ADAR2 deficiency ameliorates non‐alcoholic fatty liver disease and muscle atrophy through modulating serum amyloid A1

Abstract Background Non‐alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Sarcopenia is a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength, which is commonly associated with NAFLD. Adenosine‐to‐inosine editing, catalysed by adenosine deaminase acting on RNA (ADAR), is an important post‐transcriptional modification of genome‐encoded RNA transcripts. Three ADAR gene family members, including ADAR1, ADAR2 and ADAR3, have been identified. However, the functional role of ADAR2 in obesity‐associated NAFLD and sarcopenia remains unclear. Methods ADAR2+/+/GluR‐BR/R mice (wild type [WT]) and ADAR2−/−/GluR‐BR/R mice (ADAR2 knockout [KO]) were subjected to feeding with standard chow or high‐fat diet (HFD) for 20 weeks at the age of 5 weeks. The metabolic parameters, hepatic lipid droplet, grip strength test, rotarod test, muscle weight, fibre cross‐sectional area (CSA), fibre types and protein associated with protein degradation were examined. Systemic and local tissues serum amyloid A1 (SAA1) were measured. The effects of SAA1 on C2C12 myotube atrophy were investigated. Results ADAR2 KO mice fed with HFD exhibited lower body weight (−7.7%, P < 0.05), lower liver tissue weight (−20%, P < 0.05), reduced liver lipid droplets in concert with a decrease in hepatic triglyceride content (−24%, P < 0.001) and liver injury (P < 0.01). ADAR2 KO mice displayed protection against HFD‐induced glucose intolerance, insulin resistance and dyslipidaemia. Skeletal muscle mass (P < 0.01), muscle strength (P < 0.05), muscle endurance (P < 0.001) and fibre size (CSA; P < 0.0001) were improved in ADAR2 KO mice fed with HFD compared with WT mice fed with HFD. Muscle atrophy‐associated transcripts, such as forkhead box protein O1, muscle atrophy F‐box/atrogin‐1 and muscle RING finger 1/tripartite motif‐containing 63, were decreased in ADAR2 KO mice fed with HFD compared with WT mice fed with HFD. ADAR2 deficiency attenuates HFD‐induced local liver and skeletal muscle tissue inflammation. ADAR2 deficiency abolished HFD‐induced systemic (P < 0.01), hepatic (P < 0.0001) and muscular (P < 0.001) SAA1 levels. C2C12 myotubes treated with recombinant SAA1 displayed a decrease in myotube length (−37%, P < 0.001), diameter (−20%, P < 0.01), number (−39%, P < 0.001) and fusion index (−46%, P < 0.01). Myogenic markers (myosin heavy chain and myogenin) were decreased in SAA1‐treated myoblast C2C12 cells. Conclusions These results provide novel evidence that ADAR2 deficiency may be important in obesity‐associated sarcopenia and NAFLD. Increased SAA1 might be involved as a regulatory factor in developing sarcopenia in NAFLD.


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Suppl. Figure 3 Supplementary Figure S3.Effects of ADAR2 KO on food intake, energy intake and water intake in male and female mice.Food intake (male: a, female: d), and energy intake (male: b, female: e) and water intake (male: c, female: f) derived from WT and ADAR2 KO mice fed with ND or HFD are shown.
Data were expressed as mean±SEM.Tukey's multiple comparison test after the two-way ANOVA was conducted for (A)-(f).

Measuring fasting plasma levels of glucose and insulin
After 12-h fasting, the mice were anesthetized and blood samples were collected from the cardiac puncture with heparinized capillary tubes.Plasma was collected after centrifuging the blood at 3000 × g for 10 min.Plasma glucose levels were determined by using a commercial glucose-oxidase kit (Cat.#: 11538, BioSystems, Barcelona, Spain).Plasma insulin levels were determined by using a commercial mouse insulin ELISA kit (Cat.#: 10-1247-01, Mercodia, Uppsala, Sweden).

Calculating homeostasis model assessment insulin resistance index
The homeostasis model assessment insulin resistance (HOMA-IR) index was calculated as followed formula: fasting glucose (mM) × fasting insulin (mU/l)/22.5.

Measurement of Circulating high-sensitivity C-reactive protein
The plasma level of high-sensitivity C-reactive protein (hs-CRP) was measured according to the manufacturer's instructions (Elabscience Biotechnology CO. Ltd., China).

Real-time quantitative PCR analysis
Total RNAs were extracted from gastrocnemius muscles using TRIzol Reagent (Cat.#: 15596018, Ambion, Life Technologies Corporation).The reverse transcription reaction was performed on 2 μg of RNA using SuperScript III First-standard Synthesis System (Cat.#: 18080-051, Invitrogen) with random hexamer primers according to the manufacturer's directions.SYBR Green (Cat.#: 4385612, Thermo Fisher Scientific) was used to quantify the PCR amplification products.The expression levels of the target mRNA were measured using a real-time PCR system (Applied Biosystems, Thermo Fisher Scientific).The target mRNA levels were normalized to the levels of the housekeeping gene β-actin, which was used as the endogenous control.The sequences of the primers are listed in Supplementary Table 1.