosis. Chronic administration of liraglutide significantly reduced steatosis and hepatocellular ballooning scores in the dietary-induced obese mice but not lobular inflammation, NAS or fibrosis scores. 

 

Nutrient-deficient diet mouse models of NASH 

  

We have recently investigated the ability of choline deficient (CD) and methionine and choline deficient (MCD) diets to induce NASH in mice. The MCD diet has been employed for several years as an animal model of NASH. It produces the histological features of NASH but also marked weight-loss and reductions in plasma glucose and insulin. Thus, the MCD diet does not mirror the metabolic abnormalities observed in patients with NASH who are often obese and insulin resistant/diabetic. The CD diet was used as there is some evidence that it may produce less weight-loss than the MCD diet. The experiment included a control group of mice maintained on normal chow.  Pioglitazone was used as a standard as it has been shown to be of benefit in the treatment of NASH in man.

 

Mice were maintained on CD and MCD diets for 6 weeks. Plasma samples and livers were then taken for analysis.  Both diets significantly increased NAS and fibrosis (collagen deposition scores), liver lipids and plasma levels of the liver enzymes, ALT and AST, consistent with the development of NASH. These responses were, in general, inhibited by pioglitazone. 

NAS (NAFLD activity scores) and fibrosis (collagen deposition scores) in mice on CD and MCD diets

Liver Enzymes in CD and MCD Mouse Models of NASH

Liver Lipids in CD and MCD Mouse Models of NASH

 

The expression of genes associated with NASH (eg markers of fibrosis, inflammation and steatosis) were significantly increased in livers of mice maintained on CD and MCD diets compared to control mice on normal mouse chow. These reponses were also inhibited by pioglitazone.

 

These results demonstrate that mice on nutrient-deficient diets can be used as simple models to identify compounds with potential for the treatment of NASH. The CD diet had a clear advantage over the MCD diet as it produced much less weight-loss (see our Nutrient-Deficient Diet Mouse Models of NASH Flyer).  

 

We have continued to validate the CD diet mouse model of NASH using the dual peroxisome proliferator activated receptor alpha/delta (PPARα/δ) agonist, elafibranor, which has produced promising results (the resolution of NASH without worsening of fibrosis) in Phase 2 clinical trials and is being developed for the treatment of NASH. Elafibranor reduced liver triglycerides (concentration) and all of the histological features of NASH and fibrosis in mice on a CD diet confirming that this model can be used as an initital screen for anti-NASH activity.

 

Effects of Pioglitazone and Elafibranor on Liver Pathology in Mice on a CD Diet

Effects of Pioglitazone and Elafibranor on Liver NAS and Fibrosis Scores in Mice on a CD Diet

 

Effects of Pioglitazone and Elafibranor on Liver Triglycerides in Mice on a CD Diet

 

NB that elafibranor produced weight-loss versus the CD diet control group (13.7% on Day 43). Due to the weight-loss, the dose of elafibranor was reduced from 30 to 20 mg/kg on Day 27 (see our Elafibranor in the CD Diet Model of NASH Flyer). 

 

The ob/ob mouse H-FFC diet model of NASH

 

We have also established a genetically-obese animal model of NASH. In this model, exposure of ob/ob mice to a high-fat, high-fructose and high-cholesterol (H-FFC) diet for 12 weeks, significantly reduced body weight and increased terminal plasma ALT levels compared to ob/ob mice on a normal diet. Liver triglyceride levels were high and steatosis and hepatocellular ballooning scores were maximal in ob/ob mice on both the normal and  H-FFC diets. The H-FFC diet significantly increased liver weight, lipid deposition and lobular inflammation (and hence NAS) compared to the normal diet controls and produced a robust fibrotic response (moderate to marked liver fibrosis scores compared to minimal or slight in the control animals). Pioglitazone significantly increased body weight compared to the normal diet and H-FFC controls and significantly decreased the increase in plasma ALT and liver weight induced by the H-FFC diet. It had no effect on liver triglycerides or endpoints associated with the high liver lipid content of the ob/ob mice, but reduced lobular inflammation (and hence NAS) and markedly reduced the severity of fibrosis in this animal model of NASH. Please see our ob/ob mouse H-FFC diet model of NASH flyer. We are currently evaluating elafibranor in this model.

 

Measurement of liver lipids and assessment of liver pathology can be conducted on samples from external studies if required. 

 

Posters

 

Cheetham et al. 2017. Evaluation of elafibranor in the choline-deficient diet mouse model of NASH. NASH Summit Europe, Frankfurt, Germany, 10th-12th October 2017.