Assessment of skeletal muscle alterations and circulating myokines in metabolic dysfunction-associated steatotic liver disease: A cross-sectional study

Abstract

BACKGROUND

Skeletal muscle alterations (SMAs) are being increasingly recognized in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and appear to be associated with deleterious outcomes in these patients. However, their actual prevalence and pathophysiology remain to be elucidated.

AIM

To determine the prevalence of SMAs and to assess the significance of circulating myokines as biomarkers in patients with MASLD.

METHODS

Skeletal muscle strength and muscle mass were measured in a cross-sectional study in a cohort of 62 patients fulfilling MASLD criteria, recruited from the outpatient clinics of a tertiary level hospital. The degree of fibrosis and liver steatosis was studied using abdominal ultrasound and transitional elastography. Anthropometric and metabolic characteristics as well as serum levels of different myokines were also determined in the MASLD cohort. Statistical analysis was performed comparing results according to liver fibrosis and steatosis.

RESULTS

No significant differences were found in both skeletal muscle strength and skeletal muscle mass in patients with MASLD between different stages of liver fibrosis. Interestingly, serum levels of fibroblast growth factor-21 (FGF21) were significantly higher in patients with MASLD with advanced hepatic fibrosis (F3-F4) than in those with lower fibrosis stages (F0-F2) (197.49 ± 198.27 pg/mL vs 95.62 ± 83.67 pg/mL; P = 0.049). In addition, patients with MASLD with severe hepatosteatosis (S3) exhibited significantly higher serum levels of irisin (1116.87 ± 1161.86 pg/mL) than those with lower grades (S1-S2) (385.21 ± 375.98 pg/mL; P = 0.001).

CONCLUSION

SMAs were uncommon in the patients with MASLD studied. Higher serum levels of irisin and FGF21 were detected in patients with advanced liver steatosis and fibrosis, respectively, with potential implications as biomarkers.

Key Words: Skeletal muscle alterations; Myokines; Metabolic dysfunction-associated steatotic liver disease; Liver fibrosis; Hepatosteatosis

Core Tip: Skeletal muscle alterations (SMAs) share pathophysiology mechanisms with metabolic dysfunction-associated steatotic liver disease (MASLD), in which inflammation, insulin resistance, and physical inactivity are key factors. Previous studies describe these alterations, especially reduced muscle function, in patients with a higher degree of liver fibrosis and steatosis. We determined the prevalence of SMAs in patients diagnosed with MASLD in an outpatient setting in Spain, and analyzed the myokines as potential biomarkers of inflammation. We did not find significant SMAs, but found a marked increase in serum levels of irisin and fibroblast growth factor 21 in advanced disease stages, which deserves further assessment.

INTRODUCTION

There are clear-cut epidemiological estimates showing that metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as nonalcoholic fatty liver disease[1], is currently the most prevalent chronic liver disease globally. The new definition of MASLD includes the presence of liver steatosis along with cardiometabolic risk factors, such as obesity and type 2 diabetes among others[2]. The histological spectrum remains unchanged with the new disease definition ranging from simple steatosis to steatohepatitis. Steatohepatitis, which is now termed metabolic dysfunction-associated steatohepatitis (MASH), could lead to liver fibrosis and eventually to cirrhosis. Previously reported data revealed that, in patients with MASH, higher stages of liver fibrosis are associated with increased risk of liver-related morbidity and mortality with extensive multi-organ involvement, mainly affecting the kidneys and lungs, but also the pancreas and skeletal muscle[3]. The interplay between adipose tissue, skeletal muscle, and the liver has long been recognized as a pivotal factor in the pathogenesis and natural course of MASLD; however, the research in this field has increased only in recent years, with the aim to develop preventive measures and therapeutic interventions.

Sarcopenia is, a progressive disorder characterized by skeletal muscle alterations (SMAs) that include loss of muscle mass, loss of muscle strength or both; a significant number of meta-analyses and clinical studies have demonstrated a strong association between sarcopenia and MASLD[4,5]. The development of both conditions share a similar pathophysiology, in which inflammation, insulin resistance (IR), and physical inactivity are key factors[6,7]. In patients with MASLD, sarcopenia is a potential risk factor for the development and progression of liver fibrosis. The presence of sarcopenia in this context appears to accelerate the evolution from simple steatosis to MASH and fibrosis[8,9]. The reported prevalence of sarcopenia in patients with MASLD is approximately 46% in patients with fibrosis, and approximately 25% in those without fibrosis, with variability across studies due to differences in sarcopenia definition[10,11].

Skeletal muscle, as adipose tissue, is a target organ for insulin action. IR, highly prevalent in patients with obesity, leads to heightened protein catabolism and diminished protein synthesis in myocytes resulting in muscle loss. In adipose tissue it induces lipolysis, releasing free fatty acids and promoting a proinflammatory state known as lipotoxicity[12].

Sarcopenia, can contribute to IR regardless of total body fat. In addition, its association with obesity creates an environment suitable not only for the development of IR, but also for glucose dysregulation, lipotoxicity, and a pro-inflammatory state that ultimately triggers steatohepatitis and liver fibrogenesis[13]. Muscle-secreted cytokines, named myokines, and several signaling molecules constitute the basis of the molecular cross talk along the muscle-liver-adipose tissue axis[14]. In this regard, systemic inflammation has recently emerged as a potential link between sarcopenia and MASH, since both conditions share a proinflammatory cytokine profile[15]. Myokines appear to activate hepatic stellate cells, trigger liver fibrosis and protein catabolism, and lead to muscle loss[8,11].

To date, the majority of published clinical studies have focused on large Asian cohorts with a fatty liver disease phenotype (“lean” MASLD with body mass index [BMI] < 25 kg/m²) distinct from the Western population[16-18].

Sarcopenia is defined by a reduction in muscle mass and strength and/or impaired muscle function[19]. In patients with sarcopenia, a worse prognosis appears to be associated with reduced muscle strength and/or muscle function rather than with reduced mass. In fact, in clinical practice, assessment of muscle strength and function seems more useful for monitoring since sarcopenia may present with a “sick muscle” due to myosteatosis[20]. Myosteatosis is an early manifestation of MASLD caused by ectopic fat accumulation in skeletal muscle when available lipids exceed the adipose tissue storage capacity. This process may even affect individuals who do not meet anthropometric criteria for sarcopenia or obesity but present lower muscle strength and function[21].

Given the aforementioned context, the present study determined the prevalence of SMAs (low muscle strength and/or low muscle mass) in patients with MASLD and measured the serum levels of myokines in these patients, assessing the significance of circulating myokines as potential biomarkers of SMAs in patients with MASLD.

MATERIALS AND METHODS

Study population

The study flowchart is depicted in Figure 1. Briefly, we initially recruited 80 patients with clinically suspected fatty liver but after excluding patients based on varied reasons shown in Figure 1, the study cohort included 62 patients. They fulfilled the recently described criteria for diagnosis of MASLD: Evidence of hepatic fat accumulation (steatosis) in abdominal ultrasound and one of the following three criteria: Overweight/obesity, type 2 diabetes mellitus, or the presence of at least two metabolic risk abnormalities[2,17]. None of these patients had alcohol consumption higher than 140 g/week for males or 70 g/week for females[22]; showed analytical evidence of iron and copper overload; were seropositive for autoantibodies and/or for hepatitis B virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV); or were treated with potentially hepatotoxic drugs.

Figure 1 Study flowchart. F0-F2: Stages of liver fibrosis from 0 to 2; F3-F4: Stages of liver fibrosis from 3 to 4; GLP1: Glucagon-like peptide 1.

Clinical and laboratory assessment

Clinical examination included a detailed interview about lifestyle habits with special emphasis on both alcohol intake and medications use, history of known diabetes and arterial hypertension, as well as measurements of weight, height, blood pressure and waist and hip perimeters. BMI was calculated as the weight (kg) divided by the square root of height (m). Obesity was defined as a BMI ≥ 30 kg/m². Waist circumference was measured at the midpoint between the last rib and the iliac crest, defining central obesity as a waist circumference ≥ 102 cm in men and ≥ 88 cm in women. After a 12-hour overnight fast, venous blood samples of each participant were obtained to test serum levels of liver enzymes, metabolic, and inflammatory parameters using routine laboratory methods. In addition, plasma insulin was determined with a chemiluminescent microparticle immunoassay (ARCHITECT insulin; Abbot Park, IL, United States). IR was calculated with the homeostasis model assessment (HOMA) method[23]. Antibodies against HCV, HIV and hepatitis B virus were tested by immunoenzymatic assays (Murex, Dartford, United Kingdom).

Liver ultrasound imaging

Liver was examined with abdominal ultrasound by hepatologists experienced in the technique using an ultrasound medical device (Aplio MX Canon, Madrid, Spain) with a probe frequency of 3.5 MHz after overnight fasting. The study was carried out with the patient in the supine position, following the costal edge in the longitudinal sections and using the intercostal route in cases of significant obesity. Liver steatosis was defined as hyperechogenicity as compared to the right kidney parenchyma, distal attenuation, and the presence of areas of focal sparing. The degree of steatosis was subjectively classified as mild, moderate, and severe[24,25].

Transient elastography and controlled attenuation parameter

The FibroScan^® (Echosens, Paris, France) was used to perform transient elastography (TE) to assess liver stiffness (LS) along with controlled attenuation parameter (CAP) measurements to estimate steatosis as previously detailed[26]. Only values with at least 10 valid measurements, a success rate of at least 60%, and an interquartile range-to-median ratio of < 30% were considered reliable as suggested by previous studies[27].

TE was performed on the right lobe of the liver through the intercostal space with the participant lying in the dorsal decubitus position with the right arm in maximal abduction. All TE examinations were performed between the fifth and seventh intercostal spaces, at the mid-to-anterior axillary line. Data on the degree of liver steatosis were collected and expressed as CAP values. CAP measures the ultrasonic attenuation of hepatic steatosis at 3.5 MHz using signals acquired by TE and is calculated together with the LS value. In addition, patients with alanine aminotransferase or aspartate aminotransferase values 5-fold higher than the upper normal limits at the time of TE were excluded from the analysis due to possible overestimation of TE values as demonstrated in previous studies[28]. In the present study, the thresholds to stratify fibrosis stages were defined as follows: F2 > 8.2 kPa; F3 > 9.6 kPa; F4 > 10.7 kPa[24]. The thresholds of CAP values used to define the different grades of steatosis were: S1: 260 dB/m; S2: 285 dB/m; and S3: 294 dB/m[26].

Measurement of handgrip strength

Handgrip strength was recorded in the patient’s non-dominant hand as the average of three attempts using an electronic Jamar Hand Dynamometer (Kern Map 80K1; Kern & Sohn GmbH, Balingen, Germany). Cut-off points were established as recommended by the 2018 European consensus on definition and diagnosis of sarcopenia[19] considering values < 27 kg as low strength for men and values < 16 kg as low strength for women.

Measurement of appendicular skeletal muscle mass

Appendicular skeletal muscle mass (ASMM) was assessed using a dual-frequency bioelectrical impedance device (Akern Nutrilab™; Akern SRL, Pisa, Italia). Cut-off points were established as recommended by the 2018 European consensus on definition and diagnosis of sarcopenia[19] considering values < 20 kg as low ASMM for men and values < 15 kg as low ASMM for women.

Assessment of physical performance

Assessment of physical performance was made through the liver fragility index (LFI), incorporating values of handgrip strength, the chair rising test, and the standing balance test[29,30]. The chair rising test, was assessed as the minimum time, measured in seconds, using a stop watch, needed to complete five cycles of rising from a chair until standing fully erect and then sitting down again with arms folded across the chest. Regarding the standing balance test, this was assessed in three positions (side-by-side, semi tandem and tandem) for 10 seconds each, unsupported. Finally, LFI was calculated using the formula: LFI = (-0.330 × sex-adjusted grip strength) + (- 2.529 × number of chair stands per second) + (-0.040 × balance time) + 6.

The cut-off points for LFI categories were defined as follows, robust < 3.2, pre-frail between 3.2 and 4.4, and fragile ≥ 4.

Physical activity was estimated using the International Physical Activity Questionnaire (IPAQ) short form, and categorized according to this test as light, moderate, or intense[31].

Measurement of myokines

Circulating levels of distinct myokines were tested in serum samples from all patients with MASLD studied using the Milliplex^® Kit Human Myokine Magnetic Bead Panel (HMYOMAG-56K; Merck Millipore, Burlington, MA, United States), which is an immunoassay using the Luminex xMAP technology for simultaneous detection of up to 15 human myokines in serum samples, following manufacturer’s instructions. At least 50 beads per variable were examined in the Bio-Plex suspension array system 200 (Bio-Rad Laboratories, Hercules, CA, United States). Raw data (median fluorescence intensity) were evaluated using Bio-Plex Manager Software 6.2 (Bio-Rad Laboratories), and median fluorescence intensity from samples were interpolated to a standard curve using a five-parameter logistic equation. The intra- and inter-assay coefficients of variation were lower than 10%.

Statistical analyses

Qualitative variables are presented as absolute (n) and relative (%) frequencies. Quantitative variables are expressed as measures of central tendency (mean) and dispersion (standard deviation). Qualitative data were compared between groups using the by χ² test or the Fisher exact test as appropriate. Quantitative variables were analyzed using the t-test for data with normal distribution (Shapiro-Wilk test) and equal variances (Levene’s test), or the Mann-Whitney U test for data with non-normal distribution or heteroscedasticity variances. Significance was set at P < 0.05. Statistical analyses were performed using the Statistical Product and Service Solutions (SPSS) 26.0 statistical software (IBM SPSS Statistics; IBM Corp, Armonk, NY, United States).

Ethical considerations

This study was carried out in agreement with the Declaration of Helsinki and with local and national laws. All subjects enrolled voluntarily and gave their written consent to participate in the study. The Human Research Ethics Committee La Princesa University Hospital in Madrid, Spain, which is the reference ethics committee for all the institutions involved in the present study, approved the study procedures (No. RRN 4645, October 21, 2021).

RESULTS

Characteristics of the study population

As depicted in Figure 1, 80 patients with MASLD were initially recruited. Of them, 18 patients were excluded from the initial cohort: 10 due to loss to follow-up, 3 due to high alcohol consumption, and 5 because they started glucagon-like peptide 1 agonist therapy during the follow-up. Table 1 shows the demographic, anthropometric, and clinical characteristics of the study population stratified according to the stage of liver fibrosis measured with TE. The study cohort largely comprised middle-aged adults (60.7 ± 10.9 years), 56.45% of the participants were women, and the cohort was predominantly Caucasian. HOMA index assessed according to laboratory values was considered high (> 2.9), without differences related to sex, or between different stages of fibrosis, reflecting a state of IR in the majority of our patients. Average weight was 96.56 kg in men and 78.24 kg in women. Several of the serological scores associated with steatosis and liver fibrosis in MASLD were determined: Fatty liver index (a.u.): 85.34 ± 16.19; fibrosis 4 score (a.u.): 2.05 ± 1.26; non-invasive fibrosis score (a.u.): -0.70 ± 1.26, and aspartate aminotransferase-to-platelet ratio index (a.u.): 0.53 ± 0.55. To avoid confusion in patients with values in the indeterminate range, the study was finally performed using transitional elastography.

Table 1 Characteristics of the study population according to the stage of liver fibrosis assessed with transient elastography (n = 62), mean ± standard deviation/n (%).

Characteristics	F0-F2 (n = 44)	F3-F4 (n = 18)	P value
Age (years)	59.36 ± 11.36	63.17 ± 10.81	0.230
Men	23 (56.1)	11 (61.1)	0.780
Caucasian race	42 (95.4)	17 (94.4)	0.957
Body mass index (kg/m2)	32.32 ± 4.49	30.72 ± 4.30	0.203
Arterial hypertension	13 (29.5)	7 (38.9)	0.557
Diabetes	16 (39.0)	10 (55.6)	0.257
Glucose (mg/dL)	93.4 ± 17.5	99.2 ± 19.1	0.473
Insulin (μU/L)	12.5 ± 6.1	14.2 ± 3.5	0.235
HOMA-IR score	5.16 ± 3.12	5.27 ± 3.95	0.909
Triglycerides (mg/dL)	108.3 ± 51.4	143.1 ± 64.6	0.097
HDL-cholesterol (mg/dL)	49.5 ± 10.9	44.7 ± 12.3	0.479
ALT (IU/L)	17.1 ± 6.8	29.3 ± 5.7	0.137
AST (IU/L)	14.5 ± 4.4	24.1 ± 6.2	0.151
-GT (IU/L)	30.8 ± 29.1	36.5 ± 24.8	0.324
Ferritin (ng/mL)	65.1 ± 69.1	82.5 ± 50.6	0.079
CAP (dB/m)	323.64 ± 48.35	327.50 ± 31.49	0.756
Liver elastography (kPa)	6.01 ± 1.67	15.35 ± 7.36	< 0.001
Hand grip strength (kg)
Men	33.62 ± 6.02	32.19 ± 5.45	0.523
Women	20.18 ± 4.69	17.00 ± 2.15	0.100
ASMM (kg)	22.66 ± 5.21	22.44 ± 6.20	0.887
LFI (a.u)	3.94 ± 0.43	4.11 ± 0.39	0.173
Robust	2 (4.5)	0 (0)
Pre-fragile	36 (81.8)	14 (71.8)
Fragile	4 (9.1)	3 (16.7)
Peritoneal adipose tissue (cm2)	0.84 ± 0.41	0.98 ± 0.64	0.334
Fat mass (%)	34.89 ± 8.53	32.53 ± 6.43	0.296
Skeletal muscle mass (%)	28.92 ± 5.92	30.67 ± 5.81	0.293
Skeletal muscle index (kg/m2)	9.34 ± 1.80	9.59 ± 1.84	0.633

ALT: Alanine aminotransferase; ASMM: Appendicular skeletal muscle mass; AST: Aspartate aminotransferase; CAP: Controlled attenuation parameter; F0-F2: Lower stages of liver fibrosis; F3-F4: Higher stages of liver fibrosis; HDL: High-density lipoprotein; HOMA-IR: Homeostatic model assessment of insulin resistance; γ-GT: γ-glutamyl transpeptidase; LFI: Liver frailty index.

According to elastography values, 44 patients were compatible with F0-F2 liver fibrosis stages and 18 patients with advanced hepatic fibrosis (F3-F4) stages (6.01 ± 1.67 kPa and 15.35 ± 7.36 Kpa; P < 0.001, respectively). None of the patients with F4 fibrosis stage had decompensated cirrhosis. Interestingly, according to CAP elastography values, the majority of patients included in our study cohort had severe steatosis (S3; n = 54). A higher mean CAP value was observed in women compared to men 331 dB (range: 230-400 dB) vs 316 dB (range: 185-400 dB). Likewise, the weight and the number of patients with fibrosis > F3 (11 patients) were higher in women, although the difference did not reach statistical significance. On the other hand, no significant differences were observed in age, sex, or prevalence of obesity and diabetes in patients with MASLD between the stages of liver fibrosis. Notably, 11 of 26 diabetic patients with MASLD were receiving insulin therapy, 6 of them exhibited lower stages of liver fibrosis (F0-F2), and 5 had advanced liver fibrosis (F3-F4). In addition, both handgrip strength and ASMM were similar in patients with MASLD with low fibrosis stages (F0-F2) and in those with advanced fibrosis (F3-F4). According to the value of LFI, pre-frail patients predominated in both fibrosis groups (n = 36 [81.8%] in < F3 vs n = 14 [77.8%] in > F3; P = 0.564). Differences in impedance measurement values and peritoneal adipose tissue between both groups did not show statistical significance (Table 1).

IPAQ short version values (data not shown) showed that the majority of patients (83.5%) displayed light physical activity and none of them intense activity.

Serum levels of myokines in patients with MASLD

We determined the concentration of a wide set of myokines (n = 15) in serum samples of patients with MASLD from our cohort. Differences between different stages of liver fibrosis and between different grades of hepatosteatosis were analyzed (Table 2 and Table 3, respectively). Fibroblast growth factor-21 (FGF21) was the only myokine showing a significant increase in patients with MASLD with advanced fibrosis (F3-F4) with respect to those with lower stages of liver fibrosis (F0-F2) (197.49 ± 198.27 pg/mL vs 95.62 ± 83.67 pg/mL; P = 0.049, Table 2). Regarding hepatosteatosis, as shown in Table 3, patients with MASLD with severe steatosis (S3) had significantly higher serum levels of irisin than those with lower grades of hepatosteatosis (1116.87 ± 1161.86 pg/mL vs 385.21 ± 375.98 pg/mL; P = 0.001). In addition, interleukin 6 (IL-6) serum level were also higher in patients with a higher degree of steatosis (S3) although the difference did not reach statistical significance (1.09 ± 1.02 pg/mL (< S3) vs 3.29 ± 3.76 pg/mL [S3]; P = 0.051).

Table 2 Serum myokine levels according to the stage of liver fibrosis assessed with transient elastography (n = 62), mean ± standard deviation.

Characteristics	F0-F2 (n = 44)	F3-F4 (n = 18)	P value
Apelin (pg/mL)	155.79 ± 123.40	109.44 ± 66.48	0.138
Fractalkine (pg/mL)	511.46 ± 273.69	438.57 ± 249.46	0.333
BDNF (pg/mL)	8234.10 ± 6468.53	5858.89 ± 3471.89	0.147
Erythropoietin (pg/mL)	2822.21 ± 1209.41	2471.33 ± 1039.91	0.286
Osteonectin (ng/mL)	693.27 ± 203.90	622.93 ± 329.70	0.311
LIF (pg/mL)	6.18 ± 7.25	4.23 ± 3.98	0.825
IL-15 (pg/mL)	4.66 ± 4.76	3.72 ± 2.56	0.991
Myostatin/GDF8 (pg/mL)	1148.66 ± 2135.60	889.09 ± 1361.17	0.850
FABP3 (pg/mL)	2067.59 ± 716.21	1941.87 ± 937.38	0.569
Irisin (pg/mL)	1016.74 ± 1110.38	1036.43 ± 1169.30	0.950
FSTL-1 (pg/mL)	7368.05 ± 4937.52	8009.91 ± 9363.92	0.726
Oncostatin M (pg/mL)	10.98 ± 7.45	11.17 ± 7.45	0.927
IL-6 (pg/mL)	3.17 ± 3.92	2.59 ± 2.72	0.661
FGF21 (pg/mL)	95.62 ± 83.67	197.49 ± 198.27	0.049

BDNF: Brain-derived neurotrophic factor; F0-F2: Lower stages of liver fibrosis; F3-F4: Higher stages of liver fibrosis; FABP3: Fatty acid binding protein 3; FGF21: Fibroblast growth factor 21; FSTL-1: Follistatin-like 1; GDF8: Growth differentiation factor 8; IL: Interleukin; LIF: Leukemia inhibitory factor.

Table 3 Serum myokine levels according to the grade of hepatosteatosis assessed with controlled attenuation parameter (n = 62), mean ± standard deviation.

Characteristics	< S3 (n = 8)	S3 (n = 54)	P value
Apelin (pg/mL)	121.56 ± 155.93	145.42 ± 104.90	0.576
Fractalkine (pg/mL)	427.32 ± 270.19	499.63 ± 267.74	0.479
BDNF (pg/mL)	6999.39 ± 4117.58	7625.28 ± 6073.10	0.780
Erythropoietin (pg/mL)	2314.56 ± 1846.11	2780.46 ± 1200.06	0.295
Osteonectin (ng/mL)	690.85 ± 177.12	670.19 ± 256.11	0.827
LIF (pg/mL)	3.09 ± 2.20	5.99 ± 6.84	0.407
IL-15 (pg/mL)	2.88 ± 1.72	4.61 ± 4.46	0.405
Myostatin/GDF8 (pg/mL)	420.22 ± 797.45	1170.05 ± 2037.86	0.314
FABP3 (pg/mL)	2216.38 ± 729.02	2003.64 ± 791.00	0.477
Irisin (pg/mL)	385.21 ± 375.98	1116.87 ± 1161.86	0.001
FSTL-1 (pg/mL)	6256.33 ± 5857.72	7746.70 ± 6571.10	0.547
Oncostatin M (pg/mL)	10.83 ± 7.17	11.07 ± 7.48	0.934
IL-6 (pg/mL)	1.09 ± 1.02	3.29 ± 3.76	0.051
FGF21 (pg/mL)	88.20 ± 76.65	130.67 ± 140.63	0.437

BDNF: Brain-derived neurotrophic factor; FABP3: Fatty acid binding protein 3; FGF21: Fibroblast growth factor 21; FSTL-1: Follistatin-like 1; GDF8: Growth differentiation factor 8; IL: Interleukin; LIF: Leukemia inhibitory factor; S3: Highest grade of hepatosteatosis.

DISCUSSION

In the present study, MASLD diagnosis was performed using abdominal ultrasound and transitional elastography together with inclusion of clinical, metabolic and laboratory variables defined in previous studies[4,6-8,32]. Muscle mass was studied by impedancimetry. This technique is easier to perform and more available than the current gold standard, computed tomography. The presence of muscle dysfunction was studied using dynamometry and LFI, as described in previous studies[30,33].

In our cohort, women exhibited higher CAP values. Despite this result, no significant differences were observed in muscle strength and ASMM between different stages of liver fibrosis. However, it is important to note that, despite the homogeneity in ASMM values among our patients with MASLD, we did observe a trend to greater fragility, determined with LFI, in patients with MASLD with advanced liver fibrosis (F3-F4) than in those with lower stages (F0-F2) although the differences were not significant. A possible explanation of this lack of statistical significance could be the small sample size of our study cohort, which could preclude statistically significant differences for certain variables, particularly taking into account the few patients with advanced liver fibrosis (n = 18).

We next determined the circulating levels of a wide set of myokines in our study cohort to assess their significance as potential biomarkers of SMAs.

FGF21 is a member of a complex family of growth factors and belongs to the endocrine sub-family. Most FGFs exert both autocrine and paracrine actions and are not released into the blood circulation. In the liver, increased expression of this myokine appears in the context of obesity[34] and serum FGF21 levels seem to correlate well with MASLD[35].

A significant increase in FGF21 was found in patients with MASLD with advanced hepatic fibrosis (F3-F4). FGF21 behaves as an anti-obesity and anti-diabetic hormone, causing a reduction in liver fat content, fibrosis and inflammation. Its serum concentration seems to correlate well with intrahepatic levels and its expression has been described to be increased both with carbohydrate intake and in obesity as a compensatory effect[34-37]. Loss of FGF21 or its action, worsens lipotoxicity, thereby contributing to greater liver inflammation and fibrosis. Our data suggest that high serum concentrations of FGF21 in patients with MASLD with greater liver fibrosis may result in a state of resistance to FGF21. This effect is similar to that described for insulin in IR disorders, such as type 2 diabetes mellitus. Interestingly, previous studies have proposed that administration of analogues or modified FGF21 could play an important role in the treatment of MASLD; accordingly several ongoing clinical trials are evaluating this therapy[38,39].

IL-6 concentrations are increased in patients with steatohepatitis compared to those without this condition. Therefore, this cytokine has been proposed as a marker of steatosis and degree of liver damage[40,41]. We also found a significant increase in IL-6 levels in patients with a higher degree of steatosis (S3), although the difference did not reach statistical significance. A larger cohort along with a histological study would be necessary to confirm this association.

In addition, we found that serum levels of irisin were significantly higher in patients with MASLD with severe hepatosteatosis (S3) than in those with moderate or mild liver steatosis (< S3). Although irisin is synthesized and released predominantly at the muscular level, several cross-sectional studies have established a relationship between irisin concentrations and BMI. Two studies with non-diabetic populations described that individuals with morbid obesity have higher irisin concentrations than individuals with normal weight and patients with anorexia[42,43]. Furthermore, both studies demonstrated a close positive association between irisin concentrations and muscle mass markers. In our study, we found higher irisin values in patients with a higher degree of steatosis, associated with overweight and/or obesity but without a significant decrease in muscle mass.

Irisin appears to act as a link between skeletal muscle tissue and adipose tissue, interacting with myostatin present in skeletal muscle to regulate muscle mass and acting on glucose metabolism and insulin sensitivity. It acts as an adipokine exerting an autocrine function in adipose tissue, where it is secreted in greater quantities by subcutaneous fat than by visceral fat. Irisin seems to be related to an unfavorable metabolic state, showing higher levels in patients who develop metabolic syndrome[39,43-45]. In obesity, the greatest irisin levels are released by the adipose tissue[46]. This result is in accordance with our data because, in our cohort, the highest serum levels of irisin were observed in patients with MASLD with the highest BMI values. Therefore, our results seem to confirm the relationship between hepatic fat deposit and irisin. However, we did not find an association with the muscle function of our patients and therefore we cannot suggest the potential usefulness as a biomarker of SMAs. Furthermore, research data reported so far on circulating irisin in patients with MASLD are contradictory and further studies are needed to define its role in the pathophysiology of MASLD.

Our study had some limitations. First, the cross-sectional design, the small sample size, and the low prevalence of advanced hepatic fibrosis in the study population may have limited the statistical power of the present study. Furthermore, we used bioelectrical impedance to measure ASMM and this technique may not be sensitive enough to detect significant changes of skeletal muscle mass in the study cohort. Finally, patients included in this study were referred to the hepatology clinic to evaluate alteration of the liver profile and to determine the prognosis of the metabolic process, and the diagnosis and stratification of patients was carried out using available techniques accepted in routine clinical practice, which may have caused a certain degree of selection bias.

The increase of pro-inflammatory cytokines reported in other studies, was confirmed for IL-6, irisin and FGF21 in patients with marked steatosis and greater liver fibrosis. The study of cytokines in MASLD opens a wide field for the development of molecules acting on pro-inflammatory pathways that could help to reverse or stop the process (FGF21 analogues and myostatin inhibitors, among others). We believe that the detection of proinflammatory cytokines like FGF21 and irisin, in patients with MASLD could play a role in the future as diagnostic markers and allowing early intervention in the development of sarcopenia. Nevertheless, further multicenter prospective research studies are needed for definitively establishing the cause-and-effect relationship between SMAs and MASLD.

CONCLUSION

Serum levels of the myokines, irisin, and FGF21 were significantly higher in patients with MASLD with severe liver steatosis and in those with advanced liver fibrosis, respectively. However, no relationship was found between SMAs and the stage of liver fibrosis in patients with MASLD. Further clinical studies, including large and distinct cohorts of patients with MASLD, are needed to validate the accuracy of circulating myokines as potential biomarkers of advanced liver fibrosis and severe hepatosteatosis in these patients and their relationship with SMAs.

ACKNOWLEDGEMENTS

We would like to thank Dr. Gómez Gutierrez M for his help with the English revision of the manuscript.