Non-alcoholic fatty liver disease (NASH)/ Non-alcoholic steatohepatitis (NASH): Symptoms, causes, pathogenesis, prevention & treatment and preclinical animal models.

Non-alcoholic fatty liver disease (NASH)/ Non-alcoholic steatohepatitis (NASH)

Non-alcoholic fatty liver disease (NAFLD) is an umbrella term that comprises liver conditions disturbing individuals who drink slightly to no alcohol. As the term suggests, the key characteristic of NAFLD is additional fat deposited in liver cells. Non-alcoholic fatty liver disease (NAFLD) is a disorder in which additional fat is deposited in the liver. This accumulation of fat is not triggered by heavy alcohol use. When heavy alcohol use results in fat to accumulation in the liver, this situation is known as “Alcoholic liver disease”.

NAFLD is divided into two categories i.e. simple fatty liver and non-alcoholic steatohepatitis (NASH). These are the two different disease condition of the liver.

·         Simple fatty liver

This is also referred to as non-alcoholic fatty liver (NAFL). This form of NAFLD there is the less fat accumulation and no inflammation in the liver. NAFL also does not cause liver damage or other severe complications.

·         Non-alcoholic steatohepatitis (NASH)

This is the severe form of NAFLD in which liver inflammation & liver cell damage occur. These complications can cause fibrosis and may lead to cirrhosis & liver cancer. NAFLD comprises severe abnormalities in liver histology ranging from steatosis to NASH. This is characterized by the presence of steatosis with liver cell ballooning & inflammation with or without fibrosis.

Among developing chronic liver diseases, non-alcoholic fatty liver disease (NAFLD) and its severe condition, non-alcoholic steatohepatitis (NASH), are becoming a foremost communal health issue in developed countries. The predictable worldwide occurrence is 4-46% for NAFLD and 3%-5% for NASH. The highest occurrence of NAFLD is detected in Western nations (17% to 46%) where it is composed to become the most vital reason for morbidity and death for chronic liver disease. In the US, it is the furthermost form of chronic liver disease, disturbing about one-quarter of the residents (20-40% of US population).

Symptoms of NAFLD/NASH

The majority of people with non-alcoholic fatty liver disease (NAFLD) typically cause no sign and symptoms. When it happens, they may include fatigue and discomfort and pain in upper right abdomen.

Probable symptoms of non-alcoholic steatohepatitis (NASH) and cirrhosis comprise:

·         Abdominal swelling (fluid accumulation)

·         Enlargement of blood vessels just below the skin

·         Distended spleen

·         Redness and swelling in palms & legs.

·         Yellow discolouration of the skin and eyes (jaundice)

·         Appetite loss

Causes of NAFLS/NASH

NAFLD are the part of a metabolic syndrome characterized by insulin resistance (pre-diabetes), high fat deposition, obesity, hyperglycaemia as well as high blood pressure. The exact cause of NASH is not properly understood. Scientists are marking various factor that may help in the development of NASH. These factors are: - production of toxic inflammatory cytokines (Proteins), an imbalance between antioxidant & pro-oxidant chemicals (Oxidative stress), liver cell death (apoptosis), intestinal bacteria also cause liver inflammation and hepatocyte inflammation and permeation by WBC.

Risk factors

Fatty liver is a very common disease which is affecting one in five adults and one in ten children in the US. Obesity is the main cause of fatty liver. There is another wide type of diseases and disorders that may cause NAFLS are – metabolic syndrome, hypertension, high cholesterol, type 2 diabetes, sleep disturbances, hypothyroidism. NASH is mostly occurring in older peoples, peoples having diabetes and body fat concentrated in the abdomen. It is very difficult to differentiate the NAFLD and NASH without further testing.

Pathogenesis of NAFLD/NASH

The pathogenesis of NAFLD/NASH characterizes a composite procedure and its remains poorly understood. Different assumptions have been projected to describe NAFLD development and its progress to NASH. The preliminary hypothesis was the ‘two-hit theory’ in which the ‘first hit’ is categorized by a too much deposition of lipids in the liver. The increased invasion of free fatty acids (FFAs) within hepatocytes is because of high dietary intake of free fatty acid (FFA) as well as hepatic lipogenesis and lipolysis of triglycerides in adipose tissue.

The ‘second hit’ leads to inflammation, hepatocyte injury, and fibrosis and is introduced by numerous aspects including dysfunction of mitochondria, endoplasmic reticulum anxiety, adipocyte and gut-mediated inflammatory paths, lipotoxicity, and oxidative stress. Recently, a possible additional reasonable hypothesis, the ‘multiparallel hits’ the theory was projected to elucidate the pathogenesis of NAFLD/NASH. This theory tells that several of the aspects defined previously may occur in parallel, instead of repeatedly. The detailed involvement of each of these aspects remains to be explicated.

Progression of NAFLD to NASH

Progression of NASH from fatty liver and normal liver happens in four stages, that is: -

Stage 1: Healthy liver

The liver is the largest organ of the human body and it functions as bile production, protein synthesis, nutrient metabolism and glycogen storage. A healthy liver has a smooth surface and contains 5% or less amount of fat.

Stage 2: Steatosis or fatty liver

This is observed in peoples who consume excess calories and have an inactive routine but alcohol consumption is significantly less. Extra calories are deposited in the liver in the form of lipids causing liver fat content above 5% and pale yellow colour of the liver.

Stage 3: NASH (Non-alcoholic steatohepatitis)

When excess fat has been deposited in the liver, inflammation & ballooning (cell death) cause the development of NASH. At this stage, patients have a high chance of death from cardiovascular disease.

Stage 3: Cirrhosis

Continuous cell damage and cell death cause the formation of fibrous scar tissue (Fibrosis). Ultimately, extreme scar development causes the loss of liver function. This is the state known as stage 4 fibrosis or cirrhosis.

Outcomes:

NASH related cirrhosis is the higher risk of the last stage of liver disease. For example, liver failure, liver function loss (decompensation) and liver cancer (hepatic carcinoma). There is also a high risk of death and non-liver cancer.

 

Complications of NAFLD/ NASH

Most of the peoples with NAFLD have simple fatty liver and they don’t develop any typical complications.

NASH can lead to complications are cirrhosis which is late scaring in the liver & response to liver injury, and liver cancer. If this process is not interrupted, cirrhosis can cause ascites (Fluid build-up in abdomen), swelling of a vein in the esophagus, liver failure and hepatic encephalopathy (slurred speech). About 5 to 12 % of people suffering from NASH, progress to cirrhosis.

 Prevention and treatment of NAFLD/NASH

The risk of NAFLD can be reduced by choosing a healthy diet that is rich in fruits, vegetables, grains and healthy fats maintaining body weight and doing more exercise, keeping liver healthy (for this don’t drink alcohol, get vaccinated etc.).

In most of the cases, doctors recommended to weight loss to treat NAFLD and NASH. Weight loss may reduce, fibrosis and liver inflammation. Some researches showed that peoples with NAFLD who regularly drink coffee (2 cups/day), have a low risk of fibrosis and aerobic exercise also decrease fat in the liver and possible inflammation. 5 to 10% body weight loss might be also enough to improve liver function test and decrease liver fat and inflammation.

Drug therapy

Apart from lifestyle modifications, obeticolic acid (OCA) is a single FDA approved drug for the treatment NASH. OCA is farnesoid X receptor (FXR) agonist which controls expression of transcription factors which reduce hepatic steatosis, inflammation and bile acid synthesis. Unfortunately, there are no other FDA approved medicines to treat the NAFLD/NASH. Scientists are studying medicines that become helpful for fatty liver disease.

Some studies suggest that there are two best options acknowledged by “American Association for the study of liver disease” are Vitamin E (antioxidant, reduce ROS production and hepatic inflammation-induced liver damage) and pioglitazone (medicine for type 2 diabetes) improve NASH in the patient who doesn’t have diabetes.

Research by “National Institute of Diabetes and Digestive and Kidney Diseases” suggests that treatment with pioglitazone & Vit E improved NASH in 50% of the people.

For safety concerns, always talk with your doctor/consultant before taking these medications, dietary supplements and any other medicines.

 Preclinical models of NASH

Currently, established preclinical models are broadly categorized into three main areas i.e. dietary-induced, diet toxin-induced and diet genetically mutated models.

1. Genetically induced NASH models

Genetically induced obese mouse model of diabetes such as ob/ob, db/db, and foz/foz is also used as NASH/NAFLD models.

·         ob/ob & db/db model

The ob gene transcribes leptin that is involved in the regulation of food intake & insulin sensitivity. In ob/ob mice, there is a deficiency in the making of leptin. So, animals with this genetic alteration may progress hyperphagia and insulin resistance. ob/ob mice fed with high-fat diet (HFD), gained bodyweight rapidly and developed NASH feature. Some research has reported that leptin is also essential for liver fibrosis, so the ob/ob model is considered incompatible for studying NASH due to its paradoxical deficiency.

db/db model shows leptin resistance caused by early termination of leptin receptor transcription. Disturbance in transcription gave rise to defective leptin receptors that prohibited normal leptin signalling. Some studies suggested that db/db mice coupled with a methionine choline-deficient (MCD) diet to induce more severe liver fibrosis.

·         foz/foz model

This model also used as diabetic and obese NASH model. The foz/foz mice were generated from the recessive mutation of Alstrom syndrome 1 (Alms 1) gene that encrypts proteins involved in ciliary function. These mice usually develop hyperglycaemia, hypercholesterolemia, hyperinsulinemia, and liver inflammation.

2. Dietary induced models

Diet-induced NASH models include, but not limited to MCD diet, high-fat diet (HFD), Western diet (WS) and Amylin diet (AMLN) are the most widely used model. Methionine and choline are essential nutrients for development in humans. Feeding with MCD diet can cause fast development of hepatic lesions like steatosis, inflammation and fibrosis. There is about 40% the decrease in body weight of animals fed with MCD diet for 8 weeks. When only high-fat content is given, referred to as HFD model and when there is an addition of cholesterol with HFD, this is known as the western diet model. Animals that receive HFD or WS diet may develop insulin resistance, weight gain and steatosis. HFD and WS models are reported to cause minimum liver fibrosis. Recently, Amylin Pharmaceuticals develop ALMN diet-induced NASH model, composed of 2% cholesterol, 40% lipids and fructose water shows both systemic and liver-specific characteristic of human NASH at 28-30 weeks AMLN diet feeding models. Finally, it was concluded that NASH animal models induced by dietary interferences only need a long time to reach a mild to moderate NASH phenotype.

3. Diet & toxin-induced NASH model

To induce a more severe form of liver injury in NASH models, some toxic compounds such as streptozotocin (STZ): STAM model, corban tetrachloride (CCl₄) and diethylnitrosamine (DEN) has been added to a modified diet. In the STAM model, the single dose of streptozotocin (200µg) destroys the pancreatic β-cells and cause hyperglycaemia, coupled with HFD diet-induced liver damage. This also increases the liver steatosis, inflammation & fibrosis.

Diethylnitrosamine (DEN) induce severe hepatic injury by inducing DNA damage & increasing ROS production. When animals receive 25-30 mg/kg DEN with HFD for 4-6 weeks, it shows severe hepatic injury characterized by high inflammatory gene expression and hepatocyte ballooning. This model rapidly develops hepatocellular carcinoma (HCC) due to the carcinogenic effect of DEN.

NASH induced by the CCL4 rapidly develop severe liver inflammation and fibrosis. The use of CCL4 together with a Western diet is also reported to increase the body weight and severe liver histological changes similar to of NASH patients.

References

1.    Cheng Peng, Alastair G. Stewart, Owen L. Woodman, Rebecca H. Ritchie, Cheng Xue Qin. Non-Alcoholic Steatohepatitis: A Review of Its Mechanism, Models and Medical Treatments. Frontiers in Pharmacology. December 2020, Volume 11.

2.    Ans Jacobs, Anne-Sophie Warda, Jef Verbeek, David Cassiman, Pieter Spincemaille, An Overview of Mouse Models of Nonalcoholic Steatohepatitis: From Past to Present. Current Protocols in Mouse Biology. June 2016, Vol 6, 185-200.

3.    Nonalcoholic Fatty Liver Disease & NASH, https://www.niddk.nih.gov/health-information/liver-disease/nafld-nash.

4.    Nonalcoholic fatty liver disease, https://www.mayoclinic.org/diseases-conditions/nonalcoholic-fatty-liver-disease/symptoms-causes/syc-20354567.

5. What is nash? https://www.the-nash-education-program.com/what-is-nash/#:~:text=NASH%20stands%20for%20Non%2DAlcoholic,pre%2Ddiabetes%2C%20and%20diabetes.   

6.  Fatty liver disease: What it is and what to do about it, https://www.health.harvard.edu/blog/fatty-liver-disease-what-it-is-and-what-to-do-about-it-2019011015746.

7.    Non-Alcoholic Fatty Liver Disease, https://www.uofmhealth.org/conditions-treatments/digestive-and-liver-health/fatty-liver-disease-non-alcoholic.