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. 2024 May 31;14(1):12521.
doi: 10.1038/s41598-024-63525-7.

Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status

Patricia Marqués  1 , Jesús Burillo  2 , Carlos González-Blanco  1   2   3   4 , Beatriz Jiménez  1   2   3 , Gema García  2   3 , Ana García-Aguilar  2   3   5 , Sarai Iglesias-Fortes  1 , Ángela Lockwood  1   2   3   4 , Carlos Guillén  6   7   8   9
Affiliations

Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status

Patricia Marqués et al. Sci Rep. .

Abstract

Sirtuin1 (SIRT1) activity decreases the tuberous sclerosis complex 2 (TSC2) lysine acetylation status, inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) signalling and concomitantly, activating autophagy. This study analyzes the role of TSC2 acetylation levels in its translocation to the lysosome and the mitochondrial turnover in both mouse embryonic fibroblast (MEF) and in mouse insulinoma cells (MIN6) as a model of pancreatic β cells. Resveratrol (RESV), an activator of SIRT1 activity, promotes TSC2 deacetylation and its translocation to the lysosome, inhibiting mTORC1 activity. An improvement in mitochondrial turnover was also observed in cells treated with RESV, associated with an increase in the fissioned mitochondria, positive autophagic and mitophagic fluxes and an enhancement of mitochondrial biogenesis. This study proves that TSC2 in its deacetylated form is essential for regulating mTORC1 signalling and the maintenance of the mitochondrial quality control, which is involved in the homeostasis of pancreatic beta cells and prevents from several metabolic disorders such as Type 2 Diabetes Mellitus.

Keywords: Acetylation; Lysosome; Mitophagy; Pancreatic β cells; TSC2; mTORC1.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
TSC2 localizes in the lysosomal membrane when it is deacetylated in MEFs. (A) Immunofluorescence showing the association between LAMP1 (red channel) and TSC2 (green channel) in MEF TSC2+/+ and Sirt1+/+ cells under control conditions and after resveratrol treatment at 50 µM. The graphs represent the Manders’ colocalization coefficient M1 of TSC2-LAMP1 in either MEF Tsc2+/+ and MEF Sirt1+/+. The values represent means and SD; n = 3 independent experiments. *p ≤ 0.05 comparing MEF Tsc2+/+ control versus resveratrol treatment and MEF Sirt1+/+ control and after the addition of resveratrol. Bars, 20 μm. (B) Immunoprecipitation of TSC2 and western blotting against LAMP2A under normal conditions and after the treatment with resveratrol at 50 µM in either MEF Tsc2+/+, Tsc2−/−, Sirt1+/+ and Sirt1−/−. It is shown in the last lane a negative control using a non-specific antibody for the immunoprecipitation. It is also shown in the whole cell extracts (WCE) the mTORC1/p70S6K signalling pathway with the corresponding quantification and statistical analysis. (C,D) Representative western blots corresponding to the mTORC1 and AMPK signalling pathways using either nicotinamide (5 mM), resveratrol (50 µM) or control conditions either comparing MEF Tsc2+/+ and −/− cells (C) or MEF Sirt1+/+ and −/− cells in (D). It is shown in the graphs (in C and in D) the densitometry corresponding to the P-ACC/ACC ratio in all the cell lines analyzed. The values represent means and SD; n = 6. **p ≤ 0.01, ***p ≤ 0.001 MEF Tsc2+/+ and Sirt1+/+ vs MEF Tsc2−/− and Sirt1−/−.
Figure 2
Figure 2
Acetyl CoA disrupts the interaction between TSC2 and lysosome in MIN6 pancreatic beta cells. (A) Immunofluorescence analyzing the colocalization signal between TSC2 and LAMP1 proteins in MIN6 Scr cells. The graphs represent the Manders’ colocalization coefficient M1 of TSC2-LAMP1 after NAM or RESV treatments. The values represent means and SD; n = 4 independent experiments. Bars, 20 μm. (B) Immunofluorescence analyzing the colocalization signal between Rheb and LAMP1 proteins in MIN6 Scr cells. The graphs represent the Manders’ colocalization coefficient M1 of Rheb-LAMP1 after NAM or RESV treatments. The values represent means and SD; n = 4 independent experiments. Bars, 20 μm. (C) Immunofluorescence studying the colocalization signal between TSC2 and LAMP1 in MIN6 Scr cells after the stimulation with acetyl-CoA at either 10 or 100 µM. The values represent means and SD; n = 3 independent experiments. One-way ANOVA was performed with Tukey's multiple comparisons test as post hoc. *p ≤ 0.05 comparing control versus AcCoA 100 µM. Bars, 20 μm. (D) Representative western blots showing the acetylation status of TSC2 protein in response to increasing concentration of Acetyl-CoA and its reversion with resveratrol. The graph represents the densitometric analysis of Ac-TSC2/TSC2 and phospho-p70 (Thr 389)/p70 ratios. *p ≤ 0.05 and **p ≤ 0.01 comparing control versus either AcCoA 30 µM and AcCoA 100 µM. #p ≤ 0.05 and ###p < 0.001 comparing AcCoA at either 30 µM or 100 µM in the absence of resveratrol with the presence of resveratrol. (E) Representative western blots corresponding to the mTORC1 and AMPK signalling pathways using either nicotinamide (5 mM), resveratrol (50 µM) or control conditions in MIN6 Scr cells and MIN6 Tsc2 shRNA cells. The graph represents the densitometric analysis of the P-ACC/ACC ratio comparing both cell lines. The values represent mean and SD; n = 6. **p < 0.01, ***p ≤ 0.001 MEF Tsc2+/+ and Sirt1+/+ vs MEF Tsc2−/− and Sirt1−/−. **p ≤ 0.01, ***p ≤ 0.001 MIN6 Tsc2 Scr vs MIN6 Tsc2 shRNA.
Figure 3
Figure 3
Resveratrol induces both autophagic and mitophagic fluxes in MIN6 Scr pancreatic beta cells. (A) Representative western blots of different mitophagy markers in MIN6 Scr and MIN6 Tsc2 shRNA pancreatic beta cells treated or not with resveratrol at 50 µM using a time-course analysis. (B) The graphs represent the densitometric study showing the mean and SD; n = 4 independent experiments. One-way ANOVA was performed with Tukey's multiple comparisons test as post hoc. *p ≤ 0.05, **p ≤ 0.01 MIN6 Tsc2 Scr vs MIN6 Tsc2 shRNA; #p ≤ 0.05 MIN6 treated with resveratrol 50 µM vs control. (C) Transmission electronic microscopy (TEM) images obtained from MIN6 Tsc2 Scr and Tsc2 shRNA treated or not with resveratrol 50 µM. The bar represents 0.2 µm. *p ≤ 0.05 and **p ≤ 0.01 comparing control versus resveratrol in the MIN6 Scr cells. (D) Representative western blots for the study of autophagic and mitophagic fluxes in MIN6 Scr and Tsc2 shRNA pancreatic beta cells treated with either nicotinamide (5 mM) or resveratrol (50 µM) in the presence or in the absence of a pre-treatment with chloroquine (CQ) for 24 h. The graphs correspond to the densitometric analysis of all the blots showing the mean ± SD, n = 3. One-way ANOVA was performed with Tukey's multiple comparisons test as post hoc. **p ≤ 0.01, ***p ≤ 0.001 MIN6 Tsc2 Scr vs MIN6 Tsc2 shRNA.
Figure 4
Figure 4
Resveratrol induces mitophagy and mitochondrial biogenesis by regulating PGC1-α acetylation in MIN6 Scr pancreatic beta cells. (A) Western blotting analysis showing the study of different proteins from the different mitochondrial compartments for study mitophagy at 24 h in response to either rapamycin, CCCP or resveratrol. The graphs correspond to the quantification analysis of the protein changes in response to the different stimuli expressed in percentage (n = 3). (B) Immunoprecipitation analysis of PGC1-α for analyzing the acetylation status of the protein in response or not to resveratrol for 4 h in MIN6 Scr and Tsc2 shRNA cells. The graph corresponds to the quantification analysis showing the fold increase in the ratio of Ac-PGC1-α/PGC1-α (n = 3).
Figure 5
Figure 5
Scheme depicting the most important results obtained in the manuscript indicating the role of resveratrol in the control of acetylation status of TSC2 and its recruitment to the lysosomal membrane. It shows the involvement of resveratrol in the maintenance of a healthy pool of mitochondrial by enhancing mitophagy and mitochondrial biogenesis.
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