. 2023 Dec 18;14(1):8391.

doi: 10.1038/s41467-023-44242-7.

Cdo1-Camkk2-AMPK axis confers the protective effects of exercise against NAFLD in mice

Min Chen^{1

2

3}, Jie-Ying Zhu^{1

2

3}, Wang-Jing Mu^{1

2

3}, Hong-Yang Luo^{1

2

3}, Yang Li^{1

2

3}, Shan Li^{1

2

3}, Lin-Jing Yan^{1

2

3}, Ruo-Ying Li^{1

2

3}, Liang Guo^{4

5

6}

Affiliations

¹ School of Exercise and Health and Collaborative Innovation Center for Sports and Public Health, Shanghai University of Sport, Shanghai, 200438, China.
² Shanghai Frontiers Science Research Base of Exercise and Metabolic Health, Shanghai University of Sport, Shanghai, 200438, China.
³ Key Laboratory of Exercise and Health Sciences of the Ministry of Education, Shanghai University of Sport, Shanghai, 200438, China.
⁴ School of Exercise and Health and Collaborative Innovation Center for Sports and Public Health, Shanghai University of Sport, Shanghai, 200438, China. guoliang@sus.edu.cn.
⁵ Shanghai Frontiers Science Research Base of Exercise and Metabolic Health, Shanghai University of Sport, Shanghai, 200438, China. guoliang@sus.edu.cn.
⁶ Key Laboratory of Exercise and Health Sciences of the Ministry of Education, Shanghai University of Sport, Shanghai, 200438, China. guoliang@sus.edu.cn.

PMID: 38110408
PMCID: PMC10728194
DOI: 10.1038/s41467-023-44242-7

Cdo1-Camkk2-AMPK axis confers the protective effects of exercise against NAFLD in mice

Min Chen et al. Nat Commun. 2023.

. 2023 Dec 18;14(1):8391.

doi: 10.1038/s41467-023-44242-7.

Authors

Min Chen^{1

2

3}, Jie-Ying Zhu^{1

2

3}, Wang-Jing Mu^{1

2

3}, Hong-Yang Luo^{1

2

3}, Yang Li^{1

2

3}, Shan Li^{1

2

3}, Lin-Jing Yan^{1

2

3}, Ruo-Ying Li^{1

2

3}, Liang Guo^{4

5

6}

Affiliations

¹ School of Exercise and Health and Collaborative Innovation Center for Sports and Public Health, Shanghai University of Sport, Shanghai, 200438, China.
² Shanghai Frontiers Science Research Base of Exercise and Metabolic Health, Shanghai University of Sport, Shanghai, 200438, China.
³ Key Laboratory of Exercise and Health Sciences of the Ministry of Education, Shanghai University of Sport, Shanghai, 200438, China.
⁴ School of Exercise and Health and Collaborative Innovation Center for Sports and Public Health, Shanghai University of Sport, Shanghai, 200438, China. guoliang@sus.edu.cn.
⁵ Shanghai Frontiers Science Research Base of Exercise and Metabolic Health, Shanghai University of Sport, Shanghai, 200438, China. guoliang@sus.edu.cn.
⁶ Key Laboratory of Exercise and Health Sciences of the Ministry of Education, Shanghai University of Sport, Shanghai, 200438, China. guoliang@sus.edu.cn.

PMID: 38110408
PMCID: PMC10728194
DOI: 10.1038/s41467-023-44242-7

Abstract

Exercise is an effective non-pharmacological strategy for ameliorating nonalcoholic fatty liver disease (NAFLD), but the underlying mechanism needs further investigation. Cysteine dioxygenase type 1 (Cdo1) is a key enzyme for cysteine catabolism that is enriched in liver, whose role in NAFLD remains poorly understood. Here, we show that exercise induces the expression of hepatic Cdo1 via the cAMP/PKA/CREB signaling pathway. Hepatocyte-specific knockout of Cdo1 (Cdo1^LKO) decreases basal metabolic rate of the mice and impairs the effect of exercise against NAFLD, whereas hepatocyte-specific overexpression of Cdo1 (Cdo1^LTG) increases basal metabolic rate of the mice and synergizes with exercise to ameliorate NAFLD. Mechanistically, Cdo1 tethers Camkk2 to AMPK by interacting with both of them, thereby activating AMPK signaling. This promotes fatty acid oxidation and mitochondrial biogenesis in hepatocytes to attenuate hepatosteatosis. Therefore, by promoting hepatic Camkk2-AMPK signaling pathway, Cdo1 acts as an important downstream effector of exercise to combat against NAFLD.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. Hepatic Cdo1 is induced through the cAMP/PKA/CREB pathway during exercise in mice.**
a Treadmill training in chow diet-fed 8-week-old mice. The representing mouse model was created using BioRender.com. b–e Liver samples were isolated from the male mice after the exercise program shown in a is done. b The liver messenger RNA (mRNA) level of *Cdo1* (n = 6 mice per group). c Western blotting of mice liver lysates (n = 3 mice per group). d Quantification of western blotting results of c. e The cAMP level in mice liver (n = 6 mice per group). f CREB enrichment on the indicated gene promoters (n = 6 independent biological replicates). The β-globin gene promoter serves as a negative control. g Schematic representation of Cdo1 proximal promoter constructs used for luciferase assays. The predicted consensus of CREB-binding site is shown in the WT luciferase construct. The red letters indicate mutations of the CREB-binding site in the CREB-Mut construct. h, i Luciferase activities were measured in HEK293T cells. Data were normalized to the vector group (n = 6 independent biological replicates). h Cells were transiently transfected with WT (Wild type) reporter construct as shown in g, along with different amounts of CREB expression vector. i Cells were transiently transfected with WT or CREB-Mut reporter construct, along with control vector or CREB expression vector. j–o Primary mice hepatocytes or HepG2 cells were treated with 10 μM Forskolin for 12 h. j The mRNA level of *Cdo1* in primary hepatocytes (n = 6 independent biological replicates). k Western blotting of primary hepatocytes lysates (n = 3 independent biological replicates). l Quantification of western blotting results of k. m The *CDO1* mRNA level in HepG2 cells (n = 6 independent biological replicates). n Western blotting of HepG2 lysates (n = 3 independent biological replicates). o Quantification of western blotting results of n. Unpaired two-tailed t tests were performed in b, d, e, j, l, m and o; one-way analysis of variance plus Tukey’s post hoc tests were performed in h and i; two-way analysis of variance plus Tukey’s post hoc tests were performed in f. All data show the means ± SD. Source data are provided as a Source Data file.

**Fig. 2. Hepatocyte-specific knockout of Cdo1 (Cdo1^LKO) impairs exercise-mediated alleviation of fatty liver in mice.**
a Scatter diagram indicating the relative *Cdo1* gene expression level in the high-fat diet (HFD)-fed mice group relative to the normal chow diet (CD)-fed controls from the GEO database in a microarray assay (n = 14 mice in CD group, and n = 18 mice in HFD group). b Scatter diagram indicating the relative *CDO1* gene expression level in patients with non-NAFLD people and NAFLD patients from the GEO database in high-throughput RNA sequencing (n = 14 individuals in non-NAFLD group, and n = 15 individuals in NAFLD group). c 6-week-old male mice were fed CD or HFD for 16 weeks before being sacrificed. The mice liver lysates were analyzed by western blotting (n = 4 mice per group). d 6-week-old Cdo1^flox/flox (WT) and Cdo1^LKO male mice were fed HFD for 16 weeks, with or without exercise in the last 8 weeks. The intervention program is illustrated. The representing mouse model was created using BioRender.com. e–n Mice were treated as described in d before being sacrificed for analysis (n = 6 mice per group). e Mice liver weights. f Representative images of hematoxylin and eosin (H&E) staining and Oil Red O staining of liver sections. Experiments were performed 3 times and similar results were obtained. Scale bars, 50 μm. g, h Triglyceride (TG) and cholesterol (TC) levels in mice livers, respectively. i Glucose tolerance test (GTT) was performed in mice under 14 weeks of HFD feeding. j Analysis of the GTT data in i, with subtraction of the basal glucose to generate an area of the curve (AOC). k Insulin tolerance test (ITT) was performed in mice fed with HFD for 15 weeks. l Analysis of the ITT data in k with AOC. m, n Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels in mice, respectively. Unpaired two-tailed t tests were performed in a and b; two-way analysis of variance plus Tukey’s post hoc tests were performed in e, g, h, j and l–n. All data show the means ± SD. Source data are provided as a Source Data file.

**Fig. 3. Cdo1-regulated AMPK signaling is involved in exercise-mediated alleviation of fatty liver in mice.**
a Heatmaps representing the upregulated and downregulated genes (Cdo1^LKO vs WT) in the livers of HFD (High-fat diet)-fed mice without exercise which was obtained from the RNA sequencing (RNA-seq) data (n = 3 mice per group, p < 0.05). b Gene ontology (GO) pathway analysis based on the RNA-seq data as described in a (p < 0.05). c–g Mice were treated as described in Fig. 2d. c The mRNA levels of the indicated genes were determined in mice livers (n = 6 mice per group). d The mitochondrial DNA (mitoDNA) levels in mice livers (n = 6 mice per group). e Transmission electron microscopic analysis (HITACHI HT7800) of mice livers. Scale bar: 2μm. Arrowheads indicate the mitochondria. Experiments were performed 3 times and similar results were obtained. f Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of downregulated genes based on RNA-seq data in a (p < 0.05). “Count” means the number of genes that are in the corresponding KEGG pathway. g The mice liver lysates were analyzed by western blotting (n = 3 mice per group). Two-sided Wald tests without adjustment were performed in a, b and f. Two-way analysis of variance plus Tukey’s post hoc tests were performed in c and d. All data show the means ± SD. Source data are provided as a Source Data file.

**Fig. 4. Cdo1 deficiency exacerbates hepatocytes steatosis in vitro.**
From a–g Cdo1^flox/flox loci-containing primary hepatocytes were infected with adenovirus harboring Cre (AD-Cre) for the ablation of Cdo1. After 24 h, cells were treated with a mixture of 0.6 mM FFAs (oleate and palmitate) at a final ratio of 2:1 for 24 h. Then cells were harvested and analyzed. n = 3 independent biological replicates for western blotting. n = 6 independent biological replicates for other experiments. a Nile red and Hoechst staining of the cells. Scale bars, 50 μm. b Western blotting of the cell lysates. c The mRNA levels of the indicated genes. d MitoScene and Hoechst staining of the cells. Scale bars, 50 μm. e The mitochondrial DNA (mitoDNA) levels of the cells. f Oxygen consumption rate (OCR) of the cells. FCCP, mitochondrial uncoupler. R&A, rotenone and antimycin A. g Fatty acid oxidation (FAO) levels of the cells. h–n HepG2 cells were transfected with the indicated small interfering RNA (siRNA). After 24 h, cells were treated with a mixture of FFAs as described above for 24 h. Then cells were harvested and analyzed. n = 3 independent biological replicates for western blotting. n = 6 independent biological replicates for other experiments. h Nile red and Hoechst staining of the cells. Scale bars, 50 μm. i Western blotting of the cell lysates. j The mRNA levels of the indicated genes. k MitoScene and Hoechst staining of the cells. Scale bars, 25 μm. l The mitoDNA levels in HepG2 cells. m OCR in HepG2 cells. n Fatty acid oxidation (FAO) levels in HepG2 cells. Experiments were performed 3 times and similar results were obtained in a, b, d, **h, i** and k. Unpaired two-tailed t tests were performed in c, e and g; one-way analysis of variance plus Tukey’s post hoc tests were performed in j, l and n; two-way analysis of variance plus Tukey’s post hoc tests were performed in f and m. All data show the means ± SD. Source data are provided as a Source Data file.

**Fig. 5. Cdo1 overexpression alleviates hepatocytes steatosis in vitro.**
WT primary hepatocytes were infected with adenovirus harboring Cdo1 (AD-Cdo1^WT), AD-Cdo1^Y157F or AD-LacZ (for Vector). After 24 h, cells were treated with a mixture of 0.6 mM FFAs (oleate and palmitate) at a final ratio of 2:1 for 24 h. Then cells were harvested and analyzed. n = 3 independent biological replicates for western blotting. n = 6 independent biological replicates for other experiments. a Nile red and Hoechst staining of the cells. Scale bars, 50 μm. b TG levels in primary hepatocytes. c Primary hepatocytes lysates were analyzed by western blotting with the indicated antibodies. GAPDH serves as an internal control. d Quantification of western blotting results of c. e The mRNA levels of the indicated genes were determined. f The mitochondrial DNA (mitoDNA) levels in primary hepatocytes. g MitoScene and Hoechst staining of the cells Scale bars, 50 μm. h Respiration in primary hepatocytes. OCR, oxygen consumption rate. FCCP, mitochondrial uncoupler. R&A, rotenone and antimycin A. i Fatty acid oxidation (FAO) levels in primary hepatocytes. Experiments were performed 3 times and similar results were obtained in a, c and g. One-way analysis of variance plus Tukey’s post hoc tests were performed in b, f and i; two-way analysis of variance plus Tukey’s post hoc tests were performed in d, e and h. Source data are provided as a Source Data file.

**Fig. 6. AMPK signaling is indispensable for Cdo1-mediated amelioration of hepatocytes steatosis.**
Wild-type (WT) primary hepatocytes were infected with adenovirus harboring Cdo1 (AD-Cdo1^WT), AD-Cdo1^Y157F or Vector, together with the infection of adenoviruses harboring the indicated short hairpin RNA (shRNAs). After 24 h, cells were treated with a mixture of 0.6 mM FFAs (oleate and palmitate) at a final ratio of 2:1 for 24 h. Then cells were harvested and analyzed. n = 3 independent biological replicates for western blotting. n = 6 independent biological replicates for other experiments. a Nile red and Hoechst staining of the cells. Scale bars, 50 μm. b Triglyceride (TG) levels in primary hepatocytes. c The mRNA levels of the indicated genes were determined. d Primary hepatocytes lysates were analyzed by western blotting with the indicated antibodies (n = 3 independent biological replicates, and representative blot was shown). GAPDH serves as an internal control. e Quantification of western blotting results of d was done by using ImageJ. f MitoScene and Hoechst staining of the cells. Scale bars, 25 μm. g The mitochondrial DNA (mitoDNA) levels in primary hepatocytes. h Respiration in primary hepatocytes. OCR, oxygen consumption rate. FCCP, mitochondrial uncoupler. R&A, rotenone and antimycin A. i Fatty acid oxidation (FAO) levels in primary hepatocytes. Experiments were performed 3 times and similar results were obtained in a, d and f. Two-way analysis of variance plus Tukey’s post hoc tests were performed in b, c, e, g, h and i. All data show the means ± SD. Source data are provided as a Source Data file.

**Fig. 7. Cdo1 tethers Camkk2 to AMPK by interacting with both of them, thereby promoting AMPK phosphorylation.**
a Primary hepatocytes were infected with AD-Flag-Cdo1, and immunoprecipitation was performed using anti-Flag beads and followed by western blotting with antibodies as indicated (n = 3 independent biological replicates). WCL, whole cell lysate. b, c Hepa1-6 cells were co-transfected with plasmids encoding Flag-Cdo1^WT or Flag-Cdo1^Y157F and HA-Camkk2, and followed by confocal analyses. Scale bars, 10μm. d Cell lysates from primary hepatocytes was immunoprecipitated with antibody against Camkk2, followed by western blotting (n = 3 independent biological replicates). e Primary hepatocytes were infected with AD-Cdo1^WT or AD-Cdo1^Y157F, and cell lysates were immunoprecipitated with antibody against Camkk2 and followed by western blotting (n = 3 independent biological replicates). f Quantification of immunoprecipitation (IP) results of e. g Cdo1^flox/flox loci-containing primary hepatocytes were infected with AD-Cre for the ablation of Cdo1. Cell lysates were immunoprecipitated with antibody against Camkk2 and followed by western blotting (n = 3 independent biological replicates, and representative blot was shown). h Quantification of IP results of g. i Cell free kinase assay with purified recombinant Cdo1, Camkk2 and AMPK proteins. The mixtures were detected by western blotting with antibodies as indicated (n = 3 independent biological replicates, and representative blot was shown). j Quantification of western blotting results of i. k Primary hepatocytes were infected with AD-Cdo1 and AD-shCamkk2 for 48 h. Cell lysates were then analyzed by western blotting (n = 3 independent biological replicates, and representative blot was shown). l Quantification of western blotting results of k. m Primary hepatocytes were infected with AD-Cdo1 and AD-shLKB1 for 48 h. Cell lysates were then analyzed by western blotting (n = 3 independent biological replicates, and representative blot was shown). n Quantification of western blotting results of m. Experiments were performed 3 times and similar results were obtained in a–e, g, i, k and m. One-way analysis of variance plus Tukey’s post hoc tests was performed in j; two-way analysis of variance plus Tukey’s post hoc tests were performed in f, h, l and n. All data show the means ± SD. Source data are provided as a Source Data file.

**Fig. 8. Hepatocyte-specific overexpression of Cdo1 (Cdo1^LTG) and exercise synergistically alleviate NAFLD in mice.**
WT and Cdo1^LTG male mice under high-fat diet (HFD) feeding 16 weeks, with or without exercise for the last 8 weeks. Then mice were sacrificed for analysis. a Representative images of hematoxylin and eosin (H&E) staining and Oil Red O staining. Experiments were performed 3 times and similar results were obtained. Scale bars, 50 μm. b, c Triglyceride (TG) and cholesterol (TC) levels in mice livers (n = 6 mice per group). d Glucose tolerance test (GTT) was performed in mice under 14 weeks of HFD feeding (n = 6 mice per group). e Analysis of the GTT data in d, with subtraction of the basal glucose to generate an area of the curve (AOC). f Insulin tolerance test (ITT) was performed in mice fed with 15 weeks of HFD (n = 6 mice per group). g Analysis of the ITT data in f with AOC. h, i Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels in mice, respectively (n = 6 mice per group). j The mice liver lysates were analyzed by western blotting (n = 3 mice per group). k The mRNA levels of the indicated genes (n = 6 mice per group). l The mitochondrial DNA (mitoDNA) levels in mice livers (n = 6 mice per group). Experiments were performed 3 times and similar results were obtained in a and j. Two-way analysis of variance plus Tukey’s post hoc tests were performed in b, c, e, **g, h, i,** k and l. All data show the means ± SD. Source data are provided as a Source Data file.

**Fig. 9. A graph model for the role of hepatic Cdo1 in exercise-mediated protective effects against NAFLD.**
Exercise can promote the expression of Cdo1 through cAMP/PKA/CREB signaling pathway. CREB can bind to Cdo1 promoter and promote Cdo1 transcription. Cdo1^LKO impairs exercise-mediated effects against NAFLD in mice. On the contrary, Cdo1^LTG and exercise can cooperate to alleviate hepatocyte steatosis. Mechanistically, Cdo1 tethers Camkk2 to AMPK by interacting with both of them, thereby promoting AMPK phosphorylation. This enhances fatty acid oxidation and mitochondrial biogenesis and would promote cellular energy expenditure, thereby inhibiting hepatosteatosis. Hepatic Cdo1 could function as an exercise-responsive effector in fighting against NAFLD. The representing mouse, mitochondria and liver images in panel were created using BioRender.com.

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Cdo1-Camkk2-AMPK axis confers the protective effects of exercise against NAFLD in mice

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Cdo1-Camkk2-AMPK axis confers the protective effects of exercise against NAFLD in mice

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