Low Serum Branched-chain Amino Acid and Insulin-Like Growth Factor-1 Levels Are Associated with Sarcopenia and Slow Gait Speed in Patients with Liver Cirrhosis

Branched-chain amino acid (BCAA) and insulin-like growth factor 1 (IGF-1) are essential for muscle protein synthesis. We investigated the association of serum BCAA and IGF-1 levels with sarcopenia and gait speed in 192 patients with liver cirrhosis (LC). Sarcopenia was diagnosed according to the Japan Society of Hepatology criteria. Slow gait speed was defined as <1.0 m/s. Subjects were divided into three groups based on baseline BCAA or IGF-1 levels: low (L), intermediate (I), and high (H) groups. The L-BCAA group had the highest prevalence of sarcopenia (60.4%, p < 0.001) and slow gait speed (56.3%, p = 0.008), whereas the H-BCAA group had the lowest prevalence of sarcopenia (8.5%, p < 0.001). The L-IGF-1 group showed the highest prevalence of sarcopenia (46.9%, p < 0.001), whereas the H-IGF-1 group had the lowest prevalence of sarcopenia (10.0%, p < 0.001) and slow gait speed (18.0%, p = 0.003). Using the optimal BCAA and IGF-1 cutoff values for predicting sarcopenia (372 μmol/L and 48.5 ng/mL, respectively), the sensitivity and specificity were 0.709 and 0.759 for BCAA and 0.636 and 0.715 for IGF-1, respectively. Low serum BCAA and IGF-1 levels were associated with sarcopenia and slow gait speed in patients with LC.


Introduction
Sarcopenia, defined as a loss of skeletal muscle mass and strength, is associated with health-related quality of life, physical disability, and high mortality. Sarcopenia has now become the focus of attention in patients with liver cirrhosis (LC) [1][2][3]. Given that the liver plays a vital role in protein, glucose, lipid, and energy metabolism, LC is frequently complicated by protein-energy malnutrition (PEM), which can lead to sarcopenia [1]. In real-world clinical settings, the prevalence of sarcopenia in patients with LC ranges from 16.7% to 36.1% [4][5][6][7]. Accordingly, early comprehensive assessment and treatment intervention for sarcopenia are indispensable in patients with LC.

Statistical Analysis
Continuous variables are presented as medians and interquartile ranges in parentheses. The Mann-Whitney U test was used to evaluate differences in the distribution of continuous variables between two groups. The Kruskal-Wallis test, followed by the Steel-Dwass post-hoc test, was used for multiple comparisons among the three groups. Categorical variables are presented as numbers and percentages in parentheses. The chi-squared test was used to evaluate the group differences in categorical variables. The Cochran-Armitage trend test was used to assess the association between a variable with two categories and a variable with multiple categories. Additionally, univariate and multiple logistic regression analyses were performed to identify variables that were significantly and independently associated with sarcopenia. Correlations between two continuous variables were analyzed using the Spearman's rank correlation test. To estimate the presence or absence of sarcopenia, the area under the receiver operating characteristic (ROC) curves of BCAA and IGF-1 were depicted and the optimal cutoff values were determined by the Youden index [14]. Furthermore, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Statistical analyses were performed using SPSS version 26 (IBM, Armonk, NY, USA), with a p value < 0.05 indicating statistical significance.

Patient Characteristics
Baseline clinical characteristics of the 192 patients with LC enrolled in the present study are shown in Table 1

Comparison of Clinical Characteristics between Patients with and without Sarcopenia
The prevalence of sarcopenia among the 192 patients was 28.6% (55/192; Table 1). Patients with sarcopenia were older and had a lower body mass index (BMI) than those without sarcopenia (p < 0.001 for both). Regarding biochemical parameters, the sarcopenia group had significantly lower levels of albumin (p = 0.035), IGF-1 (p < 0.001), and BCAA (p < 0.001) than the non-sarcopenia group. The sarcopenia group showed a significantly higher prevalence of slow gait speed than the non-sarcopenia group (80.0% vs. 21.2%; p < 0.001).

Factors Associated with Sarcopenia in Patients with LC
The following five variables showed a significant relationship with sarcopenia in the univariate analysis: age, BMI, albumin, IGF-1, and BCAA (Table S1). In the multivariate analysis, the following four variables were retained as independent factors associated with sarcopenia (
The prevalence of sarcopenia and slow gait speed significantly increased stepwise with a reduction in the BCAA level (p < 0.001 and p = 0.004, respectively).
Next, we assessed the correlation between serum BCAA levels and baseline characteristics using the Spearman's rank correlation test (Table S2). Serum BCAA levels were significantly correlated with the following baseline factors: BMI, Child-Pugh score, albumin, prothrombin time, IGF-1, zinc, SMI, handgrip strength, and gait speed. The correlation coefficients for sarcopenia-related variables were highest among those for the baseline characteristics.
Next, we assessed the correlation between serum IGF-1 levels and baseline characteristics (Table  S3). Serum IGF-1 levels were significantly correlated with the following baseline factors: Child-Pugh score, albumin, prothrombin time, BCAA, SMI, handgrip strength, and gait speed. The correlation coefficient for gait speed was highest among those for the baseline characteristics. Next, we assessed the correlation between serum IGF-1 levels and baseline characteristics (Table S3). Serum IGF-1 levels were significantly correlated with the following baseline factors: Child-Pugh score, albumin, prothrombin time, BCAA, SMI, handgrip strength, and gait speed. The correlation coefficient for gait speed was highest among those for the baseline characteristics.

Optimal Cutoff Values of BCAA and IGF-1 for Predicting Sarcopenia
An ROC curve analysis was performed to determine the optimal cutoff values of BCAA and IGF-1, distinguishing between sarcopenia and non-sarcopenia ( Figure 3). The area under the ROC (AUC) value for BCAA was 0.76. The BCAA cutoff value for predicting sarcopenia was 372 µmol/L, while the sensitivity, specificity, PPV, and NPV were 0.709, 0.759, 0.542, and 0.867, respectively. Similarly, the AUC value for IGF-1 was 0.72. The optimal IGF-1 cutoff value, its sensitivity, specificity, PPV, and NPV were 48.5 ng/mL, 0.636, 0.715, 0.473, and 0.831, respectively.   An ROC curve analysis was performed to determine the optimal cutoff values of BCAA and IGF-1, distinguishing between sarcopenia and non-sarcopenia (Figure 3). The area under the ROC (AUC) value for BCAA was 0.76. The BCAA cutoff value for predicting sarcopenia was 372 μmol/L, while the sensitivity, specificity, PPV, and NPV were 0.709, 0.759, 0.542, and 0.867, respectively. Similarly, the AUC value for IGF-1 was 0.72. The optimal IGF-1 cutoff value, its sensitivity, specificity, PPV, and NPV were 48.5 ng/mL, 0.636, 0.715, 0.473, and 0.831, respectively.

Discussion
The present study is the first to focus on the relationship between baseline serum BCAA/IGF-1 levels and sarcopenia/physical performance in patients with LC. In addition, this study has the advantages of demonstrating the stepwise changes in SMI and handgrip strength along with serum BCAA/IGF-1 levels and encompassing all three aspects of the muscle (mass, strength, and function) for investigation.
In this study, we demonstrated that the prevalence of sarcopenia among the patients with LC was 28.6% and confirmed our previous findings that lower serum levels of BCAA and IGF-1 were significantly and independently associated with sarcopenia in patients with LC [4]. Furthermore, on the basis of baseline serum BCAA or IGF-1 levels, we classified the patients into three groups and investigated the association of these levels with sarcopenia and slow gait speed. Intriguingly, SMI and handgrip strength values significantly decreased stepwise with a reduction in the BCAA level.

Discussion
The present study is the first to focus on the relationship between baseline serum BCAA/IGF-1 levels and sarcopenia/physical performance in patients with LC. In addition, this study has the advantages of demonstrating the stepwise changes in SMI and handgrip strength along with serum BCAA/IGF-1 levels and encompassing all three aspects of the muscle (mass, strength, and function) for investigation.
In this study, we demonstrated that the prevalence of sarcopenia among the patients with LC was 28.6% and confirmed our previous findings that lower serum levels of BCAA and IGF-1 were significantly and independently associated with sarcopenia in patients with LC [4]. Furthermore, on the basis of baseline serum BCAA or IGF-1 levels, we classified the patients into three groups and investigated the association of these levels with sarcopenia and slow gait speed. Intriguingly, SMI and handgrip strength values significantly decreased stepwise with a reduction in the BCAA level. The L-BCAA group had a significantly higher prevalence of sarcopenia and slow gait speed, whereas the H-BCAA group had a significantly lower prevalence of sarcopenia. The prevalence of sarcopenia and slow gait speed significantly increased stepwise with a reduction in the BCAA level. Similarly, SMI and handgrip strength values decreased stepwise with a reduction in the IGF-1 level. The L-IGF-1 group showed a significantly higher prevalence of sarcopenia, whereas the H-IGF-1 group had a significantly lower prevalence of sarcopenia and slow gait speed. The prevalence of sarcopenia and slow gait speed significantly increased stepwise with a reduction in the IGF-1 level. Additionally, serum BCAA and IGF-1 levels were significantly and positively correlated with SMI, handgrip strength, gait speed, and liver functional reserve including albumin and prothrombin time. These results suggest that lower serum BCAA and IGF-1 levels are closely associated with a reduction in muscle mass and strength and physical performance and could be affected by liver functional reserve in patients with LC.
Muscle mass and function are regulated by various genes and transcription factors, which are involved in protein synthesis, differentiation and proliferation of satellite cells, and proteolysis [1,9]. Notably, both BCAA (especially leucine) and IGF-1 activate the mTOR pathway via PKB/Akt and recruitment and proliferation of satellite cells, thereby promoting muscle protein synthesis and growth. Leucine-enriched BCAA supplementation has been reported to restore impaired mTOR signaling and increase autophagy, leading to reduced muscle breakdown in patients with alcoholic LC [15]. Clinical studies on community-dwelling older adults reported that the BCAA levels among subjects with sarcopenia were significantly lower than those among subjects without sarcopenia [16,17]. Other studies reported that lower IGF-1 levels were associated with sarcopenia and deteriorated physical performance in older adults [18][19][20]. Regarding proteolysis, two major ways for skeletal muscle proteolysis exist: the ubiquitin-proteasome pathway (UPP) and the autophagy system [9]. PKB/Akt signaling inhibits proteolysis via the UPP, and its inactivation and systemic inflammation activate the UPP [9]. These basic and clinical findings suggest that decreased BCAA and IGF-1 levels could contribute to the loss of muscle mass and strength and deteriorated physical performance through the unactivated mTOR pathway, suppressing satellite cell proliferation and enhancing proteolysis.
Myostatin, a cytokine belonging to the transforming growth factor-β family, is a negative regulator of satellite cell proliferation and muscle protein synthesis [1,9]. Importantly, IGF-1 signaling has dual functions in that it inhibits myostatin and activates the mTOR pathway, thereby stimulating muscle growth. Plasma myostatin levels were found to be significantly elevated in patients with LC compared with healthy controls [21]. Reportedly, higher myostatin levels were correlated with muscle mass loss and decreased BCAA to tyrosine ratio levels in patients with LC [22]. These results suggest that decreased IGF-1 and BCAA levels in patients with LC could contribute to loss of muscle mass and strength and deteriorated physical performance through the unactivated mTOR pathway and upregulated myostatin expression levels. However, the present study did not examine the myostatin levels and the association of myostatin with BCAA, IGF-1, and sarcopenia.
This study had some limitations. First, we did not assess the patients' nutritional intake and physical exercise. Leucine-enriched nutrients with resistance exercise activate the mTOR signaling pathway and protein synthesis in skeletal muscle [23]. BCAA supplementation and walking exercise improve muscle volume and strength in patients with LC [24]. Therefore, a large-scale trial of nutritional and exercise interventions is needed to establish a treatment strategy for sarcopenia in patients with LC, especially those with low serum BCAA and IGF-1 levels. Second, patients with refractory ascites who are potentially susceptible to sarcopenia were excluded from the present study because of the unreliability of the BIA method [25]. Although SMI values estimated by the BIA method are strongly correlated with those measured using a computed tomography method with dedicated software, the former could overestimate SMI under massive ascites and thus requires careful interpretation in such cases [1]. Finally, the present study included 36 patients receiving BCAA supplementation, which could affect the BCAA levels. However, one-third of these patients were in the L-BCAA group, and there was no significant difference in the rate of BCAA supplementation among the three groups. Therefore, it could be stated that BCAA supplementation did not affect the BCAA levels in the present study (Tables 1 and 3).

Conclusions
In the present study, we demonstrated that baseline serum BCAA and IGF-1 levels were associated with sarcopenia and slow gait speed, and that they may be surrogate markers for predicting these complications in patients with LC. Comprehensive assessment including serum BCAA and IGF-1 levels and early and effective treatment intervention are crucial in patients with LC.
Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0383/9/10/3239/s1, Figure S1: Classification based on baseline serum BCAA levels, Figure S2: Classification based on baseline serum IGF-1 levels, Figure S3: Comparison of clinical characteristics among the L-BCAA, I-BCAA, and H-BCAA groups according to gender, Figure S4: Comparison of clinical characteristics among the L-IGF-1, I-IGF-1, and H-IGF-1 groups according to gender, Table S1: Univariate analysis for factors associated with sarcopenia, Table S2: Correlation between serum BCAA concentrations and baseline characteristics, Table S3: Correlation between serum IGF-1 concentrations and baseline characteristics.
Author Contributions: C.S. participated in the conception and design of the study. C.S., T.K., M.N., T.O., Y.T., and A.T. performed the acquisition, analysis, and interpretation of the data. C.S. and A.T. drafted the manuscript. M.S. and A.T. interpreted the data and revised the manuscript. A.T. substantively revised and completed the manuscript. All authors read and approved the final version of the manuscript.