Impact of isolated soy protein in non‐alcoholic fatty liver disease progression: monitored by a novel quantitative ultrasound (QUS) method
Authors: Rowles Joe L , Kelly Jamie R , Miller Rita J , Berndt Jake , Applegate Catherine C , Han Aiguo , Wallig Matthew A , O'Brien William D , Erdman John W 
Published: June 2021

BACKGROUNDNon‐alcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western world, affecting 20% of the population. If left untreated, NAFLD may progress into non‐alcoholic steatohepatitis (NASH), end‐stage liver disease or hepatocellular carcinoma. Emerging evidence indicates that soy protein may protect the liver from steatosis, which may improve health outcomes. We hypothesized that a soy protein intervention would be effective in protecting against further steatosis and disease progression in mice fed a high fat diet.METHODSIn this study, 8‐week‐old male C57BL/6J mice (n=70) were randomized onto an AIN‐93G control diet (CON, n=20) or a high‐fat atherogenic diet with a casein protein source (CAS, n=50). After 4 weeks on the CAS diet when animals were expected to develop NAFLD, CAS animals were randomized onto one of three diets: an atherogenic diet with a soy protein source (SOY, n=15), CAS (n=20) or the control diet (CAS‐CON, n=15). For this study, we utilized the novel non‐invasive QUS method developed by our lab in order to monitor hepatic fat content at various stages of steatosis. Each animal received liver ultrasound scans at baseline (in vivo) and at euthanasia (in vivo and ex vivo). At 4, 9 and 12 weeks on study, animals from each group were euthanized. At euthanasia, serum was collected and the liver was harvested. Half of the liver's left lateral lobe underwent hematoxylin and eosin (H&E) staining (3‐μm section). The right cranial lateral liver lobe was removed for ex vivo QUS scanning. The remainder of each liver was frozen for further analysis.RESULTSLiver weight, as a percentage of body weight, was significantly increased with both atherogenic diets (CAS and SOY). CAS animals had significantly higher accumulation of lipids in the liver at each respective time point (p<0.05) compared to either dietary control group (CON or CAS‐CON). CAS also had significantly higher serum cholesterol by week 12 compared to CON and CAS‐CON. The QUS was also significantly associated with the quantity of lipid in the liver. Ex vivo (p<0.0001) and in vivo (p=0.047) ultrasound attenuation was positively correlated with liver lipids. Serum alanine aminotransferase (ALT) and histology will be analyzed to determine hepatic damage and disease status. QUS attenuation and backscatter will be further correlated to histological measures of steatosis, fibrosis and inflammation in the liver. Total hepatic cholesterol will also be measured.CONCLUSIONSAtherogenic diets were associated with an increase in liver weight, liver lipids and ultrasound attenuation. Replacing casein with soy as the protein source after four weeks of feeding failed to significantly reverse hepatic lipid accumulation or liver weight. QUS attenuation was significantly associated with hepatic lipid accumulation. Serum cholesterol increased over the course of 12 weeks in the CAS group. The development of a QUS method to identify early stages of NAFLD and NASH would provide the capability to noninvasively quantify and monitor liver status and evaluate methods for intervention.Support or Funding InformationThis work was supported by NIH R37EB002641 and by the USDA National Institute of Food and Agriculture, Hatch project 228706.