Liver fibrosis is a common pathological condition that occurs because of chronic liver injury resulting from various etiological factors such as viruses, toxins, alcohol [1]. Succinate is an intermediate in the tricarboxylic acid (TCA) cycle that plays an important role in the generation of Adenosine triphosphate in the mitochondria. Different reports have shown that succinate could regulate various cell functions related to epigenetics, tumor genesis, inflammation, signal transduction, and paracrine modulation in various tissues, including the liver, kidney, heart, retina, and immune tissues [2,3,4]. Our previous study revealed that succinate increased fibrogenic markers, inflammatory cytokine expression, and the proliferation and migration of LX-2 cells, and that administration of intraperitoneal succinate in mice showed increased alpha smooth muscle actin (α-SMA) and inflammatory cytokine protein expression, apart from the histological changes [5]. A recent study demonstrated that administration of succinate with drinking water could control the activation of adipose tissue thermogenesis [6]. Although the effect of succinate on the regulation of hepatic stellate cells (HSCs) has been demonstrated, the effect of succinate on liver fibrosis and damage in mice in a time-dependent manner is not known. Further, a new mouse model for liver fibrosis using succinate is still lacking. Therefore, we conducted this research to validate whether succinate induces liver fibrosis in a mouse model.

In this study, we demonstrated that the sodium succinate diet administered for 16 weeks might induce the upregulation of α-SMA expression at 8 weeks of feeding (molecular changes) and then develop hepatic fibrosis (morphological changes) at 16 weeks of feeding in a time-dependent manner. We elucidate the possible role of GPR91 as an early fibrogenic marker, which was highly expressed at 4 weeks of feeding, even before the induction of molecular changes, such as the overexpression of α-SMA at 8 weeks of feeding.

Liver fibrosis involves the excessive accumulation of extracellular matrix proteins as the result of chronic liver injuries, such as chronic hepatitis B or C infection, alcohol, and free fatty acids in non-alcoholic fatty liver disease [7]. During the progression of hepatic fibrosis, HSCs play a major role [1].

Experimental mouse models of hepatic fibrosis can be induced by toxins (e.g., carbon tetrachloride and thioacetamide), diets (e.g., high fat diet and methionine- and choline-deficient diet), and surgery (e.g., bile duct ligation) [8]. In the present study, we focused on the effect of succinate within the liver to establish a new mouse model of liver fibrosis based on the sodium succinate diet. In our previous study, we found that daily administration of an intraperitoneal injection of 100 mg/kg succinate in C57BL/6J mice for four weeks could lead to increased α-SMA expression, but no histo-morphological changes, such as hepatic fibrosis, were seen [5]. The present study was designed to demonstrate that an oral succinate diet containing 2% sodium succinate instead of an intraperitoneal succinate injection, which was lethal over four weeks, may induce hepatic fibrosis through prolonged oral succinate feeding. In this study, we demonstrated for the first time that a sodium succinate diet leads to hepatic fibrosis changes in histology at 16 weeks of feeding, including the overexpression of the α-SMA protein and gene at 8 weeks of feeding. In addition, we clarified that the sodium succinate diet increased serum ALT and hepatic triglyceride levels in the mouse model. Our study suggests that the oral succinate diet mouse model could be a model of hepatic fibrosis or hepatic damage in future research.

The succinate receptor, GPR91, is expressed in quiescent rat HSCs, and the induction of ischemia in an isolated liver by interrupting portal flow leads to a marked increase in succinate release, thereby activating HSCs [9]. However, our previous study showed that the expression of GPR91 is significantly increased in activated HSCs, which is contrary to our findings in the present study [9]. Additionally, our previous study showed that GPR91 knockdown with small interfering RNA deactivates HSCs, demonstrating that succinate induces the activation of HSCs through GPR91 signaling [10].

Succinate accumulation and GPR91 signaling seems to be a key early step for cells to sense and respond to hyperglycemic conditions in the kidney, which leads to the activation of the intra-renal renin-angiotensin system [11,12]. Recently, another study showed that deletion of the GPR91 gene protected high-fat diet-fed mice from obesity only during the initial period; however, in later stages, body weight differences and weight curves were not different between GPR91 knockout and wild-type mice [13], suggesting GPR91 as an early sensor for dietary energy intake. Notably, during the development of liver fibrosis by the consumption of a sodium succinate diet, a marked overexpression of the GPR91 protein and gene in the liver occurred at 4 weeks of feeding, following which the expression of GPR91 declined rapidly from 8 weeks to 16 weeks. This might be explained by the compensatory response of GPR91 to sustained exposure to high concentration of succinate in long-term feeding.

Mice fed with sodium succinate showed increased expression of the α-SMA protein and gene in the liver at 8 weeks of feeding and demonstrated hepatic fibrosis histology at 16 weeks of feeding. However, the expression of the GPR91 protein and mRNA increased earlier at 4 weeks of feeding and then decreased rapidly from 8 weeks to 16 weeks. In addition, mice orally administered sodium succinate lost body weight and showed an increase in serum ALT and hepatic triglyceride levels compared to control mice. These results suggest that succinate could induce liver damage and fibrosis in the mouse model, and that GPR91 signaling might be an early marker or sensor of hepatic fibrosis development. Thus, GPR91 signaling could serve as a potential therapeutic target to prevent or delay the progression of liver fibrosis.