Brain-derived neurotrophic factor (BDNF) is a protein involved in the modulation of numerous biological processes occurring in the central and peripheral nervous systems in mammals (Li et al., 2022). BDNF has been shown to have the ability to activate transmembrane tyrosine kinase B (Trk-B) receptors in neurons (Guo et al., 2014; Jin, 2020), which triggers various intracellular signaling pathways to alter specific neuronal functions (Yoshii and Constantine-Paton, 2010). However, BDNF and its high-affinity receptors have also been found in skeletal (Mousavi and Jasmin, 2006) and smooth muscles (Aravamudan et al., 2016), the endocrine system (Podyma et al., 2021), adipose tissue (Nakagomi et al., 2015) as well as some inner organs such as the heart (Pius-Sadowska and Machaliński, 2017), pancreas (Fulgenzi et al., 2020) and liver (Genzer et al., 2017), which plays a vital role in many biological processes, including metabolism (Han et al., 2016), detoxification (Grant, 1991), and the production of various hormones and proteins (Canbay et al., 2007).

In the liver, the sources of several neurotrophins and their receptors (including BDNF and Trk-B) are hepatic stellate cells (Cassiman et al., 2001) and hepatocytes (Genzer et al., 2017), which are the main cell types in this organ. The hepatic BDNF/Trk-B axis has been shown to play a key role in maintaining liver innervation and haematopoietic function (García-Suárez et al., 2006) and biliary remodelling during cholestasis (Vivacqua et al., 2014). In addition, it has been shown that hepatic BDNF levels can be useful for diagnosing liver function in patients with cirrhosis (Shu et al., 2019), and associated with liver fibrosis in alcohol use disorders (Girard et al., 2021). Studies in rodents and humans (Chaldakov et al., 2004; Motamedi et al., 2017) have shown that low levels of circulating BDNF are associated with metabolic dysfunctions such as obesity, metabolic syndrome and related disorders (e.g., diabetes, cardiovascular disease). Experiments on mice lacking the gene encoding BDNF (Lyons et al., 1999; Kernie et al., 2000) have shown that exogenous administration of BDNF can reverse the effects of metabolic syndrome. Furthermore, Genzer et al. (2017) showed that BDNF binding to specific receptors in rat liver hepatocytes activates catabolic pathways, such as fatty acid oxidation, in parallel with inhibition of gluconeogenesis and increased glycogen storage. All these reports indicate an important regulatory role for BDNF/TrkB axis in liver tissue (Hang et al., 2021).

It is well known that physical activity, especially of endurance nature, can modulate serum/plasma concentration of BDNF (Szuhany et al., 2015; Dinoff et al., 2016). The results of most human studies suggest considerable elevation of BDNF concentration in blood serum after acute endurance training lasting several weeks (Tang et al., 2008; Rasmussen et al., 2009; Arazi et al., 2021; Ribeiro et al., 2021). Moreover, Belviranlı and Okudan (2018) showed that voluntary wheel training in rats increased not only BDNF levels in plasma but also in liver tissue. In addition, another study in rats showed that the application of moderate to high-intensity training leads to both increased plasma BDNF levels and insulin tolerance, which are suppressed by daily intraperitoneal administration of the Trk-B blocker (Jiménez-Maldonado et al., 2014).

Hence, existing evidence suggests that there may be a strong association between BDNF/Trk-B axis and metabolic products in the liver and physical activity. The purpose of this study was to investigate the potential link between changes in the hepatic BDNF/TrkB axis induced by different BDNF genotype (normal vs. knockout rats) and endurance training (trained vs. untrained rats) and the activity and concentration of metabolic biomarkers in the liver. Knockout rats were selected due to their reduced serum BDNF levels ranging from 20% (Grzelak et al., 2023) to 73% (Harris et al., 2016), while 5-week endurance training was used to increase BDNF levels in the rat liver.

For this reason, two types of rats were used in this study: wild-type and BDNF heterozygous knockout (HET, SD-Bdnf), some of which were subjected to an endurance training procedure. The following four groups of rats were studied in the experiments conducted: 1) with typical hepatic BDNF levels (control with a normal genotype, Bdnf+/+), 2) with altered hepatic BDNF levels [control with BDNF knockout genotype (HET, SD-BDNF, Bdnf+/−)], and (3, 4) trained with normal (Bdnf+/+T) and knockout genotype (Bdnf+/-T) that underwent 5-week endurance training procedure.

In liver homogenates of these rats levels of BDNF, Trk-B as well as concentration/activity of some lipids: total cholesterol (CHOL), low-density lipoprotein (LDL) and triglycerides (TG), enzymes: lactate dehydrogenase (LDH), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) and hormones: insulin (INS) and leptin (LEPT) were measured. In addition, levels of hepatic interleukin-6 (IL-6) which is an essential factor indicating liver homeostasis, regeneration, infection defence and fine tuning of metabolic functions (Schmidt-Arras and Rose-John, 2016) were also measured in all four studied groups of animals.

Relationships between hepatic amounts of both BDNF/Trk-B and the aforementioned specific metabolic factors were determined separately for each group and then compared among all studied populations. We hypothesised that this approach would allow us to demonstrate how genotype and physical activity affect the association between BDNF/Trk-B axis and metabolic factors in the rat liver.

In the current study, there was an increase in body and liver weight, as well as a decrease in liver BDNF and Trk-B concentrations in BDNF knockout rats (Bdnf+/− and Bdnf+/−T) compared to normal genotype rats (Bdnf+/+ and Bdnf+/+T) (Figures 1A,B; Table 1). Moreover, our previous research (Grzelak et al., 2023) demonstrated that BDNF knockout rats had significantly lower serum BDNF levels than rats with normal genotype. Taken together, these results showed that rats with hemizygous deletion of the BDNF gene exhibited reduced serum BDNF levels and impaired hepatic BDNF/TrkB signaling in the liver. These both findings provide evidence to support the use of these rats as a genetic model to study metabolic syndrome. Moreover, this seems to be consistent with the report by Belviranli and Okudan (2018), which suggests a strong connection between BDNF levels in the liver and circulating blood.

Additionally, our study indicated that reduced BDNF and Trk-B signaling in the liver, resulting from the absence of one BDNF allele, has a particularly important impact on lipids metabolism in hepatocytes. Differences in lipid concentrations, including CHOL, LDL and TG, were evident when comparing the knockout with normal genotype rat populations. In particular, hepatic CHOL and LDL levels were significantly reduced in BDNF knockout rats (Bdnf+/− and Bdnf+/−T) compared to those with normal genotypes (Bdnf+/+ and Bdnf+/+T). CHOL is synthesised directly by hepatocytes in the liver (Zhou and Sun, 2021), while LDL is formed in the liver from particles of very low-density lipoproteins (Feingold, 2022). LDL plays a key role in transporting cholesterol from the liver to peripheral cells, according to previous research (Eckardstein, 2020). Moreover, several studies have demonstrated an inverse relationship between the concentration of CHOL (Abdel-Gayoum and Ahmida, 2017) and LDL (Ogunmoyole et al., 2019) in the liver and their corresponding levels in serum. Thus, our results suggest that CHOL accumulation and storage in the liver may be impaired in rats with hemizygous BDNF deletion. This also indicates that knockout rats may be subject to CHOL accumulation in the arteries, which may lead to the formation of atherosclerotic plaques—a major contributor to atherosclerosis (Subczynski and Pasenkiewicz-Gierula, 2020). It is therefore reasonable that knockout rats may have an increased risk of developing cardiovascular. Such a suggestion is in line with previous human studies that have linked reduced serum or plasma BDNF levels to a higher incidence of various cardiovascular diseases (Chaldakov et al., 2004; Sustar et al., 2019).

We also found that liver TGs levels were significantly higher in knockout rats compared to normal genotype rats. TGs are obtained from dietary sources as well as synthesised in the liver as part of the process of lipid metabolism, where they are stored in hepatocytes (Alves-Bezerra and Cohen, 2017). Elevated levels of TGs in the liver caused by increase food intake, as observed in knockout rats, can lead to increase liver weight and eventually induce fatty liver (Garnol et al., 2016). This may explain the considerable increase in liver weight observed in knockout rats. Furthermore, it can be supposed that elevated hepatic TGs levels may be responsible for increased body weight in these rats, as excess TGs are stored in adipose tissue, leading to obesity (Ahmadian et al., 2009). Based on all the aforementioned observations, it can be strongly inferred that the significant reduction in BDNF/Trk-B signaling in the liver, observed in genetically modified rats, plays a key role in inducing substantial changes in hepatic lipid metabolism. This finding is also supported by the study conducted by Tsuchida et al. (2002), which demonstrated that subcutaneous administration of BDNF could lead to significant reductions in liver weight, TG concentrations, and the development of fatty liver in obese diabetic mice. Therefore, it can be concluded that a decrease in BDNF/Trk-B signaling in the liver is a crucial factor that contributes to the onset of metabolic syndrome symptoms.

The moderate-intensity endurance training applied in our research did not alter either the BDNF and Trk-B content (Figure 1 A and B; Table 1) or the CHOL and LDL concentrations in the liver of the studied rats. Long-term aerobic exercise, particularly at high intensity, is recommended as an effective therapy for improving lipid profiles (Mann et al., 2014) or to counteract metabolic syndrome (Mrówczyński, 2019). However, 5-weeks endurance training of graduate increasing intensity, utilized in the present study, did not result in elevated levels of BDNF and Trk-B in the liver, suggesting that it may have been insufficient to induce significant improvement in the lipid profile. It is important to emphasise that our previous research has shown that training at such intensity can lead to an increase in both BDNF and Trk-B levels in some fast hindlimb muscles (Grzelak et al., 2023). However, there is no a significant effect on circulating levels of BDNF what seems to be in line with results of Gaitan et al. (2021) who found no increase of BDNF level in the blood of adult humans even after 26 weeks of aerobic exercise. However, it cannot be ruled out that the implementation of high-intensity or prolonged endurance activity may evoke increase in BDNF levels in both the serum and the liver which could improve of the lipid profile. It has been reported that high-intensity interval training has a particularly desirable effect on lipid metabolism by reducing their concentrations in the blood, including levels of CHOL, TGs and others (Cao et al., 2022).

Knockout rats (Bdnf+/− and Bdnf+/−T) showed a decrease in liver metabolic enzyme activities, specifically ALAT and GGT compared to rats with a normal genotype (Bdnf+/+ and Bdnf+/+T). ALAT is a specific liver enzyme crucial for amino acid metabolism (Glinghammar et al., 2009) while GGT is a key enzyme in the metabolism of glutathione (Hanigan et al., 2015). Higher activities of these enzymes in the liver indicate more efficient enzyme function, as their levels in the liver are inversely proportional to those in the blood. However, liver disorders can cause the release of these enzymes from hepatocyte cytoplasm into the blood, where they serve as indicators of impaired metabolic function in liver disorders (Vermeulen et al., 1992; Deoras et al., 1997; Ogunmoyole et al., 2019). ALAT and GGT levels are routinely measured in serum or plasma to investigate liver disease (Green and Flamm, 2002; Carobene et al., 2013) due to these both markers are particularly relevant in the context of pathogenesis of various liver diseases related to fat accumulation in liver such as obesity, metabolic syndrome and the cardiovascular disease (Sookoian and Pirola, 2015). For example, elevated levels of these markers have been observed in children as a consequence of liver damage associated with overweight and obesity (Johansen et al., 2020). Therefore, it is reasonable to speculate that knockout rats would exhibit increased activities of ALAT and GGT in circulation, indicating disruptions in metabolic processes within the liver.

Our results demonstrated that endurance training led to an increase in hepatic ALAT activity and a significant elevation in ASAT activity in knockout rats (Bdnf+/− and Bdnf+/−T). However, this effect was mainly observed in the group of trained knockout rats (Figures 5B,C,E; Table 1). It is well-established that endurance training can improve liver function and reduce liver enzyme levels in the bloodstream, such as ALAT and ASAT, particularly in individuals with obesity or metabolic syndrome (Haukeland et al., 2008; Skrypnik et al., 2016). Therefore, our findings demonstrate an increase in ALAT and ASAT levels in the liver of knockout rats, confirmed previous observations. The implemented training regimen led to a significant reduction in hepatic LDH activity in the trained rat population (Bdnf+/+T and Bdnf+/−T), an enzyme crucial for cellular metabolism, particularly anaerobic glycolysis (Farhana and Lappin, 2023). Studies by Lupa et al. (1994), have previously demonstrated a decrease in hepatic LDH activity in response to endurance training in young rats. This finding suggests that endurance training may have a positive impact on liver health and metabolic function, potentially reducing the risk of liver-related disorders. Importantly, our results indicate that the decrease in hepatic LDH activity observed after endurance training is independent of the BDNF genotype.

No significant differences were observed in INS and LEPT hormone concentrations in the livers of rats with knockout (Bdnf+/− and Bdnf+/−T) and normal (Bdnf+/+ and Bdnf+/+T) genotypes. These findings indicate that liver functions, including the regulation of glucose metabolism (Chadt and Al-Hasani, 2020) and maintenance of energy balance (Martínez-Uña et al., 2020) remain unchanged in knockout rats. This can be attributed to the fact that this liver function is closely related to hormones produced by other organs and transported to the liver via the circulation. However, it is worth noting that liver INS and LEPT levels were significantly higher after endurance training in our study. This observation is therefore consistent with previous reports emphasising the importance of regular exercise in regulating body weight and blood sugar levels in humans (Karacabey, 2009). Interestingly, our results indicate that these training effects are not dependent on BDNF genotypes, as observed for LDH activity.

It is important to note that a significant reduction in BDNF and Trk-B levels in liver of knockout rats (Bdnf+/− and Bdnf+/−T) seems to be also associated with a higher concentration of hepatic IL-6 compared to rats with the normal genotype (Bdnf+/+ and Bdnf+/+T). However, such difference is caused by significant increase in IL-6 concentration in the group of trained animals with BDNF gene deficiency (see Figure 2 and Table 1). Cressman et al. (1996) discovered that even a single dose of IL-6 can restore gene expression in mice lacking IL-6 gene, resulting in hepatocyte proliferation and inhibition of liver damage. Garnol et al. (2016) observed heightened levels of IL-6 in the liver after partial hepatectomy, considering it as an essential factor for initiating liver regeneration. Therefore, elevated levels of IL-6 play a crucial role in promoting hepatocyte proliferation and are commonly regarded as a key factor in driving liver regeneration. Accordingly, our findings have demonstrated a significant increase in hepatic IL-6 levels, particularly in the trained knockout rat group (Figure 2; Table 1). This suggests that endurance training has the potential to enhance hepatocyte proliferation and initiate liver regeneration processes, especially in animals with reduced levels of BDNF and Trk-B in the liver. Additionally, it is well-established that muscle cells are a major source of IL-6 production in response to exercise. Steinbacher and Eckl (2015) proposed that IL-6 produced by muscles during exercise exerts an anti-inflammatory effect, leading to a reduction in pro-inflammatory cytokine levels. Moreover, it has been observed that muscles release a substantial amount of IL-6 into the bloodstream even after a single exercise session (Fischer, 2006), and this IL-6 can be captured by the liver (Febbraio et al., 2003). Therefore, our results suggest that the production of IL-6 during training plays a crucial role in promoting liver regeneration, particularly in animals with metabolic liver disorders like knockout rats. Furthermore, the elevated IL-6 levels in BDNF-deficient rats may be a potential factor contributing to the increase in ALAT and ASAT activity. This suggests that IL-6 might play a regenerative role by enhancing the efficiency of some metabolic pathways.

The study revealed that BDNF knockout rats loss exhibited a significant reduction in BDNF/Trk-B content within the liver. This reduction was accompanied by lower levels of lipids, such as CHOL and LDL, and a simultaneous increase in TGs levels. In addition, there was a reduction in the activity of two liver enzymes—ALAT and GGT. These alterations in lipid metabolism and enzyme activity contributed to increase in obesity, as evidenced by elevated liver weight and body weight in the rats. Our findings suggest that a reduction in BDNF/Trk-B signaling contributes to changes in lipid metabolism that lead to hepatic steatosis and obesity, which are symptoms of the metabolic syndrome.

Moderate endurance training induced an increase in hepatic ALAT and ASAT activity in knockout rats, suggesting improved metabolic pathway efficiency in the liver. Furthermore, a significant increase in hepatic IL-6 levels was demonstrated in trained animals with the BDNF knockout genotype, suggesting that the initiation of liver regeneration is mediated by IL-6 produced by contracting skeletal muscle. It cannot be excluded that it is responsible for improving the efficiency of metabolic pathways by increasing ALAT and ASAT activity. On the other hand, the effect of endurance exercise on LEPT and INS concentrations, as well as LDH activity, was not dependent on a specific BDNF genotype.

Future studies should focus on investigating the effects of the BDNF/Trk-B axis on the pathways of the above-mentioned enzymes, which play a key role in liver metabolic functions. Furthermore, upcoming studies should aim to provide a more complex analysis of the role of IL-6 in liver regeneration.

Measurement of the biomarkers tested in blood was not possible due to the fact that blood samples were collected to assess neurotrophin levels, as shown in our previous work performed on the same group of animals (Grzelak et al., 2023). The volume of blood samples collected was also limited due to the in vivo electrophysiology experiment being conducted in parallel. In addition, we did not conducted immunochemistry research in liver.