In this study, the effect of chronic alcohol intake for 4 weeks on the muscular atrophy factors of rat skeletal muscle was studied using 6-week-old growing Sprague-Dawley rats. Experimental animals were classified into a control group and an alcohol intake group. The alcohol intake group consumed alcohol orally at a concentration of 3-g/kg body weight every day for 4 weeks. The control group consumed tap water in the same way. After 4 weeks alcohol ingestion, glucose, total cholesterol, triglyceride, and high-density lipoprotein cholesterol in serum levels were measured. Western blot was performed to detect the expressions of muscle RING-finger protein-1 (MuRF1), protein kinase B (Akt), phosphorylated Akt (p-Akt), forkhead box O (FoxO), phosphorylated FoxO (p-FoxO), p38, and phosphorylated p38 (p-p38). Results of this experiment showed that chronic alcohol intake enhanced triglyceride concentration. Chronic alcohol intake increased MuRF1 expression to promote muscle proteolysis and decreased p-Akt/Akt ratio and p-FoxO/FoxO ratio to inhibit skeletal muscle growth. Therefore, alcohol consumption has been shown to cause muscle atrophy.
It has been reported that alcohol consumption in adolescence inhibits normal brain development, leading to a decrease in attention and concentration, as well as severe cognitive loss and visual impairment (
Alcohol administration at a concentration of 6 g/kg increased the concentration of triglyceride in the mouse liver rapidly within 8 hours after ingestion, indicating the possibility of future fatty liver lesion expansion (
Muscle RING-finger protein-1 (MuRF1) is upregulated during skeletal muscle atrophy. Therefore, degradation of the myosin heavy chain, a major component of sarcomere, is an important mechanism for degradation of skeletal muscle under atrophic conditions. MuRF1 has been shown to be upregulated during denervation, glucocorticoid administration, immobilization, and casting (when a limb is cast for immobilization). All these treatments cause skeletal muscle atrophy (
Protein kinase B, also known as Akt, is the collective name of a set of three serine/threonine-specific protein kinases that play key roles in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. There are three different genes that encode isoforms of protein kinase B. These three genes are referred to as Akt1, Akt2, and Akt3. Akt1 is involved in the cell survival pathway by inhibiting the process of apoptosis. Akt1 can also induce protein synthesis pathways and thus is a key signaling protein in cellular pathways leading to skeletal muscle hypertrophy and general tissue growth. A mouse model in which the Akt1 gene is completely deleted exhibits growth retardation and increased spontaneous apoptosis in tissues such as the testis and thymus (
The forkhead box O (FoxO) transcription factor plays a key role in an evolutionarily conserved pathway. FoxO proteins are involved in a variety of cellular functions and have clinical significance, including cell cycle arrest, cell differentiation, tumor suppression, DNA repair, lifespan, diabetic complications, immunity, wound healing, metabolic regulation and consequent treatment of several types of disease. Akt is a signaling pathway as a key regulator of FoxO that performs a variety of functions in organisms (
Chronic alcoholic myopathy is one of the most frequent and profound signs of chronic alcoholism. The disease is characterized by marked atrophy of motor muscles containing predominantly fibers expressing the myosin isoform of type I fast (
In this experiment, male Sprague-Dawley rats (Samtaco Bio Korea, Hwaseong, Korea) were used. A total of 18 experimental animals were purchased, and they were bred for 4 weeks in an animal breeding room, classified into a control group (n=9) and an alcohol intake group (n=9). The alcohol intake group was orally administered with alcohol (3-g/kg body weight) at 9 am every day, and the same amount of tap water was orally administered in the control group. This experiment was approved by the Research Ethics Committee of Eulji University (EU-2021-4-15).
After breeding for 4 weeks, the animals were anesthetized using ether, and blood was collected from the left ventricle using a disposable syringe and placed in a tube for serum separation. Blood was centrifuged at 700×g for 15 min using a refrigerated centrifuge at 4°C to separate serum. The concentrations of glucose, total cholesterol, triglyceride and high-density lipoprotein cholesterol in serum were analyzed by enzymatic method using a commercial kit (Asan Pharm., Seoul, Korea) with a spectrophotometer (Biochrom Ltd., Hollston, MA, USA).
After removing all tendon tissues of the soleus muscle, cytoplasmic extract buffer was added 9 times the muscle tissue weight, and then homogenization was performed for about 10 sec using a polytron homogenizer (Biospec Products, Bartlesville, OK, USA). After homogenization, the supernatant was extracted by centrifugation at 10,000×g for 5 min. Meanwhile, the precipitate was centrifuged at 10,000×g for 5 min after adding nuclear extract buffer to extract the supernatant and used as a sample for protein expression. Protein concentration was quantified using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard concentration.
Western blot was performed using the soleus muscle. Proteins were electrophoresed on a 12% running gel. After electrophoresis, the gel was transferred to polyvinylidene diflouride membranes (Millipore, Bedford, MA, USA) at 4°C for 12 hr using a blotting device. Blocking was performed for 1 hr with 5% bovine serum albumin diluted with Tris-buffered saline in Tween-20 at room temperature. Primary antibodies against Akt, phosphorylated Akt (p-Akt), FoxO, phosphorylated FoxO (p-FoxO) (Cell Signaling, Beverly, MA, USA), MuRF1, p38, and phosphorylated p38 (p-p38) (Abcam, Cambridge, MA, USA) were prepared using 5% bovine serum at room temperature. The secondary antibody was reacted with horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) for 1 hr, respectively. Thereafter, each protein band was identified using an enhanced chemiluminescence kit (GE Healthcare, Buckinghamshire, UK).
The data processing was analyzed using SPSS ver. 16.0 (SPSS Inc., Chicago, IL, USA) and all data was shown as the mean standard error of the mean. Independent specimens were performed to verify the difference between groups, and the statistical significance level was set to
As a result of ingestion of alcohol for 4 weeks, the changes of glucose, total cholesterol, triglyceride, and high-density lipoprotein cholesterol in serum are presented in
As a result of ingestion of alcohol for 4 weeks, MuRF1 protein expression in the muscle was high as 132.4%±6.02% in the alcohol intake group when the control group was set at 100% (
As a result of ingesting alcohol for 4 weeks, the change in p-Akt/Akt ratio of the muscle was low as 0.73±0.03 in the alcohol intake group when the control group was set to 1 (
As a result of ingesting alcohol for 4 weeks, the change in p-FoxO/FoxO ratio of the muscle was as low as 0.51±0.04 in the alcohol intake group when the control group was set to 1 (
As a result of ingesting alcohol for 4 weeks, the change in p-p38/p38 ratio of the muscle was 0.98±0.11 in the alcohol intake group when the control group was set to 1, indicating no difference (
In the present study, alcohol intake for 4 weeks increased the triglyceride concentration in the blood. In an animal experiment on rats chronically ingesting 36% alcohol for 4 months, fatty acid metabolism in the liver was reduced by an increase in serum triglyceride concentration (
In the present study, MuRF1 protein expression in the alcohol intake group was significantly increased compared to the control group. It was reported that MuRF1 and MAFbx expression increased in an experiment in which skeletal muscle atrophy was induced (
In the present study, p-Akt/Akt ratio in the alcohol intake group was significantly decreased compared to the control group. Akt has been reported to play an important role in the growth of tissues such as bone and muscle tissue (
In the present study, p-FoxO/FoxO ratio in the alcohol intake group was significantly decreased compared to the control group. Insulin inhibits FoxO transcription factors, which down-regulate the expression of genes involved in metabolism, cell cycle arrest, and apoptosis. FoxO turns on a feedforward loop that is relayed by p38 and serves to amplify both Akt activation and FoxO repression. In conclusion, key signaling pathways are activated in hepatocytes via nuclear FoxO (
In the present study, p-p38/p38 ratio in the alcohol intake group was not changed compared to the control group. Phosphorylation of p38 was increased in the skeletal muscle of rats weighing 400 g, in which 36% of daily total calories were replaced with alcohol for 12 weeks, and at the same time, the muscle cross-sectional area and muscle weight were decreased (
As a result of this experiment, chronic alcohol intake in growing rats promoted muscle protein degradation by increasing MuRF1 protein expression and inhibited skeletal muscle growth by decreasing the p-Akt/Akt ratio and p-FoxO/FoxO ratio. Therefore, it was shown that alcohol intake induces muscle atrophy.
The authors received no financial support for this article.
No potential conflict of interest relevant to this article was reported.
Effect of alcohol intake on muscle-specific RING finger protein 1 (MuRF1) expression. Data are mean±standard error of the mean. *
Effect of alcohol intake on ratio of phosphorylated protein kinase B (p-Akt)/Akt expression. Data are mean±standard error of the mean. *
Effect of alcohol consumption on ratio of phosphorylated Forkhead box protein O (p-FoxO)/FoxO expression. Data are mean±standard error of the mean. *
Effect of alcohol consumption on ratio of phosphorylated p38 (p-p38)/p38 expression. Data are mean±standard error of the mean. CG, control group; AG, alcohol intake group.
Blood composition after alcohol intake
Variable | Control group | Alcohol intake group |
---|---|---|
Glucose (mg/dL) | 148.3±3.47 | 155.6±2.87 |
Triglyceride (mg/dL) | 58.7±6.29 | 71.3±13.45 |
Total cholesterol (mg/dL) | 43.1±1.09 | 44.5±1.40 |
High-density lipoprotein cholesterol (mg/dL) | 29.9±1.13 | 33.5±0.80 |
Values are presented as mean±standard deviation.