Non-Alcoholic Fatty Liver Disease (NAFLD) - Biomarkers

• Non-alcoholic fatty liver disease (NAFLD) refers to the accumulation of fat in the liver that is not caused by excessive alcohol consumption. It can progress to more severe diseases including nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, liver cancer and end-stage liver disease
• Metabolic syndromes such as obesity and insulin resistance and genetic mutations can increase the likelihood of developing NAFLD.
• NAFLD reversal can be achieved by losing weight and implementing a healthier lifestyle.

Background of NAFLD

- What is NAFLD?

- NAFLD vs NASH

- What are the Symptoms of NAFLD

- Risk Factors of NAFLD

Diagnosis and Biomarkers for NAFLD

- NAFLD Biomarkers used for Detection

- Analytical Methods for Determining NAFLD Severity

- Treatment for NAFLD

- NAFLD Pharmacological Treatment

Final Notes

Nonalcoholic fatty liver disease (NAFLD) is becoming one of the major causes of liver disease worldwide with an estimated prevalence of around 24% (Younossi, 2018). This disease encompasses simple steatosis, or the abnormal retention of fats in a cell or an organ, to more progressive steatosis characterised by diseases such as nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma (liver cancer) and end-stage liver disease which may potentially require a liver transplant (Benedict & Zhang, 2017). NAFLD is considered a growing epidemic due to the increasing incidence of obesity and insulin resistance which lead to the excess deposition of triglycerides, cholesterol and free fatty acids in the liver (Benedict & Zhang, 2017).

NASH is the more severe stage of NAFLD as it leads to the accumulation of lipids in the liver which can trigger liver cell death, inflammation and fibrosis (Pierantonelli & Svegliati-Baroni, 2019). Therefore, the difference between NAFLD and NASH is that NAFLD is characterized by excess fat in the liver, whereas NASH causes inflammation, hepatocyte damage and death due to a build up of lipid droplets in the liver (National Institute of Diabetes and Digestive and Kidney Disease, 2020).

NAFLD is commonly referred to as a silent disease because there are few identifiable symptoms. The risk factors of those suffering with non-alcoholic fatty liver disease are being overweight or obese and suffering from insulin resistance,mostly characterised by type 2 diabetes, NAFLD symptoms include hypertension, tiredness, lethargy and pain or dullness in the right upper abdomen (Basaranoglu & Neuschwander-Tetri, 2006).

Another genetic mutation that has been associated with NAFLD is the SNP rs58542926 in the TM6SF2 (transmembrane protein 6 superfamily 2) which is associated with increased hepatic triglyceride content, a key characteristic of NAFLD (Li et al., 2019). This gene has been documented to influence hepatic fibrosis progression in patients with NAFLD (Liu et al., 2014).

Lifestyle factors also have a major effect on the likelihood of the developing NAFLD. As a significant proportion of the population suffering from NAFLD also has various metabolic syndromes such as obesity, insulin resistance and hypertension, the first line of NAFLD treatment is to implement a healthier lifestyle by following a healthy and balanced diet.

NAFLD can be diagnosed by imaging, liver biopsies and blood tests. Histological markers seen in patients with NAFLD are the ballooning of hepatocytes, which indicates hepatocellular death, lobular inflammation characterized by the significant presence of mononuclear and polymorphonuclear cells and portal granulocytic inflammation, which is a hallmark of acute inflammation (Dumitrascu & Neuman, 2018). Liver biopsies allow the grading and staging the disease. Blood tests that show high serum triglycerides and low high density lipids (HDL) in the serum, as well as elevated liver enzymes such as ALT (alanine aminotransferase), ASF (aspartate aminotransferase) and GGT (gamma-glutamyl transferase) are indicative of NAFLD (Neuman et al., 2014).

Analytical methods for measuring hepatotoxicity can be carried out by using an assay kit that measures triglyceride accumulation in hepatocytes, and a staining reagent that can visualize lipid droplet formation in the cells. This is a useful assay as it can screen for candidates who are at risk of developing steatosis.

Additionally, another method to measure the severity of NAFLD in patients is to measure the concentration of fatty acid uptake by adipocytes. Inability of adipocytes to successfully uptake fatty acids and abnormalities in lipid metabolism is a contributing factor to non-alcohol fatty liver related diseases. The use of an assay provides the rapid determination of fatty-acid uptake kinetics in live cells expressing fatty acid transporter proteins.

Certain dietary factors that need to be considered for those implementing a NAFLD diet include a reduction of saturated fats, fructose and simple carbohydrates and an increase in unsaturated fats, fresh produce and complex carbohydrates (Perdomo et al., 2019). In addition, it is important to take measures to reduce stress on the liver such as over-consumption of alcohol and pain-killers (Kahali et al., 2015).

Vigorous exercise as opposed to moderate exercise was also seen to have a more significant effect of histological improvement in patients with NAFLD ((Beaton, 2012). When there are no significant changes in the severity of the liver disease, pharmacological treatments are considered (Kahali et al., 2015). NAFLD reversal can be done by a slight reduction in weight and a decrease in caloric intake.

Pharmacological treatment is usually implemented when the patient suffering from steatohepatitis has a NAS of 4 or higher. As NASH can lead to advanced fibrosis and cirrhosis, it is important that it is treated when first diagnosed (Francque & Vonghia, 2019). There are several drugs that are not licensed for NASH specifically however; they have some therapeutic benefit (Francque & Vonghia, 2019). One of the treatments available are insulin sensitizers.

Insulin resistance is a hallmark of NAFLD, therefore targeting it can provide some therapeutic benefit. Insulin sensitizers including metformin and thiazolidinediones (TZD’s) have beneficial effects on improving the severity of NAFLD (Ozturk & Kadayifci, 2014). Metformin has been shown to improve transamine levels, excess adipose tissue deposition in the liver and hepatocyte ballooning, however, it is ineffective at reducing fibrosis and inflammation in the liver (Beaton, 2012). TZD’s have beneficial effects on insulin resistance, hepatocyte fatty acid metabolism and adiponectin levels. However, similarly to metformin, TZDs have not been proven to reduce fibrosis in patients with liver disease (Omer et al., 2010, Belfort et al., 2006).

Lipid lowering agents are also a common treatment given to patients with NAFLD. Statins, fibrates and omega-3 fatty acids are commonly used to treat abnormal amounts of lipids in the blood, and have beneficial effects on adiponectin levels (Beaton, 2012). Statins, such as simvastatin and lovastatin have been documented to lower total cholesterol, LDL cholesterol, triglycerides and free fatty acids in NAFLD patients (Nascimbeni et al., 2019). Additionally, they can downregulate several pro-inflammatory modulators which are the culprits for inflammation in the liver leading to fibrosis and cirrhosis (Nascimbeni et al., 2019).

Vitamin E is a strong antioxidant potent and anti-inflammatory properties and has been documented to have beneficial effects on NAFLD. It has been noted to significantly lower serum transamine levels and improve hepatic steatosis and necroinflammation (Nan et al., 2009). The administration of vitamin E was also associated with suppression of expression of pro-fibrotic genes such as TGF-β and MMP-2, COX-2 and pro-apoptotic genes such as Bax. In addition, it downreguated expression of NF-kB, a vital transcription factor for expression of various pro-inflammatory cytokines (Nan et al., 2009).

In conclusion, NAFLD is a growing epidemic partly to do with the dramatic increase of obesity and insulin resistance resulting in liver accumulation of triglycerides and free fatty acids. Although NAFLD is not fatal, if precautionary measures such as weight loss and the adoption of a healthier lifestyle aren’t implemented, the development of more serious fatty-liver associated diseases such as NASH, cirrhosis and fibrosis will occur.

Angulo, P., Hui, J.M., Marchesini, G., Bugianesi, E., George, J., Farrell, G.C., Enders, F., Saksena, S., Burt, A.D., Bida, J.P., Lindor, K., Sanderson, S.O., Lenzi, M., Adams, L.A., Kench, J., Therneau, T.M., and Day, C.P. (2007) The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 45: 846-854.

Basaranoglu, M., and Neuschwander-Tetri, B.A. (2006) Nonalcoholic Fatty Liver Disease: Clinical Features and Pathogenesis. Gastroenterol Hepatol (N Y) 2: 282-291.

Beaton, M.D. (2012) Current treatment options for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Can J Gastroenterol 26: 353-357.

Belfort, R., Harrison, S.A., Brown, K., Darland, C., Finch, J., Hardies, J., Balas, B., Gastaldelli, A., Tio, F., Pulcini, J., Berria, R., Ma, J.Z., Dwivedi, S., Havranek, R., Fincke, C., DeFronzo, R., Bannayan, G.A., Schenker, S., and Cusi, K. (2006) A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 355: 2297-2307.

Benedict, M., and Zhang, X. (2017) Non-alcoholic fatty liver disease: An expanded review. World J Hepatol 9: 715-732.

Brunt, E.M., Janney, C.G., Di Bisceglie, A.M., Neuschwander-Tetri, B.A., and Bacon, B.R. (1999) Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 94: 2467-2474.

Dumitrascu, D.L., and Neuman, M.G. (2018) Non-alcoholic fatty liver disease: an update on diagnosis. Clujul Med 91: 147-150.

Foroughi, M., Maghsoudi, Z., Khayyatzadeh, S., Ghiasvand, R., Askari, G., and Iraj, B. (2016) Relationship between non-alcoholic fatty liver disease and inflammation in patients with non-alcoholic fatty liver. Adv Biomed Res 5: 28.

Francque, S., and Vonghia, L. (2019) Pharmacological Treatment for Non-alcoholic Fatty Liver Disease. Adv Ther 36: 1052-1074.

Hadizadeh, F., Faghihimani, E., and Adibi, P. (2017) Nonalcoholic fatty liver disease: Diagnostic biomarkers. World J Gastrointest Pathophysiol 8: 11-26.

Kahali, B., Liu, Y.L., Daly, A.K., Day, C.P., Anstee, Q.M., and Speliotes, E.K. (2015) TM6SF2: catch-22 in the fight against nonalcoholic fatty liver disease and cardiovascular disease? Gastroenterology 148: 679-684.

Li, Y., Liu, S., Gao, Y., Ma, H., Zhan, S., Yang, Y., Xin, Y., and Xuan, S. (2019) Association of TM6SF2 rs58542926 gene polymorphism with the risk of non-alcoholic fatty liver disease and colorectal adenoma in Chinese Han population. BMC Biochem 20: 3.

Liu, Y.L., Reeves, H.L., Burt, A.D., Tiniakos, D., McPherson, S., Leathart, J.B., Allison, M.E., Alexander, G.J., Piguet, A.C., Anty, R., Donaldson, P., Aithal, G.P., Francque, S., Van Gaal, L., Clement, K., Ratziu, V., Dufour, J.F., Day, C.P., Daly, A.K., and Anstee, Q.M. (2014) TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease. Nat Commun 5: 4309.

Maleki, I., Aminafshari, M.R., Taghvaei, T., Hosseini, V., Rafiei, A., Torabizadeh, Z., Barzin, M., and Orang, E. (2014) Serum immunoglobulin A concentration is a reliable biomarker for liver fibrosis in non-alcoholic fatty liver disease. World J Gastroenterol 20: 12566-12573.

Nan, Y.M., Wu, W.J., Fu, N., Liang, B.L., Wang, R.Q., Li, L.X., Zhao, S.X., Zhao, J.M., and Yu, J. (2009) Antioxidants vitamin E and 1-aminobenzotriazole prevent experimental non-alcoholic steatohepatitis in mice. Scand J Gastroenterol 44: 1121-1131.

Nascimbeni, F., Pellegrini, E., Lugari, S., Mondelli, A., Bursi, S., Onfiani, G., Carubbi, F., and Lonardo, A. (2019) Statins and nonalcoholic fatty liver disease in the era of precision medicine: More friends than foes. Atherosclerosis 284: 66-74.

National Institute of Diabetes and Digestive and Kidney Disease, N., (2020) Nonalcoholic Fatty Liver Disease & NASH. In., pp.

Neuman, M.G., Cohen, L.B., and Nanau, R.M. (2014) Biomarkers in nonalcoholic fatty liver disease. Can J Gastroenterol Hepatol 28: 607-618.

Nomura, F., Ohnishi, K., Koen, H., Hiyama, Y., Nakayama, T., Itoh, Y., Shirai, K., Saitoh, Y., and Okuda, K. (1986) Serum cholinesterase in patients with fatty liver. J Clin Gastroenterol 8: 599-602.

Omer, Z., Cetinkalp, S., Akyildiz, M., Yilmaz, F., Batur, Y., Yilmaz, C., and Akarca, U. (2010) Efficacy of insulin-sensitizing agents in nonalcoholic fatty liver disease. Eur J Gastroenterol Hepatol 22: 18-23.

Ozturk, Z.A., and Kadayifci, A. (2014) Insulin sensitizers for the treatment of non-alcoholic fatty liver disease. World J Hepatol 6: 199-206.

Perdomo, C.M., Fruhbeck, G., and Escalada, J. (2019) Impact of Nutritional Changes on Nonalcoholic Fatty Liver Disease. Nutrients 11.

Pierantonelli, I., and Svegliati-Baroni, G. (2019) Nonalcoholic Fatty Liver Disease: Basic Pathogenetic Mechanisms in the Progression From NAFLD to NASH. Transplantation 103: e1-e13.

Romeo, S., Kozlitina, J., Xing, C., Pertsemlidis, A., Cox, D., Pennacchio, L.A., Boerwinkle, E., Cohen, J.C., and Hobbs, H.H. (2008) Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 40: 1461-1465.

Speliotes, E.K., Butler, J.L., Palmer, C.D., Voight, B.F., and Hirschhorn, J.N. (2010) PNPLA3 variants specifically confer increased risk for histologic nonalcoholic fatty liver disease but not metabolic disease. Hepatology 52: 904-912.

Tesfay, M., Goldkamp, W.J., and Neuschwander-Tetri, B.A. (2018) NASH: The Emerging Most Common Form of Chronic Liver Disease. Mo Med 115: 225-229.

Younossi, Z.M. (2018) The epidemiology of nonalcoholic steatohepatitis. Clin Liver Dis (Hoboken) 11: 92-94.