Alteration of the serum myostatin level following L‑carnitine treatment in patients with chronic liver disease: A pilot study
- Authors:
- Published online on: August 4, 2020 https://doi.org/10.3892/ijfn.2020.4
- Article Number: 4
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Copyright: © Nakano et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Sarcopenia is the loss of muscle strength and muscle mass, which can be caused by various factors, including ageing, inflammation, malnutrition and chronic disease (1,2). Sarcopenia is observed in approximately 30% of patients with chronic liver disease and in approximately 40% of patients with liver cirrhosis (3). Sarcopenia is associated with an increased risk of falls (4) and muscle cramps (5). Sarcopenia is also a risk factor for hepatic encephalopathy (6), decompensated liver cirrhosis (7) and hepatocellular carcinoma (8). In addition, Hanai et al reported that sarcopenia impairs the prognosis of patients with liver cirrhosis (9).
One of causative factor for sarcopenia is carnitine deficiency and carnitine deficiency is frequently observed in patients with liver cirrhosis (10). Carnitine transports long-chain fatty acids into the mitochondria, leading to the production of energy through fatty acid oxidation. Carnitine deficiency is involved in the development of sarcopenia in patients with liver cirrhosis (10). On the other hand, L-carnitine treatment has been reported to suppress the loss of skeletal muscle mass in patients with liver cirrhosis (10). Furthermore, in cirrhotic patients, L-carnitine has been reported to improve hyperammonemia, which is associated with the loss of skeletal muscle mass (11). In addition, it has been demonstrated that L-carnitine treatment prevents the loss of skeletal muscle mass (12). Thus, L-carnitine treatment is beneficial for the prevention of sarcopenia in patients with liver cirrhosis. However, the mechanisms through which L-carnitine treatment affects muscle mass remain unclear.
Recently, skeletal muscle has been shown to secrete cytokines and proteoglycan peptides named ‘myokines’ by muscle contraction (13). Myokines perform various biological functions through the activation of receptors on the liver, bone, fat, brain and heart, as well as in muscle (14). Myostatin, irisin and decorin are myokines regulating muscle mass (15-17). In particular, serum levels of myostatin are known to be significantly elevated in patients with liver cirrhosis compared with healthy controls (18). In addition, higher serum myostatin levels have been shown to be associated with hyperammonemia and muscle atrophy in patients with liver cirrhosis (19,20). Moreover, high serum myostatin levels are associated with a high mortality rate of patients with liver cirrhosis (19,20). However, alterations in the serum levels of myokines following L-carnitine treatment in patients with chronic liver disease have yet to be determined, at least to the best of our knowledge. The aim of the present study was to investigate alterations in the serum levels of myokines following L-carnitine treatment in patients with chronic liver disease.
Patients and methods
Study design
The present study was a retrospective observational study that aimed to investigate alterations in the serum levels of myokines following L-carnitine treatment in patients with chronic liver disease.
Ethics
The study protocol conformed to the ethical guidelines of the Declaration of Helsinki as reflected in the prior approval granted by the Institutional Review Board of Kurume University. An opt-out approach was used to obtain informed consent from the patients, and personal information was protected during data collection. None of the patients were institutionalized.
Patients
From July, 2017 to January, 2018, 7 patients were enrolled who met following the following inclusion and exclusion criteria. The inclusion criteria were patients with liver cirrhosis who: i) Were ≥20 years of age; ii) treatment with L-carnitine for 3 months; and iii) had undergone biochemical examination. The exclusion criteria were patients with: i) Hepatocellular carcinoma; and ii) severe heart, pulmonary, renal, or brain failure.
For stratification analysis, patients were classified into the low FIB-4 index (n=3) or high FIB-4 index groups (n=4) according to the cut-off value of >3.25 of the FIB-4 index. The cut-off value of the FIB-4 index for advanced fibrosis was based on a previous study (21).
Laboratory determinations
Venous blood samples were drawn before and 12 weeks after treatment of L-carnitine. Red blood cell count, hemoglobin, white blood cell count, lymphocytes, platelet count, prothrombin activity and serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transpeptidase, total protein, albumin, total bilirubin, total cholesterol, triglycerides, α-fetoprotein, des-γ-carboxy prothrombin, blood urea nitrogen, creatinine, sodium, creatine kinase (CK) and ammonia were measured using standard clinical methods as previously described (9,11). The Child-Pugh score and FIB-4 index were calculated as previously described (22). The estimated glomerular filtration rate (eGFR) was also calculated as previously described (23).
Evaluation of myokines
In the present study, 3 myokines, namely as myostatin, irisin and decorin were evaluated. The serum levels of myostatin, irisin and decorin were evaluated by enzyme-linked immunosorbent assay (ELISA) (Irisin, recombinant-ELISA kit, EK-067-29, Phoenix Pharmaceuticals Inc.; Decorin, Human Decorin ELISA, ab99998, Abcam; Myostatin, GDF-8/Myostatin Quantikine ELISA kit, DGDF80, Research and Diagnostic Systems Inc.).
Statistical analysis
Data are expressed as the median (interquartile range), range, or number. Changes in variables before and after treatment of L-carnitine were analyzed by Wilcoxon signed rank tests. The correlation between the FIB-4 index and changes in serum myostatin level was analyzed by Spearman's rank correlation analysis. The level of statistical significance was set at P<0.05.
Results
Patient characteristics
The patient characteristics are summarized in Table I. The median age was 66 years, and the ratio of female to male was 6:1. The median body mass index was 24.7 kg/m2. Child-Pugh class B was observed in 71.4% of patients. The median level of blood ammonia and FIB-4 index were 79.5 µg/dl and 5.35, respectively. The median level of serum CK was 94.5 U/l.
Alterations in biochemical examinations before and after L-carnitine treatment
Alterations in biochemical examinations before and after L-carnitine treatment are summarized in Table I. No significant differences were observed in the serum levels of aspartate aminotransferase, and lactate dehydrogenase before and after L-carnitine treatment (Fig. 1A and B). In addition, no significant differences were observed in the blood ammonia level before and after L-carnitine treatment (Fig. 1C). The serum CK level was significantly increased after L-carnitine treatment (Fig. 2).
Alterations in serum levels of myostatin, irisin, and decorin after L-carnitine treatment
Alterations in the serum levels of, irisin, decorin and myostatin after L-carnitine treatment were investigated. No significant differences were observed in the serum levels of irisin, decorin and myostatin before and after L-carnitine treatment (Table II).
Alterations in serum levels of irisin, decorin and myostatin after L-carnitine treatment in the high FIB-4 index group
In the stratification analysis according to the FIB-4 index, patients were classified into the low FIB-4 or high FIB-4 groups based on the cut off value of >3.25 of the FIB-4 index, which corresponds to the F4 stage. No significant differences were observed in the serum levels of irisin and decorin before and after L-carnitine treatment in the high FIB-4 group (Fig. 3A and B). In the high FIB-4 group, the serum level of myostatin was significantly decreased after L-carnitine treatment (Fig. 3C).
Correlation between the FIB-4 index and changes in the serum myostatin level after L-carnitine treatment
The correlation between the FIB-4 index and changes in the serum myostatin level was analyzed by Spearman's rank correlation analysis. A significant negative correlation was observed between the FIB-4 index and changes in the serum myostatin level (Fig. 4).
Discussion
The present study investigated the association between L-carnitine and the serum levels of myostatin, irisin and decorin in patients with chronic liver disease. However, no significant differences in the serum levels of myostatin, irisin and decorin were observed before and after L-carnitine treatment in patients with chronic liver disease. A a stratification analysis was also performed according to the FIB-4 index and it was found that the serum myostatin levels were decreased after L-carnitine treatment in patients with a high FIB-4 index. Thus, L-carnitine treatment may suppress the serum myostatin level in patients with liver cirrhosis.
Blood ammonia levels did differ significantly after L-carnitine treatment in the present study. L-carnitine is known to enhance ammonia detoxification in the urea cycle by increasing N-acetylglutamate, leading to a decrease in blood ammonia levels in patients with liver cirrhosis (24). Abbasnezhad et al performed a meta-analysis on 799 patients from 9 randomized clinical trials and demonstrated that L-carnitine treatment significantly reduced the blood ammonia level (25). Thus, the results of the present study differ from those of that previous study. Although the reason for this discrepancy remains unclear, the small sample size may be a possible reason. Another possible reason may be renal dysfunction. The kidneys are involved in ammonia metabolism by removing a significant amount of ammonia in urine in cirrhotic patients with hyperammonemia (26). The meta-analysis by Abbasnezhad et al demonstrated an improvement of renal function by L-carnitine treatment (25). However, in the present study, the tendency of an increase in the serum creatinine level and a decrease in eGFR was observed during the study period. Thus, an impairment of removing ammonia in the urine is a possible reason for the blood ammonia level exhibiting no change in the present study.
The serum CK level was significantly increased by L-carnitine treatment in the present study. However, the serum aspartate aminotransferase and lactate dehydrogenase levels were not significantly increased by L-carnitine treatment, indicating that the increase in the serum CK level was not due to muscle damage. L-carnitine is known to improve fatigue and muscle cramps (27,28), which can result in an increase in physical activity, and the serum CK level is known as a maker for physical activity (29). In addition, CK is an enzyme, which catalyzes the reversible phosphorylation of creatine to phosphocreatine and of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) (30). The expression of CK is known to be increased during muscle hypertrophy in order to adapt to energy demands (30). Although the present study did not evaluate the effect of L-carnitine on muscle mass, L-carnitine has been reported to cause muscle hypertrophy (10,11). Thus, an increase in the serum CK level may be a response to an increase in physical activity and/or muscle hypertrophy in the present study.
L-carnitine has been reported to cause muscle hypertrophy even in patients with no improvement of hyperammonemia (10). The present study examined the effects L-carnitine treatment on serum myokine levels, which are associated with muscle hypertrophy. It was found that the serum level of myostatin, but not that of irisin and decorin, was decreased 12 weeks after L-carnitine treatment in patients with a high FIB-4 index. In addition, there was a significant negative correlation between the FIB-4 index and the changes in the myostatin level after L-carnitine treatment. Since myostatin is a negative regulator of muscle hypertrophy (17), L-carnitine may suppress myostatin, leading to muscle hypertrophy. The mechanisms responsible for the L-carnitine-induced suppression of myostatin remain unclear; the expression of myostatin is regulated by AMP-activated protein kinase (AMPK) (31). L-carnitine transports long-chain fatty acids from the cytosol to the mitochondrial matrix, leading to an increase in ATP production through β-oxidation (32). An increase in intracellular ATP level is known to downregulate AMPK (33). Thus, L-carnitine may suppress myostatin expression via the suppression of AMPK.
There are several limitations to the present study. First, this was a retrospective pilot study with a small sample size, which conducted in a single center. Second, the etiology and severity of liver cirrhosis vary, suggesting the heterogeneity of the enrolled subjects. Third, no information was available for dietary habits, as L-carnitine is an abundant nutrient found in red meat (34). Thus, a multi-center randomized controlled trial with information on dietary habits is required to prove the effects of L-carnitine on the serum myostatin level.
In conclusion, the present study investigated an association between L-carnitine treatment and the serum levels of myostatin, irisin and decorin in patients with chronic liver disease. No significant differences were observed in the serum levels of these 3 myokines. However, it was found that the serum level of myostatin, but not that of irisin and decorin, was decreased 12 weeks after L-carnitine treatment in patients with a high FIB-4 index. Thus, L-carnitine may downregulate myostatin, leading to improved muscle atrophy in patients with liver cirrhosis.
Acknowledgements
Not applicable.
Funding
The present study was supported by the JSPS Grant-in-Aid for Scientific Research (C) JP20K08395.
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
DN and TK participated in the study conception and design, data acquisition and interpretation, and manuscript drafting. TTs, SY, KS and RH participated in data acquisition and interpretation. TTs, SY, KS, HK and TTo participated in the analysis and interpretation of data. HK and TTo participated in revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study protocol conformed to the ethical guidelines of the Declaration of Helsinki, as reflected in the prior approval given by the institutional review board of Kurume University. An opt-out approach was used to obtain informed consent from patients, and personal information was protected during data collection. None of the patients were institutionalized.
Patient consent for publication
Not applicable.
Competing interests
TK discloses honoraria (lecture fees) from Mitsubishi Tanabe Pharma Corporation, MSD K.K, and Otsuka Pharmaceutical Co., Ltd. The other authors disclose no competing interests.
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