The liver plays an essential role in lipid homeostasis. Lipid uptake, de novo synthesis, metabolism, and export contribute to overall hepatic lipid content. Failure to maintain a balance among these processes results in steatosis [1,2]. Hepatic steatosis is the first stage of metabolic dysfunction-associated steatotic liver disease (MASLD), and is characterized by intrahepatic lipid, primarily as triglyceride (TG), that exceeds 5% of total organ weight [[1], [2], [3]]. MASLD is considered to be the primary cause of chronic liver diseases worldwide, and is precipitated by factors other than alcohol consumption. The prevalence of MASLD worldwide has exceeded 32%, with a higher incidence among males compared to females [4]. In most cases, MASLD coexists with other metabolic disorders, especially obesity and insulin resistance [[1], [2], [3], [4]]. Since there is currently no effective therapy for MASLD, aside from liver transplantation and the recently approved Resmetirom for individuals with MASH and stage 2–3 fibrosis [5], weight loss, including weight loss achieved via GLP-1 receptor agonists [6], remains the only feasible approach for reducing liver fat and managing the early stages of MASLD and its associated conditions [2,3].

A recent expert consensus statement for the prevention and management of MASLD has recommendations for personalized balanced diets high in protein, low in carbohydrate, and with controlled or restricted energy intakes [7]. Specific dietary advice for protein encourages consumption of healthy sources of protein (primarily plant-based), regular intake of fish and seafood, low-fat or fat-free dairy products, and lean or unprocessed forms of meat and poultry [7]. These recommendations are based on evidence indicating that high-protein diets (HPDs) reduce body weight, improve insulin resistance, and decrease hepatic fat content [7,8]. Clinical trials have demonstrated that HPDs (containing 30% energy from protein) reduce liver fat and improve glycemic parameters in individuals with type 2 diabetes or MASLD, however, outcomes vary on whether diets rich in animal protein (meat, dairy) or diets rich in plant protein (primarily legume protein) are equally effective or not, and whether small reductions in body weight might be responsible for lowering liver fat [[9], [10], [11], [12], [13]]. Pre-clinical studies have demonstrated that diets containing soy protein reduce hepatic fat and improve insulin resistance independent of body weight in obese rats compared to diets comprised of casein, the protein source in the American Institute of Nutrition semi-purified diet for rodents [8,14,15]. Interestingly, HPDs (35% energy from protein) containing a mixture of animal and plant protein sources were as effective as HPDs comprised of soy protein for reducing hepatic fat and improving glycemic parameters in obese rats [8]. These findings raise questions about the effects of specific dietary protein sources, and mixtures of protein sources, on hepatic lipid metabolism in the context of MASLD and in the context of HPDs within the upper limit of 35% of energy from protein based on the Acceptable Macronutrient Distribution Ranges (ADMR) [16].

To date, studies have not examined how lipid metabolic pathways are altered by HPDs (within the AMDR for protein), and for this reason, the significance of dietary protein sources in HPDs for the treatment of MASLD remains unknown. The objective of this study was to examine the effects of HPDs containing animal and/or plant protein sources on hepatic steatosis and lipid-related metabolic pathways in fa/fa Zucker rats which exhibit hepatic steatosis, insulin resistance, and obesity. It was hypothesized that HPDs containing plant proteins or a mixture of plant and animal protein sources, but not animal protein sources alone, would lead to a reduction in liver fat and insulin resistance, compared to normal-protein control diet (15% of energy from protein) containing casein (protein source in the American Institute of Nutrition-93 diet), via decreased lipid synthesis and increased lipid export from the liver.