In contrast to the documented beneficial effect of BCAA supplementation in cell culture models, higher BCAA circulatory levels were found in NAFLD patients [47,48,49]. The rise in BCAA levels has also been positively associated with insulin resistance (IR) and total cholesterol and glycerol levels in type 2 diabetes (T2D) and obese patients [50,51]. Since T2D and obesity are known to be risk factors for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) [52], this raised the question of whether the BCAA level is influenced by these underlying risk factors.

A large-scale clinical study on NAFLD subjects without T2D provided insight into the synergistic effect of NAFLD and the elevated BCAA levels on the development of type 2 diabetes. The total plasma BCAAs were positively correlated with a high fatty liver index (FLI), which was calculated from the levels of blood triglycerides, blood gamma-glutamyl-transferase, BMI, and waist circumference. In the 7.3-year follow-up analysis, nearly 20% of patients with elevated FLI were found to develop T2D. This elevation was suggested to be linked with the impaired hepatic mitochondrial function and increased mitochondrial lipid β-oxidation in NAFLD [47]. Although there is no causative relationship between T2D and the high BCAA circulatory levels in NAFLD, BCAA levels were positively correlated with T2D incidence [53]. The activity and expression of BCAA catabolic enzymes were previously reported to be altered in pathologic conditions involving metabolic disorders, in which they are downregulated in patients with type 2 diabetes [54].

Patients with both NAFLD and obesity have a higher BCAA circulatory level than non-obese NAFLD patients. In an Italian cohort with non-obese NAFLD patients, obese NAFLD patients, and healthy subjects, the rise in BCAA levels was more profound in the obese NAFLD group when compared to the healthy control, while valine and isoleucine levels were only significantly higher in the obese NAFLD group [55]. These findings aligned with observations from a study on NAFLD patients with severe obesity, where plasma BCAAs were positively correlated with steatosis stages and liver fat content [10]. A recent study illustrated the metabolic differences between obese subjects with and without progression to NAFLD. BCAAs were found to be increased in NAFLD-obese patients, but not in obese or lean healthy subjects. Further univariate analysis identified isoleucine as one of the factors that discriminates between obese patients vs. obese NAFLD patients. This study highlighted the crucial association between impaired BCAA metabolism and the manifestation of NAFLD [56]. In obese NAFLD patients, a higher consumption of BCAAs was associated with worse hepatic health in terms of liver fat content [57].

The elevated BCAAs levels were also found to contribute to IR. IR was also found to be positively correlated with the rise in BCAA levels in NAFLD and fibrosis patients [47,58]. The circulatory levels of BCAAs were positively correlated with the insulin-resistance index, HOMA-IR [58]. It was suggested that BCAAs may lead to IR through activating the mTORC1 signaling pathway, which produces the chronic phosphorylation of mTOR and IRS1_Ser307 [9]. However, recent findings found that an increase in mTORC1 signaling from BCAA consumption alone would not affect insulin sensitivity in the long term [59].

The rise in plasma BCAA levels in NAFLD patients was also found to be sex-dependent. Male subjects were found to have significantly higher BCAA levels than female subjects. Plasma BCAA levels in female subjects were correlated with NAFLD and fibrosis stages, while the opposite result was found in male subjects. Leucine and valine were inversely correlated with NAFLD stages in males. Nevertheless, without considering the gender differences, leucine and isoleucine were significantly associated with NAFLD stages [60].

Since circulatory BCAA levels were consistently found to be significantly increased in liver diseases, the possibility of using circulating BCAAs’ concentration as a diagnostic tool was suggested. A study on obese children found a high area under the curve (AUC), 0.92 (95% confidence interval 0.83–1.00), for using BCAA to discriminate between severe steatosis and a healthy obese subject, while an AUC of 0.82 (95% CI 0.67–0.97) could be used for the discrimination of any steatosis [10]. The elevation in BCAAs was not limited to their systematic levels. BCAA level was elevated in liver tissue in NASH patients vs. healthy subjects. However, the liver BCAA levels were found to be unchanged in simple steatosis/NAFLD patients vs. healthy subjects [61]. Although the study only included data from a limited number of patients, it suggested that the change in systematic levels is aligned with the local level in NASH patients, which might contribute to the activation of the aforementioned mTOR pathway [62].

Plasma BCAA levels were found to be significantly increased and have been identified as a biomarker of progression to HCC [63]. A low BCAAs/tyrosine ratio (≤4.4) was found to be a prognostic factor for HCC patients with chronic liver diseases. The BCAAs/tyrosine ratio was significantly negatively correlated with the liver function marker, albumin albumin-bilirubin (ALBI) [64,65].

The rise in BCAAs was not limited to their systemic level. A recent study found an increase in tissue BCAA level in HCC patients with severe fibrosis and cirrhosis. In 52 paired HCC tumor and nontumor tissues, BCAAs were found to be elevated in HCC tissue when compared with adjacent non-tumoral tissues [66]. The same finding was also found in another study with paired HCC tumor and nontumor tissues from 48 of their patients [67]. The team took a further look into the transcriptomic profile of HCC tumors and adjacent tissues of patients in both Singapore General Hospital and data from the Cancer Genome Atlas [67]. They found that the BCAA degradation pathway was a significantly enriched KEGG pathway in the tumors of both their 48 HCC patients and the HCC cohort from the Cancer Genome Atlas. More than 40 BCAA catabolic enzymes, including BCKDH and acyl-CoA dehydrogenase enzymes (ACADs), were suppressed in tumors. The accumulation of BCAA in the tumor activated mTORC1 signaling. A higher expression of the catabolic enzyme of BCAA was, therefore, linked to better survivability for patients. The group further investigated the impact of BCAAs on tumor development by using diethylnitrosamine (DEN)-injected high-fat diet-fed mice. Tumor number and size were elevated in the BCAA-fed group. Consistent with their findings in human subjects, BCAA catabolic enzymes were suppressed in BCAAs/DEN-injected mice, while they were enhanced in control mice fed with BCAAs.

In livers of HCC patients, and animal models, including high-fat diet-induced obesity and HCC tumor models, BCKDH activity and expression were found to be downregulated, and BCKDH kinase (BCKDK), the enzyme responsible for suppressing the activity of BCKDH, was found to be upregulated [23]. The consequence of this is an inability to fully oxidize BCKAs. The accumulation of BCKAs, especially from valine and isoleucine metabolism, may lead to mitochondrial dysfunction. It was previously reported that increased BCKA levels suppress the expression of succinate dehydrogenase, which affects the TCA cycle and the electron transport chain [68]. As a result, acylcarnitine byproducts were formed instead of the complete TCA cycle, and this elevation of plasma acylcarnitine is considered a marker of IR, type 2 diabetes, and cardiovascular diseases [23]. Meanwhile, in an animal and human HCC tumor model, the dysregulation of BCAA oxidation was found to induce chronic mTORC1 activation [67].

Despite the association between elevated blood BCAA levels and negative conditions in liver diseases, the consumption of BCAA supplements was previously linked to a beneficial outcome in various liver diseases, especially during advanced fibrosis or cirrhosis, and especially hepatic encephalopathy. BCAA supplementation is recommended to cirrhotic patients according to the guidelines of the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) [69].

In three separate studies, the supplementation of BCAAs in the diet of patients with advanced liver cirrhosis resulted in a significant improvement in major cirrhosis-related events, including improvements in Child–Pugh (CP) score, MELD score, and/or a significantly higher number of patients with event-free survival [70,71,72]. The beneficial effect of BCAA supplementation was not limited to cirrhotic patients. BCAA supplementation was also found to be useful in preventing the occurrence of HCC in cirrhotic patients [73]. The majority of HCC patients (80–90%) were diagnosed with underlying cirrhotic conditions [74]. Although there have been few human trials on BCAA supplementation in HCC patients, increasing evidence from animal studies provides an indication of the potential beneficial effect of BCAAs.

A DEN-injected rat liver injury model showed that BCAAs significantly lowered dysplastic nodules. Although BCAAs could not prevent progression to malignant tumors, the supplementation prevented liver neoplasm lesions [17]. This effect was due to the suppression of tumor angiogenesis as a result of the low secretion of VEGF. BCAA(s) was also previously found to boost the efficacy of the chemotherapy drug, cisplatin, which is widely used for the treatment of cancers. The supplementation of leucine increased cisplatin sensitivity by activating the mTOR pathway [78].

Over the course of NAFLD/NASH progression, cirrhosis may also develop; therefore, BCAA supplementation has also been increasingly investigated to treat these diseases, and/or prevent them from progressing to cirrhosis. Although there is a lack of human studies utilizing BCAAs to treat NAFLD or NASH patients, some animal studies have pointed to a potential positive outcome of its utilization, although the results are controversial and not conclusive. In a choline-deficient, high-fat diet-induced NASH mice model, BCAA lowered serum ALT levels and hepatic triglyceride, while the liver histology showed that the lipid droplet area and fatty acid synthase (FAS) were lowered [79]. Similar results were obtained from high-fat (45%) diet NAFLD rat and obese mice models, where BCAA supplementation decreased fat accumulation and triglyceride concentration in the liver, and significantly lowered the steatosis score [46,80]. However, several studies highlighted that while BCAA supplementation reduced hepatic triglycerides, body weight, and food intake, hepatic IR could not be improved and a persistent induction of mTORC1 activation was observed, implying that the supplementation of BCAAs worsens the underlying metabolic disorder [15,37,81]. The persistent mTOR activation arose from the combination of both high-fat and BCAA supplementation, and this IR could be reversed using the mTORC1 inhibitor, rapamycin [9]. In contrast, rapamycin could not reverse high-fat diet-induced IR. Furthermore, BCAA-supplemented normal chow-feeding in rats did not induce increased mTOR activation [9]. This indicates that BCAA-high-fat-induced IR is likely to be more reversible compared to only high-fat-induced IR upon adopting a healthier diet. Furthermore, a previous survey conducted on the typical human Western diet found that the diet only contains around ~35% fat; hence, a review article suggested the use of diets with ~45% fat in rodents to confer a better rodent and human inter-study agreement [37]. Contradictory observations were found in the studies by Muyyarikkandy et al. and Zhao et al., who adopted a 60% fat rodent diet; thus, these observations may not necessarily be duplicated in humans. Indeed, a clinical trial of 102 NAFLD patients found that BCAA supplementation significantly lowered both liver disease markers (i.e., ALT and keratin-18 (K18)) and fibrosis markers [16]. With this, and the abundant evidence that BCAAs could help in liver cirrhosis, it should not be of great concern that BCAAs may exacerbate the disease condition if a healthy balanced diet is adopted during the intervention. On the other hand, the overall impression of these studies highlights the complex relationship between diet, BCAA, liver health, and IR, while also bringing attention to the gut–liver axis.

The full mechanism of how BCAAs prevent further deterioration in chronic liver diseases remains largely unclear. An explanation for this may be that the supplementation of BCAAs could elevate its catabolism via directly affecting the levels of its catabolizing enzyme. In particular, the increase in PPAR-α expression by BCAAs, through AMPK and an increase in serum-free fatty acid levels, could prevent the increase in BCKDK activity, preventing the suppression of BCKDH activity in catabolizing BCKAs [82,83]. The contribution of PPAR-α to lipid homeostasis was found to be crucial to preventing steatosis-induced NASH development [84]. It is also important to note that the loss of muscle mass, the major BCAA catabolic site, is usually accompanied by chronic liver diseases [85,86]. Improving the muscle mass [87] could potentially benefit muscle BCAA catabolism and its subsequent glutamine synthesis. The increase in plasma glutamine (GLN) was observed via the supplementation of BCAAs, along with a lowering of plasma glutamate (GLU) [88]. This increase in GLN availability was found to be beneficial to the immune system and the production of the natural antioxidant glutathione, which is beneficial to liver health [89,90].