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. 2023 Mar 28;5(7):100746.
doi: 10.1016/j.jhepr.2023.100746. eCollection 2023 Jul.

Human umbilical cord mesenchymal stem cell-derived exosomes ameliorate liver steatosis by promoting fatty acid oxidation and reducing fatty acid synthesis

Fuji Yang  1   2 , Yanshuang Wu  2   3 , Yifei Chen  2 , Jianbo Xi  4 , Ying Chu  1   4 , Jianhua Jin  4 , Yongmin Yan  1   4
Affiliations

Human umbilical cord mesenchymal stem cell-derived exosomes ameliorate liver steatosis by promoting fatty acid oxidation and reducing fatty acid synthesis

Fuji Yang et al. JHEP Rep. .

Abstract

Background & aims: Non-alcoholic fatty liver disease (NAFLD) affects nearly a quarter of the population with no approved pharmacological therapy. Liver steatosis is a primary characteristic of NAFLD. Recent studies suggest that human umbilical cord mesenchymal stem cell-derived exosomes (MSC-ex) may provide a promising strategy for treating liver injury; however, the role and underlying mechanisms of MSC-ex in steatosis are not fully understood.

Methods: Oleic-palmitic acid-treated hepatic cells and high-fat diet (HFD)-induced NAFLD mice were established to observe the effect of MSC-ex. Using non-targeted lipidomics and transcriptome analyses, we analysed the gene pathways positively correlated with MSC-ex. Mass spectrometry and gene knockdown/overexpression analyses were performed to evaluate the effect of calcium/calmodulin-dependent protein kinase 1 (CAMKK1) transferred by MSC-ex on lipid homoeostasis regulation.

Results: Here, we demonstrate that MSC-ex promote fatty acid oxidation and reduce lipogenesis in oleic-palmitic acid-treated hepatic cells and HFD-induced NAFLD mice. Non-targeted lipidomics and transcriptome analyses suggested that the effect of MSC-ex on lipid accumulation positively correlated with the phosphorylation of AMP-activated protein kinase. Furthermore, mass spectrometry and gene knockdown/overexpression analyses revealed that MSC-ex-transferred CAMKK1 is responsible for ameliorating lipid accumulation in an AMP-activated protein kinase-dependent manner, which subsequently inhibits SREBP-1C-mediated fatty acid synthesis and enhances peroxisome proliferator-activated receptor alpha (PPARα)-mediated fatty acid oxidation.

Conclusions: MSC-ex may prevent HFD-induced NAFLD via CAMKK1-mediated lipid homoeostasis regulation.

Impact and implications: NAFLD includes many conditions, from simple steatosis to non-alcoholic steatohepatitis, which can lead to fibrosis, cirrhosis, and even hepatocellular carcinoma. So far, there is no approved drug for treating liver steatosis of NAFLD. Thus, better therapies are needed to regulate lipid metabolism and prevent the progression from liver steatosis to chronic liver disease. By using a combination of non-targeted lipidomic and transcriptome analyses, we revealed that human umbilical cord mesenchymal stem cell-derived exosomes (MSC-ex) effectively reduced lipid deposition and improved liver function from HFD-induced liver steatosis. Our study highlights the importance of exosomal CAMKK1 from MSC-ex in mediating lipid metabolism regulation via AMPK-mediated PPARα/CPT-1A and SREBP-1C/fatty acid synthase signalling in hepatocytes. These findings are significant in elucidating novel mechanisms related to MSC-ex-based therapies for preventing NAFLD.

Keywords: AMPK; CAMKK1; Exosomes; MSC; NAFLD; Steatosis.

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Conflict of interest statement

The authors declare no competing financial interest. Please refer to the accompanying ICMJE disclosure forms for further details.

Figures

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Graphical abstract
Fig. 1
Fig. 1
Therapeutic efficacy of MSC-ex in the HFD-induced NAFLD model. (A) Representative result of NTAs of MSC-ex and HFL1-ex. (B) Representative results of nanosized vesicles (MSC-ex and HFL1-ex) photographed by TEM and AFM. Scale bar, 100 nm. (C) Exosome markers CD9, CD63, Alix, and TSG101 were positive in MSC-ex and HFL1-ex by immunoblotting. (D) C57BL/6 mice were fed a HFD (40%) for 10 weeks and then were i.v. administered PBS as a control or 10 mg/kg MSC-ex or 10 mg/kg HFL1-ex or deMSC-ex from the 10th week to the 14th week of HFD feeding. (E) Representative Images of gross liver appearance from mice. (F and G) Representative images of H&E (scale bars, 50 μm) (F) and Oil Red O (scale bars, 20 μm) staining (G) of liver sections from mice from each treatment group. (H) Liver contents of total TG and TC in each group. (I) Liver indexes (liver index = liver wet weight/body weight) and body weight changes of mice from each group. (J) i.p. ITTs evaluated individual insulin tolerance by injecting insulin at a dose of 0.75 IU/kg body weight; blood glucose levels were detected at 0, 30, 60, 90, and 120 min and compared with that at 0 min. Individual glucose tolerance was assessed by IPGTT; fasted mice were administered 1 g of glucose/kg body weight by i.p. injection, and blood glucose levels were determined at 0, 30, 60, 90, and 120 min. (K) Fasting glycaemia (after 16-h fast). (L and M) Serum insulin (L), and TG, TC, AST, and ALT (M) levels in each group. Data are represented as the mean ± SEM. Statistical analyses were performed by a one-way ANOVA (H–M). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. normal chow diet group; #p <0.05, ##p <0.01, ###p <0.001 vs. HFD group. AFM, atomic force microscopy; ALT, alanine transaminase; AST, aspartate transaminase; deMSC-ex, MSC-ex-free conditional medium supernatant; HFD, high-fat diet; HFL1, human lung fibroblast 1; HLF1-ex, foetal HFL1-derived exosomes; IPGT, i.p. glucose tolerance test; ITT, insulin tolerance test; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; NAFLD, non-alcoholic fatty liver disease; NTA, nanoparticle tracking analysis; TC, total cholesterol; TFM, transmission electron microscopy; TG, triglycerides; TSG101, tumour susceptibility gene 101.
Fig. 2
Fig. 2
MSC-ex inhibit lipid accumulation in vitro by promoting the β-oxidation of fatty acids and suppressing the fatty acid synthesis. (A and B) Intracellular lipid droplets in L02 cells stimulated with OPA (2.0 mM, 2:1 ratio) in combination with different concentrations of MSC-ex or PBS for 24 h were visualised by Nile red staining (A) and quantified by ImageJ for five random areas (B). Scale bars, 100 μm. (C) Cell viability was measured by FDA staining. (D) The effect of MSC-ex on the viability of L02 cells was determined by CCK-8 assay. (E) Mitochondrial fatty acid β-oxidation of L02 cells treated with a combination of OPA and MSC-ex (800 μg/ml) or deMSC-ex for 24 h, n = 3 biological replicates per group. (F and G) Lipogenic metabolites in L02 cells subjected to stimulation with OPA (2.0 mM, 2:1 ratio) combined with MSC-ex (800 μg/ml) or PBS treatment for 24 h were observed by non-targeted lipidomics (n = 6 per group), followed by Venn diagram (F) and heat map (G) analyses of fatty acid metabolites. Data are represented as mean ± SEM. Statistical analyses by a one-way ANOVA (B, D, and E). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. normal group; #p <0.05, ##p <0.01, ###p <0.001 vs. PBS group. CCK-8, Cell Counting Kit-8; deMSC-ex, MSC-ex-free conditional medium supernatant; FDA, fluorescein diacetate; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; OPA, oleate and palmitate.
Fig. 3
Fig. 3
MSC-ex activate AMPK-mediated PPARα/CPT-1A and the SREBP-1C/FASn signalling pathway in hepatocytes and liver tissues. (A and B) mRNA-seq analyses were performed on L02 cells that were untreated (normal group) or subjected to OPA stimulation (2.0 mM, ratio of 2:1) combined with MSC-ex (800 μg/ml) (MSC-ex group) or PBS treatment (PBS group) for 24 h, n = 3 biological replicates per group. Histogram (A) and Venn diagram (B) analyses of differentially expressed genes. (C) Gene ontology analyses of differentially activated biological processes. (D) The top 10 enriched pathways as determined by the KEGG database. (E) Heat map of differentially expressed genes in the AMPK signalling pathway. (F and G) Evaluation of protein expression by immunoblotting (F) and quantification of the results (G). n = 3 biological replicates per group; ∗p <0.05, ∗∗∗p <0.001 vs. normal group; #p <0.05, ##p <0.01, ###p <0.001 vs. PBS group. (H and I) mRNA-seq analyses were performed on liver tissues in mice placed on a HFD for 10 weeks followed by 10 mg/kg MSC-ex or PBS treatment for 4 weeks. n = 3 biological replicates per group. The top 10 enriched pathways as determined by the KEGG database (H) and heatmap (I) of differentially expressed genes in the AMPK signalling pathway. (J and K) Immunoblotting analyses of AMPK signalling proteins in mice placed on a HFD for 10 weeks followed by 10 mg/kg MSC-ex or 10 mg/kg HFL1-ex or deMSC-ex and PBS treatment for 4 weeks (J) and quantification of the results (K). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. normal chow diet group; #p <0.05, ##p <0.01, ###p <0.001 vs. HFD group. (L) Immunofluorescence images of staining for p-AMPK, PPARα, CPT-1A, SREBP-1C, FASn, and p-IRS1 (green) in liver sections of mice. Nuclei were labelled with DAPI (blue). Scale bars, 100 μm. Data are represented as the mean ± SEM. Statistical analyses by a one-way ANOVA (G and K). AMPK, AMP-activated protein kinase; CPT-1A, carnitine palmitoyltransferase 1A; deMSC-ex, MSC-ex-free conditional medium supernatant; FASn, fatty acid synthase; HFD, high-fat diet; HFL1-ex, foetal HFL1-derived exosomes; HFL1, human lung fibroblast 1; IRS1, insulin receptor substrate 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; mRNA-seq, mRNA sequencing; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; NAFLD, non-alcoholic fatty liver disease; OPA, oleate and palmitate; p-AMPK, phosphorylated AMPK; p-IRS1, phosphorylated IRS1; PPARɑ, peroxisome proliferator-activated receptor alpha; SREBP-1C, sterol regulatory element-binding protein-1C; TCA, tricarboxylic acid cycle; TNF, tumour necrosis factor.
Fig. 4
Fig. 4
MSC-ex transport CAMKK1 into liver tissues and hepatocytes. (A and B) Proteomic profiling of MSC-ex as compared with HFL1-Ex was analysed by LC-MS/MS. (A) Protein classification diagram and (B) Biological process enrichment analyses. (C) A partial list of proteins involved in metabolic biological processes. (D) CAMKK1 expression in MSC-ex was examined by immunoblotting. (E and F) Representative confocal microscopy images of CD81 (green) and CAMKK1 (red) colocalisation in L02 cells subjected to OPA stimulation (2.0 mM, 2:1 ratio) in combination with MSC-ex (800 μg/ml), deMSC-ex, or PBS treatment for 24 h (E) and liver sections of mice fed a normal chow diet (n = 10) or HFD and injected with PBS (n = 10), 5 mg/kg MSC-ex (n = 10), or 10 mg/kg MSC-ex (n = 10) (F); nuclei were labelled with DAPI (blue). Scale bars, 20 μm. CAMKK1, calcium/calmodulin-dependent protein kinase 1; de-MSC-ex, MSC-ex-free conditional medium supernatant; HFD, high-fat diet; HFL1-ex, foetal HFL1-derived exosomes; HFL1, human lung fibroblast 1; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; OPA, oleate and palmitate.
Fig. 5
Fig. 5
Overexpression of CAMKK1 activates the AMPK signalling pathway and attenuates lipid accumulation in OPA-treated L02 and 293T cells. (A and B) The expression of AMPK signalling proteins in L02 and 293T cells transfected with pCAMKK1 (2 and 4 μg) or pCtr control vector before OPA stimulation (2.0 mM, 2:1 ratio) for 24 h was detected by immunoblotting (A) and quantified (B). (C and D) Intracellular lipid droplets in L02 and 293T cells were visualised by Nile red staining (C) and quantified by ImageJ for five random areas (D). Scale bars, 100 μm. (E and F) The expression of AMPK signalling proteins in L02 and 293T cells after transfection with pCAMKK1 (4 μg) or pCtr vector and OPA stimulation (2.0 mM, 2:1 ratio) with or without compound C (2 and 4 μM) treatment for 24 h was examined by immunoblotting (E) and quantified (F). (G and H) Intracellular lipid accumulation was visualised by Nile red staining (G) and quantified by ImageJ for three random areas (H). Data are represented as the mean ± SEM. Statistical analyses by unpaired two-tailed Student’s t test (B and D) and a one-way ANOVA (F and H). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001 vs. pCtr group; #p <0.05, ##p <0.01 vs. pCAMKK1 (4 μg) group. AMPK, AMP-activated protein kinase; CAMKK1, calcium/calmodulin-dependent protein kinase 1; CPT-1A, carnitine palmitoyltransferase 1A; FASn, fatty acid synthase; OPA, oleate and palmitate; p-AMPK, phosphorylated AMPK; PPARα, peroxisome proliferator-activated receptor alpha; SREBP-1C, sterol regulatory element-binding protein-1C.
Fig. 6
Fig. 6
Knockdown of CAMKK1 in MSC-ex inactivates the AMPK signalling pathway and increases lipid accumulation. (A) CAMKK1 expression in MSC-exshCtr and MSC-exshCAMKK1 was examined by immunoblotting. (B) Representative confocal microscopy images of CD63 (red) and CAMKK1 (green) colocalisation in L02 cells treated with OPA in combination with PBS, MSC-exshCtr, or MSC-exshCAMKK1; nuclei are labelled with DAPI (blue). Scale bars, 20 μm. (C and D) The expression of AMPK signalling proteins in L02 cells stimulated with OPA (2.0 mM, 2:1 ratio) and PBS, MSC-exshCtr (800 μg/ml), or MSC-exshCAMKK1 (800 μg/ml) for 24 h was detected by immunoblotting (C) and quantified (D). (E and F) Intracellular lipid droplets in L02 cells were visualised by Nile red staining (E) and quantified by ImageJ for three random areas (F). Scale bars, 100 μm. Data are represented as the mean ± SEM. Statistical analyses by a one-way ANOVA (D and F). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. PBS group; #p <0.05, ##p <0.01 vs. MSC-ex (800 μg/ml) group. AMPK, AMP-activated protein kinase; CAMKK1, calcium/calmodulin-dependent protein kinase 1; CPT-1A, carnitine palmitoyltransferase 1A; FASn, fatty acid synthase; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; OPA, oleate and palmitate; p-AMPK, phosphorylated AMPK; PPARα, peroxisome proliferator-activated receptor alpha; SREBP-1C, sterol regulatory element-binding protein-1C.
Fig. 7
Fig. 7
Knockdown of CAMKK1 in MSC-ex increases hepatic lipid accumulation and injury in HFD-fed mice. (A) C57BL/6 mice were placed on a HFD (40%) and administered 10 mg/kg of MSC-exshCtr or MSC-exshCAMKK1 i.v. from the 10th week to the 14th week of HFD feeding. As a control, the same volume of PBS was injected. (B) Immunoblotting analyses of CAMKK1 proteins in mice and quantification of the results. (C) Representative immunofluorescence confocal microscopy images of CD63 (green) and CAMKK1 (red) colocalisation; nuclei were labelled with DAPI (blue). Scale bars, 20 μm. (D) Representative images of gross liver appearance for mice fed a normal chow diet (normal group) or HFD for 10 weeks and then injected with PBS, MSC-exshCtr, or MSC-exshCAMKK1 for 4 weeks. (E) Representative images of H&E (top; scale bars, 50 μm) and Oil Red O (bottom; Scale bars, 20 μm) staining of liver sections. (F) Liver contents of total TG and TC, n = 10 per group. (G) Body weight changes of mice. (H) Changes in the liver indexes of mice (liver index = liver wet weight/body weight). (I) i.p. ITTs evaluated individual insulin tolerance by injecting insulin at a dose of 0.75 IU/kg body weight; blood glucose levels were detected at 0, 30, 60, 90, and 120 min and compared with that at 0 min. Individual glucose tolerance was assessed by IPGTT; fasted mice were administered 1 g of glucose/kg body weight by i.p. injection, and blood glucose levels were determined at 0, 30, 60, 90, and 120 min. (J) Fasting glycaemia (after 16-h fast) and serum insulin levels, n = 10 per group. (K and L) Serum TG and TC (K), and AST and ALT (L) levels, n = 10 per group. (M) Immunoblotting analyses of AMPK signalling proteins in livers and quantification. Data are represented as the mean ± SEM. Statistical analyses by a one-way ANOVA (F–M). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. HFD group; #p <0.05, ##p <0.01, ###p <0.001 vs. MSC-exshCtr (10 mg/kg) group. ALT, alanine transaminase; AMPK, AMP-activated protein kinase; AST, aspartate transaminase; CAMKK1, calcium/calmodulin-dependent protein kinase 1; CPT-1A, carnitine palmitoyltransferase 1A; FASn, fatty acid synthase; HFD, high-fat diet; IPGTT, i.p. glucose tolerance test; IRS1, insulin receptor substrate 1; ITT, insulin tolerance test; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; p-AMPK, phosphorylated AMPK; p-IRS1, phosphorylated IRS1; PPARα, peroxisome proliferator-activated receptor alpha; SREBP-1C, sterol regulatory element-binding protein-1C; TC, total cholesterol; TG, triglycerides.
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