Aerobic exercise improves hepatic steatosis by modulating miR-34a-mediated PPARα/SIRT1-AMPK signaling pathway

doi:10.21203/rs.3.rs-2967131/v1

The role of aerobic exercise in preventing and ameliorating non-alcoholic fatty liver disease (NAFLD)has been widely demonstrated. MicroRNA-34a (MiR-34a) , a small non-coding RNA that regulates gene expression, has received much attention for its essential role in the progression of metabolic liver disease. However, it is unclear whether exercise can prevent and ameliorate hepatic lipid degeneration by targeting miR-34a and its underlying molecular mechanisms. In this study, normal or high-fat diet -induced male C57BL/6J mice underwent an 8-week running program (6 days/week, 18 m/min, 50 min, 6% incline) or remained sedentary. Histomorphometric examination and biochemical analysis were performed to evaluate intrahepatic lipid deposition. Adeno-associated viral vectors were injected into mice to construct miR-34a liver-specific overexpression mice. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of genes and proteins related to lipid metabolism in the liver. After exercise intervention, the liver weight/body weight, liver TG, and HE of mice in the High-fat diet with aerobic exercise group (HCE) indicated significant improvement in hepatic steatosis, and mir-34a levels were significantly suppressed. This study identified aerobic exercise improves hepatic lipid degeneration by increasing the expression of its target genes PPARα and SIRT1 through mir-34a and activating the expression of AMPK and changes in genes related to lipid metabolism downstream of the PPARα/SIRT1-AMPK pathway.

Health sciences/Endocrinology/Endocrine system and metabolic diseases

Biological sciences/Molecular biology/Epigenetics

Aerobic exercise

liver steatosis

miR-34a

PPARα

SIRT1

Excessive storage of intrahepatic fat, i.e., hepatic steatosis, is recognized as a significant metabolic and hepatic pathology associated with the ongoing obesity epidemic. NAFLD, or as recently proposed, 'metabolic-associated fatty liver disease' (MAFLD), is a direct result of hepatic steatosis, which can progress to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma [1, 2]. Due to the high prevalence globally (more than 32.4%) and its serious complications, including hyperlipidemia, type 2 diabetes, insulin resistance (IR), and obesity, NAFLD has become the primary health issue [3]. In addition, factors that an imbalance between lipid acquisition (free fatty acids uptake and de novo lipogenesis) and disposal (β-oxidation and very low density lipoprotein export) can contribute to the development of hepatic steatosis [4,5].

MiRNAs (MicroRNAs) is a small single-stranded non-coding RNA of about 19-25 nucleotides in length that can regulate the expression of its target gene through reverse complementary pairing with the partial sequence of the 3' untranslated region of its target gene [6]. In human diseases, miRNAs have received increasing attention due to their deregulation and their potential as both diagnostic and therapeutic targets [7, 8]. MiR-34a, a specific miRNA that regulates liver diseases, has a high expression level in the liver of ob/ob obese mice and hepatic steatosis patients [9,10,11]. Besides, studies have reported that miR-34a can increase the levels of its downstream targets, silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor-α (PPARα), to adjust the genes related to fatty acid oxidation, thereby alleviating liver steatosis [12].

As a nuclear receptor superfamily member, PPARα is a downstream target gene of miR-34a [13, 14]. After PPARα binds to the ligand, it forms a heterodimer with the retinoid X receptor (RXR), and the formed PPARα/RXR heterodimer binds to the PPAR response element (PPRE) of the target genes, promoting the transcription of its downstream genes related to fatty acid oxidation [12]. Thus PPARα has excellent effects on the regulation of hepatic lipid metabolism.

Nicotinamide adenine dinucleotide (NAD+) deacetylase, SIRT1 that regulates DNA expression, apoptosis, senescence, and involvement in physiological or pathological processes of organisms by deacetylating substrate proteins, and is the best characterized direct target of miR-34a, which regulated the activity of AMP-activated protein kinase (AMPK), a known regulator of energy metabolism that has a significant role in the development and progression of metabolic diseases [15, 16]. After phosphorylation, AMPK could regulate fatty acid oxidation genes and transport-related proteins, thereby reducing liver steatosis. Therefore, the SIRT1-AMPK phosphorylation signaling pathway regulates lipid metabolic homeostasis and may be a therapeutic target for hepatic steatosis.

Aerobic exercise is a low-cost, low-risk intervention that can effectively reduce the occurrence and development of liver steatosis and has been adopted by most people [17, 18]. In addition, numerous studies have shown that miRNA expression can be affected by many factors, such as exercise, drugs, hormones, and others. However, the potential mechanisms by which aerobic exercise prevents and ameliorates lipid accumulation in hepatocytes via miRNA remain unknown[19,20,21]. Therefore, this study aims to determine whether aerobic exercise can improve liver steatosis by adjusting the miR-34a-PPARα/Sirt1 signal pathway.

2.1 Ethical Permission

The Shanxi University Ethics Committee for Scientific Research has approved this protocol. All experiments conformed to local and international guidelines on the ethical use of animals, and all efforts were made to minimize the number of animals used and their suffering. This study adhered to the ARRIVE guidelines. Approval Number: CIRP-IACUC-(R)2019014.

2.2 Animal experiments

Forty SPF-grade male C57BL/6J mice (8 weeks old), weighing 21.4 ± 0.92 g, were bought from Beijing Life River Laboratory Animal Technology Co. (animal license no. SCXK (Beijing) 2016-0006). After one week of adaptive feeding, the experimental subjects were randomly selected into four groups: normal control group (NC, n = 10 ), normal diet with aerobic exercise group (NE, n = 10), high-fat diet group (HFD, 60% fat, 20% carbohydrate, 20% protein, n = 10) and high-fat diet with aerobic exercise group (HCE, n = 10). Details of the formulation and calories of the mouse chow are shown in Supplementary information file 1. Next, all groups continued their dietary regimens for 13 weeks, respectively. For the last nine weeks of feeding, mice in the NE and HCE groups underwent one week of acclimatization exercise and eight weeks of formal aerobic exercise intervention based on the original dietary intervention. All experimental subjects were housed under standard laboratory conditions with 12/12 dark light cycles, 20–26 ºC, 40–60% relative humidity, and free access to food and water. The body weight of mice was recorded weekly.

To specifically overexpress miR-34a in mice, we used a mouse miR-34a sequenced adenovirus (Sangon Biotech, Shanghai, China). The virus was assayed in 293A cells, and the virus titer was measured. The miR-34a sequence was correct, and the virus was successfully packaged at a titer of 9 × 10¹¹ PFU/ml. Meanwhile, the packaging of an unrelated sequence control adenovirus was assayed, and the virus was successfully packaged at a titer that met the experimental requirements. Adenoviral vectors (100 µl) were injected into the tail vein of mice for five weeks and divided into three groups: virus empty vector as a negative control group (EV, n = 10), miR-34a overexpression group (over-expression mir-34a, n = 10), mir-34a overexpression combined plus aerobic exercise group (over-expression mir-34a + EX, n = 10). During the whole experimental period, all mice were fed a standard diet.

2.3 Aerobic exercise protocol

After four weeks of a normal or high-fat diet, mice performed aerobic exercise on an animal treadmill (SA101, Jiangsu Saionce Biotechnology Co., China). During week 1, NE, HCE, and overexpression mir-34a + Ex groups performed moderate exercise in the first week, and the intensity, time, and incline were gradually increased. The final aerobic exercise intensity was determined to be 18 m/min, 45 minutes, 6% incline, and each time the mice had a 5-min warm-up before exercise, six days per week for eight weeks. Mouse exercise protocols refer to our previous studies and the work of Linden MA et al. [30, 45]. The specific exercise protocol is shown in Fig. 1 and Table 1.

2.4 Tissue Collection

After the last treadmill exercise, mice were fasted overnight and anesthetized intraperitoneally with sodium pentobarbital at a dose of 80 mg/Kg, followed by the spinal dislocation method of execution. Blood samples were collected in EDTA-containing tubes, centrifuged at four ºC and 3500 rpm for 15 min, and the supernatant was transferred to a new tube and stored at -80ºC. The mouse livers were rapidly taken out and washed with cold phosphate-buffered saline (PBS), then some of the liver tissues were cut into the fixative, and the remaining liver tissues were frozen in liquid nitrogen and saved at -80 ºC until analysis.

2.5 Triglyceride Assay

Frozen livers were weighted and homogenized in lysis buffer (140 mM NaCl, 50 mM Tris, 0.1% Triton-X) using a tissue laser (Qiagen). The homogenates were centrifuged at 3,000×g for 10 min at 4°C. Next, the supernatant was incubated at 37℃ for 10 minutes, left to cool, and colorimetric at 420 nm on a spectrophotometer (UV-6100s, Mapada, Shanghai, China) according to the instructions of the Triglyceride (TG) assay kit (Sangon Biotech, Nanjing, China), and the TG content in the liver of each group of mice was calculated.

2.6 Histologic analysis

The fresh livers were laid flat in the embedding box and immersed in the prepared 10% formalin solution; after 24–48 h, the box was removed, rinsed in 50% alcohol solution, and immersed in 70% alcohol overnight. The cassette was transferred from the 70% alcohol solution to the 80% alcohol solution for two hours. The wax was dehydrated and preserved for embedding, then sliced, spread retrieved, and dried in a warm oven. Filter hematoxylin for Hematoxylin-eosin staining (HE). The tissue was completely coated with drops of neutral gum, covered with a coverslip, and spread out to dry naturally. The degree of hepatic steatosis and lipid accumulation was observed with a light microscope (Zeiss, German).

2.7 RNA extraction and quantitative real-time polymerase chain reaction

The RNA was extracted by the Trizol method, and the air-dried RNA was precipitated for 5–10 min after discarding the supernatant, and the precipitate was dissolved in 20 µl of DEPC water. Take 2 µl of the dissolved RNA and measure the OD260, OD280, and OD260/OD280 values with a microspectrophotometer (Allsheng Instruments Co., Ltd, Hangzhou, China) to calculate the purity and concentration of RNA. The concentration of sample RNA was calculated according to the following formula based on the absorbance value: Total RNA concentration (µg/µl) = OD260 × 40 × 10^− 3. Quantitative PCR was performed on a LightCycler480 system (Roche, Switzerland) using TB Green premix Ex Taq II mix (TaKaRa, Dalian, China). mRNA expression was calculated by the 2^−△△CT method. GAPDH was used as an internal control for RNA analysis. The primers used for quantitative PCR are shown in Table 2.

2.8 Western blot

Liver tissue was lysed in RIPA buffer, and protein concentration was determined by the BCA method (Beyotime, Shanghai, China). During the assay, 100 mg of liver tissue was taken, and the protein concentration was measured in a ratio of 1:10 with RIPA lysis buffer. Proteins (40 µg) were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto PVDF membranes (Millipore, Billerica, USA). Soak PVDF membranes in TBST (sealing solution) containing 5% skimmed milk powder and seal with vigorous shaking for 2h at room temperature. And they were incubated overnight at 4C with 1:1000-fold diluted specific primary antibodies (1:1000 anti-PPARα, anti-SIRT1, anti-AMPK, anti-CPT1, anti-CPT2, anti-SLC27A1, anti-SLC27A4), all the antibodies were purchased from Bioss Biological Inc. (Beijing, China). β-actin was used as an endogenous control. After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated (HRP-conjugated) secondary antibodies (1:5000) (Boster Biotech, Wuhan, China) for one hour, with the signals detected by an ECL detection kit (Applygen Technologies Inc., Beijing, China) and imaged in a chemiluminescence-measuring instrument (ChemiDoc XRS+, Bio-Rad, USA).

3.1 Aerobic exercise alleviated hepatic steatosis in mice.

Histological analyses of the livers by HE staining revealed a significant increase in the number and size of hepatic lipid droplets with inflammatory cell infiltration in the HFD group. In contrast, mice in the HCE group showed reduced hepatic lipid droplets and neatly aligned hepatocytes, and hepatic steatosis was significantly improved (Fig. 2A). Besides, as observed in the 13-week diet regimens, both body and liver weight of the HCE group compared to that of mice fed HFD alone were significantly less and near the mean weight gain of mice fed the standard diet (Figs. 2B-C). Consistently, the HFD group also had a higher liver weight/body weight ratio than the NC mice, while the ratio decreased in the HCE group (Fig. 2D). Consistent with the above results, the ELISA assay also found that the TG level in the HFD group was significantly higher than in other groups (Fig. 2E). These observations suggested that HFD-induced hepatic steatosis could be attenuated by eight weeks of treadmill exercise.

3.2 Aerobic exercise inhibits miR-34a expression.

Considering the principal role of miR-34a in liver lipid metabolism, we examined the effects of aerobic exercise on hepatic miR-34a expression through an 8-week treadmill training program. The results showed that miR-34a expression was significantly increased in the liver of the HFD group compared to the NC group (P < 0.05). In contrast, the expression of miR-34a in the liver of HCE mice under the intervention of aerobic exercise was significantly suppressed ( Fig. 2F). Further, to verify the effect of aerobic exercise on miR-34a, we overexpressed miR-34a by tail vein injection using a mouse miR-34a sequenced adenovirus and performed eight weeks of treadmill intervention. The findings of qRT-PCR analysis showed that miR-34a expression in the liver of over-expression mice was reversed by such training( Fig. 2G). These data indicate that aerobic exercise may play an essential role in improving hepatic steatosis by suppressing miR-34a expression.

3.3 Aerobic exercise upregulated hepatic PPARα expression and activity via mir-34a

The lipid pathway transcriptional regulator PPARα is known to be a direct target of miR-34a in the liver. On this basis, this study further explored whether aerobic exercise would affect PPARα through this pathway. As shown in Figs. 3 A and B, both mRNA and protein levels of PPARα were decreased in the liver of the HFD group. At the same time, after an 8-week treadmill training intervention, PPARa expression increased significantly. To further validate the effected of aerobic exercise on miR-34a-PPARα pathway. We examined the expression of PPARα in the liver of mir-34a overexpressing mice. As expected, aerobic exercise increased the mRNA and protein expression levels of the lipid metabolism-related gene PPARα in the liver of mice. The mRNA and protein expression levels of PPARα were significantly lower in the miR-34a + overexpression group compared to the EV group. However, after eight weeks of treadmill training, mRNA and protein levels of PPARα were elevated (P < 0.05, Fig. 3C-D). This result suggests that aerobic training can improve PPARα expression by suppressing miR-34a.

3.4 Aerobic exercise promoted the SIRT1/AMPK pathway via mir-34a.

SIRT1 has been extensively studied as a critical factor in regulating lipid metabolism. In this study, we observed no significant changes in SIRT1 mRNA and protein levels in NE group mice compared to NC mice, whereas SIRT1 mRNA and protein levels were significantly decreased in mice fed a high-fat diet. However, this decrease was reversed after aerobic exercise intervention. (Fig. 4A-B). SIRT1 can regulate the AMPK, a regulator of energy metabolism. Increased levels of AMPK phosphorylation are closely associated with enhanced lipid metabolism. As shown in Fig. 4C, the level of p-AMPK (THR-172) in the HFD group was lower than that in the NC group, while eight weeks of treadmill training could rescue the adverse effects caused by HFD, resulting in the upregulation of P-AMPK (THR-172). On this basis, we further explored whether exercise would affect this pathway via miR-34a, thereby alleviating hepatic lipid degeneration. As shown in Fig. 4D-E, both mRNA and protein levels of SIRT1 were decreased in the livers of the miR-34a + overexpression group and reduced P-AMPK levels(Figure F). In contrast, eight weeks of treadmill training had a facilitative effect on this pathway. These observations suggest that the miR-34a-SIRT1-AMPK pathway is another mechanism by which aerobic exercise improves hepatic lipid degeneration.

3.5 Aerobic exercise ameliorated hepatic steatosis via reduced mir-34a expression altering PPARα/SIRT1-AMPK-regulated fatty acid oxidation and transport gene expression.

Next, to gain insight into the mechanism by which aerobic exercise improves hepatic lipid degeneration through the miR-34a-PPARα/SIRT1-AMPK pathway, we examined the expression of several essential target genes involved in fatty acid oxidation and transport, namely carnitine palmitoyl transterase-1 (CTP1), carnitine palmitoyl transterase-2 (CTP2) and solute carrier family 27 member 1 (SLC27A1), solute carrier family 27 member 4 (SLC27A4), which are all regulated by PPARα, and AMPK regulation. In the present study, we found that the expression levels of downstream target genes in NE group mice were similar to those in NC group mice. In contrast, the expression of CTP1 and CTP2 was lower in HFD mice than in NC mice, and the expression of SLC27A1 and SLC27A4 was higher in HFD mice than in NC mice, but the expression of all these genes was reversed in the HCE group. In addition, we found that the mRNA and protein expression levels of fatty acid oxidation-related target genes CPT1 and CPT2 were significantly reduced in liver samples from miR-34a overexpressing mice. The expression levels of fatty acid transport-related genes SLC27A1 and SLC27A4 were significantly elevated, and these changes were reversed after exercise intervention (P < 0.05, Fig. 5F-G). Based on these findings, we demonstrated that aerobic exercise ameliorated hepatic steatosis by suppressing mir-34a expression and was closely associated with fatty acid oxidation and transport regulated by the PPARα/SIRT1-AMPK pathway.

In this experiment, after eight weeks of treadmill exercise, high-fat/high-cholesterol-fed mice showed significant improvements in body weight, liver weight, and liver pathology. Moreover, we believe that one mechanism by which aerobic exercise ameliorates hepatic steatosis is through the downregulation of miR-34a expression, which consequently impacts vital target genes that are closely related to lipid metabolism.

MiRNAs, a popularly studied regulatory growth factor, regulate the expression of 30% of essential human genes, most essential for everyday survival and development [22]. In both in vitro and in vivo models, miRNAs regulate cell growth, differentiation, energy, and liver metabolic functions, regulating fatty acid and cholesterol metabolism [8]. Among them, miR-34a, one of the most widely studied miRNAs in the molecular pathways regulating the development and progression of NAFLD, has been demonstrated in several studies to be differentially expressed in hepatic steatosis [23]. Hepatic miR-34a expression is elevated in people with NAFLD and appears to increase with disease progression [24, 25–26]. In an experiment in cells and a high-fat diet-induced mouse model, miR-34a was shown to regulate progressive NAFLD by acting on several metabolic pathways, including activation of lipid absorption, lipogenesis, inflammation, and apoptosis as inhibition of fatty acid oxidation [27]. Similarly, this study model of hepatic steatosis showed increased hepatic miR-34a expression and miR-34a overexpression may lead to disturbances in lipid metabolism, consistent with previous findings.

NAFLD is a complicated disease that is caused by a mix of genetic and environmental factors. As of today, no specific medication has been made to treat fatty liver other than diet and exercise. Regular exercise is considered the most effective strategy to overcome the liver pathology associated with fatty liver [28, 29]. As a potent regulator of epigenetic modifications, exercise may improve the onset and progression of hepatic steatosis by affecting miRNAs. For example, Xiao et al. showed that exercise prevents NAFLD by downregulating miR-212 and affecting the FGF-21 signaling pathway [21]. In addition, our previous study showed that aerobic exercise suppresses target genes closely related to lipid metabolism by enhancing the expression of miR-21a-5p [30]. In conclusion, exercise can exert a wide range of physiological regulatory effects through miRNAs, and miR-34a may also be another key target for exercise to improve hepatic steatosis. However, no studies have shown that exercise improves hepatic steatosis by affecting miR-34a and its downstream target genes. This study results identified a negative regulatory effect of aerobic exercise on miR-34a expression in hepatic steatosis in mice and further explored the downstream mechanisms of exercise intervention in mir-34a.

Several studies have shown that miR-34a controls hepatic lipid metabolism pathways by targeting key transcription factors [23]. miR-34a directly reduces SIRT1 protein levels by interacting with the 3' untranslated region, and antagonizing miR-34a normalizes SIRT1 activity in obese mice, leading to deacetylation of several regulators of hepatic lipid metabolism and its downstream β-oxidation gene upregulation and downregulation of adipogenic genes, an effect that parallels the reduction in the degree of hepatic steatosis [31]. In addition, experimental evidence suggests a reciprocal regulatory relationship between SIRT1 and AMPK, and SIRT1 can activate AMPK by activating liver kinase B1 (LKB1) through deacetylation [32, 33]. In our study, aerobic exercise increased the expression of SIRT1 through inhibition of miR-34a, which increased the level of AMPK phosphorylation in mouse liver. Moreover, the exercise-activated AMPK may regulate the body's lipid metabolic process and reverse the development of hepatic lipid degeneration by inhibiting lipid synthesis and increasing fatty acid oxidation. The PPARα is the major transcription factor that promotes FAs (Fatty acids) uptake and mitochondrial oxidation (β-oxidation) by stimulating the mitochondrial carnitine palmitoyl transferase enzyme family [34]. However, in the case of NAFLD, the expression of PPARα is significantly diminished, leading to impaired lipogenesis and fatty acid oxidation [35, 36]. Ding et al. demonstrated that miR-34a could potentially post-transcriptionally regulate PPARα through specific binding with the PPARα wild-type luciferase construct [12]. This study confirmed that PPARα is a downstream target of miR-34a, consistent with previous studies, and the overexpression of miR-34a abolished aerobic exercise-induced activation of PPARα. Since PPARα is a mediator of the fatty acid β-oxidation and transport pathway, our results suggest that aerobic exercise activates PPAR, enhances fatty acid β-oxidation, and reduces fatty acid accumulation, decreasing TG accumulation and hepatic steatosis.

The mitochondrial carnitine system, which includes CPT1 and CPT2, plays a crucial role in fatty acid oxidation. CPT1 catalyzes the esterification of long-chain acyl-CoA into L-carnitine. L-carnitine is used as a carrier to transfer long-chain fatty acids from outside the mitochondria to the inside of the mitochondria as an acyl-carnitine conjugate. Acyl-carnitine conjugates are transformed back to acyl-CoA esters by CPT2 in the mitochondria to promote the oxidation and decomposition of fatty acids [37]. This study identified after aerobic exercise inhibited miR-34a, AMPK was activated to monitor the fuel meter of cell energy status and activate the gene related to fatty acid oxidation. Mechanistically, the high consumption of ATP during exercise increases the AMP/ATP ratio and increases AMPK phosphorylation levels, which in turn enhances AMPK activity in the liver. The activation of AMPK increased the levels of CPT1 and CPT2, leading to increased transport of fatty acids into the mitochondria and facilitating the oxidation of mitochondrial palmitoyl-CoA. These observations helped us understand the enhanced fatty acid oxidation mechanism after aerobic exercise inhibits miR-34a. It is determined that AMPK is a medium for the increased hepatic oxidation of miR-34a after aerobic exercise inhibition.

Disturbances in the synthesis, transport, and elimination systems of long-chain fatty acids (LCFAs) and TG are the leading causes of fatty liver [38]. Critical actors of LCFAs transport and activation are the solute carrier family 27 (SLC27, also known as fatty acid transport proteins [FATPs]), which has six members (SLC27A1-6) expressed in a highly tissue-specific pattern in mammals [39]. Among these members, SLC27A1 and SLC27A4 are the only two evolutionarily conserved genes from invertebrates to vertebrates. They may have overlapping biological functions in the plasma and intracellular membranes of cells such as hepatocytes and small intestinal mucosa [40]. It has been demonstrated that overexpression of SLC27A1 promotes FA esterification and lipid accumulation in adipocytes and tissues. In contrast, resveratrol reduces the accumulation of total free fatty acids and triglycerides in macrophages by inhibiting Fatp1 expression through activation of PPARα signaling [41]. Similarly, HFD feeding increased SLC27A4 expression in mouse liver, and SLC27A4 activation of LCFAs may lead to increased LCFA uptake and subsequent metabolism that may result in hepatocyte lipid accumulation [42]. This study further supports the high expression of SLC27A1 and SLC27A4 in a high-fat diet pattern and attenuates this effect by aerobic exercise intervention. Aerobic exercise improves intrahepatic lipid metabolism by increasing insulin sensitivity in adipose tissue, decreasing plasma and transported FFA levels to the liver, and decreasing hepatic fat content [43, 44]. We speculate that aerobic exercise could affect lipid accumulation by activating PPARα and inhibiting hepatic fatty acid uptake of protein.

Our results confirm the negative regulatory effect of aerobic exercise on miR-34a expression in mouse liver steatosis and further validate our conjecture by constructing an in vivo miR-34a overexpression model. However, there are some limitations. First, the present study only analyzed whether aerobic exercise could affect hepatic lipid degeneration in mice by regulating mir-34a expression, however the deep mechanism of miR-34a regulation by exercise is still unclearh, which will be the next direction of our study; second, the effect of PPARα on lipid metabolism could also be indirectly mediated by activating AMPK when interfering with miR-34a expression in high-fat diet (HFD)-fed mice, suggesting that mir-34a mediated regulation of hepatic lipid metabolism by PPARα and SIRT1-AMPK pathways may be two cross-regulatory mechanisms of hepatic steatosis, but how the two pathways interact with each other was not further analyzed in this study [12]; finally, because the solute carrier family 27A (SLC27A) contains both fatty acid transport motifs and fatty acid acyl coenzyme a synthase (ACS), the its function has been controversial [33], and current studies on how exercise affects changes in the expression of SLC271 and SLC274 are scarce, and how exercise interferes with changes in the levels of both remain to be further investigated and confirmed.

In conclusion, our study suggests that aerobic exercise is an important component in regulating hepatic lipid metabolism by controlling the level of miR-34a. Mechanistically, aerobic exercise may activate PPARα and SIRT1 by suppressing miR-34a expression, which further activates the AMPK pathway. PPARα and AMPK ameliorate steatosis by inhibiting fatty acid uptake and promoting fatty acid oxidation (Fig. 5E). whether the role of mir-34a differs in different exercise intensities remains to be elucidated by further studies. Pharmacological inhibition of miR-34a using antagonists (chemically engineered oligonucleotides used to silence endogenous miRNAs) may be a novel therapeutic strategy that mimics the benefits of exercise in the treatment of NAFLD.

AMPK	AMP-activated protein kinase
ANOVA	Analysis of variance
CPT1	Carnitine palmitoyl transterase-1
CPT2	Carnitine palmitoyl transterase-2
EV	Virus empty vector group
FA	Fatty acids
HCE	High-fat diet with aerobic exercise group
HE	Hematoxylin-eosin staining
HFD	High-fat diet group
IR	Insulin resistance
LCFAs	Long-chain fatty acids
LKB1	Liver kinase B1
MAFLD	Metabolic-associated fatty liver disease
MiR-34a	MicroRNA-34a
MiRNAs	MicroRNAs
NC	Normal control group
NE	Normal diet plus aerobic exercise group
NAFLD	Non-alcoholic fatty liver disease
NAD+	Nicotinamide adenine dinucleotide
Over-expression mir-34a	MiR-34a overexpression group
Over-expression mir-34a+EX	Mir-34a overexpression plus aerobic exercise group
PPARα	Peroxisome proliferator-activated receptor-α
PPRE	Peroxisome Proliferator Response Element
PBS	Phosphate-buffered saline
qRT-PCR	Quantitative real-time PCR
RXR	Retinoid X receptor
SIRT1	Silent information regulator 1
SLC27A1	Solute carrier family 27 member 1
SLC27A4	Solute carrier family 27 member 1
TG	Triglyceride

Data Availability

All data generated or analyzed during this study are included in this published article [and its supplementary information file 1 ].

Acknowledgements

The authors thank the Institute of Biological Sciences Shanxi University for professional technical assistance and the China Institute of Radiation Protection for animal care.

Author information

Authors and Affiliations

Department of Sports and Education, Sports and education College, No. 92, Wucheng Road, Xiaodian District, Taiyuan City 030000, Shanxi Province, China,

Baoai Wu, YiMing Tian, Chong Xu, Longpeng Li, Yue Guan, Yinghua Chen, Jinfeng Zhao

Contributions

Baoai Wu: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing-review & editing. Yiming Tian: Conceptualization, Methodology, Investigation, Writing - original draft. Chong Xu: Investigation, Writing-review & editing. Longpeng Li: Investigation, Writing-review & editing. Yue Guan: Investigation, Writing-review & editing. Yinghua Chen: Investigation, Writing-review & editing. Jinfeng Zhao: Conceptualization, Formal analysis, Writing-review & editing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jinfeng Zhao.

Competing interests

The authors declare that they have no competing interests.

Additional Information

Ethics approval and consent to participate

The protocol of animal experiments was complied with the Guide for the Care and Use of Laboratory Animals formulated by the US National Institutes of Health (NIH Publication No. 85–23, Revised 1996) and approved by the Shanxi University Ethics Committee for Scientific Research, china.

Funding

This research was supported by the National Natural Youth Science Foundation of China (grant number 31500962).

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Table 1. Training protocol

Week	Day	Velocity (m·min-1)	Duration (min)	Slope (°)
1 week	1	8	10	0
	2	10	20	2
	3	12	30	3
	4	14	40	4
	5	16	50	5
	6	18	50	6
	7	Rest
2-9weeks	1-6	18	50	6

Table 2. Primers for mRNA expression analysis in RT-qPCR

Genes

Forward(F) primer

Reverse(R) primer

miR-34a

PPARα

SIRT1

CTP1

CTP2

SLC27A1

SLC27A4

GAPDH

5′-AATCAGCAAGTATACTGCCCT-3′

5′-AGA GCC CCA TCT GTC CTC TC-3′

5′-CCT GAC TTC AGA TCA AGA GAC GGT A-3′

5’-CTC CGC CTG AGC CAT GAA G -3′

5′-CAG CAC AGC ATC GTA CCC A -3′

5′-CGC TTT CTG CGT ATC GTC TG -3′

5’-ACT GTT CTC CAA GCT AGT GCT -3′

5′-TCA ACG ACC ACT TTG TCA AGC TCA-3′

5′-ACT GGT AGT CTG CAA AAC CAA A-3′

5′-CTG ATT AAA AAT GTC TCC ACG AAC AG-3′

5′-CAC CAG TGA TGA TGC CAT TCT-3′

5′-TCC CAA TGC CGT TCT CAA AAT -3′

5′-GAT GCA CGG GAT CGT GTC T -3′

5’-GAT GAA GAC CCG GAT GAA ACG -3’

5′-GCT GGT GGT CCA GGG GTC TTA CT -3′

No competing interests reported.

Supplementaryfile1..docx

Aerobic exercise improves hepatic steatosis by modulating miR-34a-mediated PPARα/SIRT1-AMPK signaling pathway

Status:

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Ethical Permission

2.2 Animal experiments

2.3 Aerobic exercise protocol

2.4 Tissue Collection

2.5 Triglyceride Assay

2.6 Histologic analysis

2.7 RNA extraction and quantitative real-time polymerase chain reaction

2.8 Western blot

3. Result

3.1 Aerobic exercise alleviated hepatic steatosis in mice.

3.2 Aerobic exercise inhibits miR-34a expression.

3.3 Aerobic exercise upregulated hepatic PPARα expression and activity via mir-34a

3.4 Aerobic exercise promoted the SIRT1/AMPK pathway via mir-34a.

4. Discussion

Abbreviation

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

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