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. 2022 Jun 17;23(12):6764.
doi: 10.3390/ijms23126764.

SAMM50 Regulates Thermogenesis of Beige Adipocytes Differentiated from Human Adipose-Derived Stem Cells by Balancing Mitochondrial Dynamics

Se-Jun Park  1 , Dong-Hyun Shon  1   2 , Jae-Hyun Kim  1 , Yang-Hwan Ryu  1   3 , Yong Ko  1
Affiliations

SAMM50 Regulates Thermogenesis of Beige Adipocytes Differentiated from Human Adipose-Derived Stem Cells by Balancing Mitochondrial Dynamics

Se-Jun Park et al. Int J Mol Sci. .

Abstract

Brown/beige adipocyte thermogenesis is a process that is important for energy balance. The thermogenesis of brown/beige adipocytes occurs in the mitochondria, which is modulated by the dynamic balance between mitochondrial fusion and fission. Mitophagy is also involved in mitochondrial dynamics. The sorting and assembly machinery (SAM) complex protein, SAMM50, plays a key role in mitochondrial dynamics and quality control through regulating mitophagy. However, the roles of SAMM50 in the thermogenesis of beige adipocytes remain unknown. Thus, the objective of this study was to conduct functional analyses of SAMM50. The expression of mitochondrial fusion genes was repressed by SAMM50 knockdown but was not altered by SAMM50 overexpression. These results agreed with the distribution of the fluorescence-stained mitochondria and an mtDNA copy number. In contrast, the expression of mitochondrial fission genes showed an opposite outcome. As a result, suppression by the SAMM50 shRNA inhibited the expression of thermogenic genes (UCP1, PPARGC1A, DIO2, ELOVL3, CIDEA, and CIDEC) and mitochondrial-related genes (CYCS, COX7A1, TFAM, CPT1B, and CPT2). Conversely, SAMM50 overexpression promoted the expression of the thermogenic genes and mitochondrial genes. Thus, SAMM50 links the balance between the mitochondrial dynamics and thermogenesis of beige adipocytes.

Keywords: SAMM50; UCP1; adipose-derived stem cells; mitochondrial dynamics; obesity; thermogenic adipocytes.

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Conflict of interest statement

The authors declare that they have no competing interest or personal relationships that could have appeared to influence this paper.

Figures

Figure 1
Figure 1
The adipogenic differentiation of human adipose-derived stem cells. (A) Schematic images of human adipose-derived stem cell differentiation into white and beige adipocytes. (B) The cells were stained with Oil Red O. The lipid accumulation levels were measured by absorbance at 490 nm by extracting Oil Red O dye with isopropanol. (C) The expression levels of adipogenic genes were assayed by qPCR. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the hADSC group.
Figure 2
Figure 2
The establishment of beige adipocytes differentiated from human adipose-derived stem cells and SAMM50 expression in beige adipocytes. (A) qPCR analysis assessed UCP1 mRNA levels in the human adipose-derived stem cells and differentiated cells. (B,C) The expression levels of thermogenic (B) and beige-specific (C) genes were investigated by qPCR. (D,E) qPCR and Western blot analyses were performed to examine mRNA and protein levels of SAMM50 in the human adipose-derived stem cells and the differentiated cells. The protein levels of SAMM50 were quantified using ImageJ software. Results are shown as mean ± SEM. *** p < 0.005 compared to the hADSC and white group.
Figure 3
Figure 3
Knockdown and overexpression of SAMM50 in human adipose-derived stem cells. (A,B) qPCR and Western blot analyses were performed to determine mRNA and protein levels of SAMM50 in the shSAMM50-infected human adipose-derived stem cells using lentivirus. (C,D) qPCR and Western blot analyses were performed to assess mRNA and protein levels of SAMM50 in the human adipose-derived stem cells infected with FLAG-tagged SAMM50 using lentivirus. The protein levels of SAMM50 were quantified using ImageJ software. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the controls (NT and FLAG group, respectively).
Figure 4
Figure 4
Effects of SAMM50 knockdown and overexpression on the mitochondrial dynamics and biogenesis of beige adipocytes. (A,B) Relative mRNA levels of mitochondrial fusion (A) and fission (B) genes in beige adipocytes infected with shSAMM50 and FLAG-tagged SAMM50 were analyzed by qPCR. (C) The expression ratio of fusion/fission was calculated as the ratio of fusion genes to fission genes. (D,E) The expression levels of mitochondrial genes in beige adipocytes infected with shSAMM50 (D) and FLAG-tagged SAMM50 (E) were assayed by qPCR. (F) Representative fluorescence images of beige adipocytes infected with shSAMM50 and FLAG-tagged SAMM50 were stained with MitoTracker (red). The fluorescence levels of MitoTracker were quantified using ImageJ software. (G) Relative mtDNA copy numbers in beige adipocytes infected with shSAMM50 and FLAG-tagged SAMM50 were investigated by qPCR. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the controls.
Figure 4
Figure 4
Effects of SAMM50 knockdown and overexpression on the mitochondrial dynamics and biogenesis of beige adipocytes. (A,B) Relative mRNA levels of mitochondrial fusion (A) and fission (B) genes in beige adipocytes infected with shSAMM50 and FLAG-tagged SAMM50 were analyzed by qPCR. (C) The expression ratio of fusion/fission was calculated as the ratio of fusion genes to fission genes. (D,E) The expression levels of mitochondrial genes in beige adipocytes infected with shSAMM50 (D) and FLAG-tagged SAMM50 (E) were assayed by qPCR. (F) Representative fluorescence images of beige adipocytes infected with shSAMM50 and FLAG-tagged SAMM50 were stained with MitoTracker (red). The fluorescence levels of MitoTracker were quantified using ImageJ software. (G) Relative mtDNA copy numbers in beige adipocytes infected with shSAMM50 and FLAG-tagged SAMM50 were investigated by qPCR. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the controls.
Figure 5
Figure 5
Effects of SAMM50 knockdown or overexpression on the expression of UCP1 in beige adipocytes. (A,B) The expression levels of UCP1 in beige adipocytes infected with shSAMM50 (A) and FLAG-tagged SAMM50 (B) were analyzed using qPCR. (C,D) Representative fluorescence images of beige adipocytes infected with shSAMM50 (C) and FLAG-tagged SAMM50 (D) were stained with DAPI (blue) and anti-UCP1 (green), respectively. The fluorescence levels of UCP1 were quantified using ImageJ software. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the controls.
Figure 5
Figure 5
Effects of SAMM50 knockdown or overexpression on the expression of UCP1 in beige adipocytes. (A,B) The expression levels of UCP1 in beige adipocytes infected with shSAMM50 (A) and FLAG-tagged SAMM50 (B) were analyzed using qPCR. (C,D) Representative fluorescence images of beige adipocytes infected with shSAMM50 (C) and FLAG-tagged SAMM50 (D) were stained with DAPI (blue) and anti-UCP1 (green), respectively. The fluorescence levels of UCP1 were quantified using ImageJ software. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the controls.
Figure 6
Figure 6
The effects of SAMM50 knockdown and overexpression on the thermogenesis of beige adipocytes. (A,B) The expression levels of thermogenic genes in beige adipocytes infected with shSAMM50 (A) and FLAG-tagged SAMM50 (B) were analyzed by qPCR analysis. (C,D) Western blot analysis was performed to determine the protein levels of PPARGC1A and UCP1 in the shSAMM50 (C) and FLAG-tagged SAMM50 (D) -infected beige adipocytes. The protein levels of PPARGC1A and UCP1 were quantified using ImageJ software. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the controls.
Figure 6
Figure 6
The effects of SAMM50 knockdown and overexpression on the thermogenesis of beige adipocytes. (A,B) The expression levels of thermogenic genes in beige adipocytes infected with shSAMM50 (A) and FLAG-tagged SAMM50 (B) were analyzed by qPCR analysis. (C,D) Western blot analysis was performed to determine the protein levels of PPARGC1A and UCP1 in the shSAMM50 (C) and FLAG-tagged SAMM50 (D) -infected beige adipocytes. The protein levels of PPARGC1A and UCP1 were quantified using ImageJ software. Results are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005 compared to the controls.
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