The global prevalence of nonalcoholic fatty liver disease (NAFLD) is approximately 15–30% [1]. Unhealthy dietary habits and insufficient physical activity are risk factors for NAFLD [2]. NAFLD is characterized by a hepatic triglyceride (TG) accumulation of more than 5% of the liver weight, and its pathological symptoms are associated with metabolic disorders, including obesity, insulin resistance, and hypertriglyceridemia [3].

Inhibiting lipid synthesis or increasing lipid degradation are effective methods for lowering lipid accumulation in livers, and it is vital to balance lipid metabolism to reduce NAFLD incidence [4]. An increase in de novo lipogenesis (DNL) and reduction of fatty acid oxidation leads to hepatic steatosis [5]. The liver also exports TGs to extracellular tissues through very low-density lipoprotein (VLDL). Therefore, when VLDL cannot effectively transport TGs out of the liver, NAFLD progression may be exacerbated [6]. In NAFLD patients with hepatic steatosis, an excessive amount of free fatty acids can increase cytochrome P-450 2E1 expression, leading to an elevation in reactive oxygen species (ROS) and oxidative stress levels; it can also increase the concentrations of lipid peroxides such as malondialdehyde (MDA) [7]. Moreover, recent studies have indicated that ferroptosis, an iron-induced form of cell death caused by iron-dependent lipid peroxidation, can accelerate NAFLD progression. Excessive accumulation of ferrous ions within tissues induces ROS generation through the Fenton reaction, leading to oxidative damage and cell death; it is also associated with the progression of various diseases [8]. In individuals with NAFLD, fatty liver accompanied by oxidative stress can lead to liver injury and the release of damage-associated molecular patterns (DAMPs), activating the toll-like receptor 4 (TLR4) pathway. The downstream targets of TLR4 include nuclear factor kappa B (NF-κB) and the inflammasome pathway, which further activates proinflammatory cytokines, leading to liver inflammation [9]. The gut-liver axis, which connects the liver and intestines via the hepatic portal vein, is another crucial component influencing NAFLD development. Because of dietary factors, imbalances in the gut microbiota, such as a decrease in microbial diversity and an alteration in microbial composition, may further exacerbate the inflammatory response and accelerate NAFLD progression [10].

Due to the lack of relevant approved drug therapies, the American Association for the Study of Liver Diseases’ treatment guidelines for NAFLD recommend focusing on achieving >10% weight loss as the primary treatment approach [11]. Regular exercise and dietary restriction can lower weight by about 10%, which is adequate to reverse NAFLD in most patients; however, this reduction is rarely realized in practice [12]. The very low-carbohydrate diets (VLCD) have recently drawn considerable attention. It involves an extreme reduction in carbohydrate intake, leading to the increase of fatty acids breakdown into acetyl-CoA via β-oxidation. However, if the amounts of acetyl-CoA generated through β-oxidation challenge the processing capacity of the TCA cycle, acetyl-CoA will be shifted to the biosynthesis of ketone bodies as an essential energy source and ultimately to rapid weight loss [13]. Moreover, ketosis is achieved when less than 50 g of carbohydrates per day are introduced in humans [14]. Although VLCD may be effective for weight loss, its effects on hepatic steatosis, oxidative stress, and inflammation remain controversial. Some studies have reported that VLCD may reduce liver fat levels by preventing DNL [15]. However, some other studies have demonstrated that the extremely low carbohydrate content in the VLCD may significantly increase hepatic lipid accumulation [16]. A study in which mice were fed a VLCD for 4 weeks also observed considerable inflammation in the liver [17].

The relative abundance of proteins in the VLCD may also affect ketosis and metabolism in vivo. Bielohuby et al. indicated that in mice consuming very low-carbohydrate diets with different protein ratios, despite weight reduction and primarily utilizing fat as the primary source of metabolic energy, only the group with a lower protein ratio exhibited ketosis. Moreover, the study also mentioned that providing a high-fat, low-protein ratio diet is more likely to induce ketosis [18]. Van Wyk et al. also reported that extreme dietary patterns such as the VLCD may be challenging for some individuals to adapt to, possibly because the VLCD is primarily composed of fats (>80%), and the effects of increasing protein content in the VLCD in terms of weight loss and metabolism warrant further research [19]. Furthermore, altering the protein content in the VLCD can improve hepatic lipid accumulation. In a study, mice fed a balanced diet demonstrated significantly lower VLDL receptor (VLDLR) expression and TG levels in the liver than those fed a low-protein diet [20]. Moreover, in mice with dyslipidemia, a high-protein diet with 35% energy from protein resulted in decreased NF-κB expression than a low-protein diet [21].

Although consuming the VLCD may facilitate rapid weight loss, its effects on hepatic lipid metabolism and inflammatory responses remain controversial, and the possible underlying mechanisms also remain unclear. In patients with NAFLD intending to consume the VLCD for weight loss, its safety is a crucial consideration. Therefore, in this study, we investigated the effects of VLCD with different protein contents and under isocaloric control on weight loss, hepatic lipid metabolism, oxidative stress, and inflammatory responses in mice with diet-induced NAFLD and examined the possible underlying mechanisms.