The impact of cannabinoid receptor 1 absence on mouse liver mitochondria homeostasis: insight into mitochondrial unfolded protein response

Abstract

Introduction: The contribution of Cannabinoid type 1 receptor (CB1) in mitochondrial energy transduction mechanisms and mitochondrial activities awaits deeper investigations. Our study aims to assess the impact of CB1 absence on the mitochondrial compartment in the liver, focusing on both functional aspects and remodeling processes.

Methods: We used CB1^-/- and CB1^+/+ male mice. Cytochrome C Oxidase activity was determined polarographically. The expression and the activities of separated mitochondrial complexes and supercomplexes were performed by using Blue-Native Page, Western blotting and histochemical staining for in-gel activity. Key players of Mitochondrial Quality Control processes were measured using RT-qPCR and Western blotting. Liver fine sub-cellular ultrastructural features were analyzed by TEM analysis.

Results and discussion: In the absence of CB1, several changes in the liver occur, including increased oxidative capacity, reduced complex I activity, enhanced complex IV activity, general upregulation of respiratory supercomplexes, as well as higher levels of oxidative stress. The mitochondria and cellular metabolism may be affected by these changes, increasing the risk of ROS-related damage. CB1^-/- mice show upregulation of mitochondrial fusion, fission and biogenesis processes which suggests a dynamic response to the absence of CB1. Furthermore, oxidative stress disturbs mitochondrial proteostasis, initiating the mitochondrial unfolded protein response (UPR^mt). We noted heightened levels of pivotal enzymes responsible for maintaining mitochondrial integrity, along with heightened expression of molecular chaperones and transcription factors associated with cellular stress reactions. Additionally, our discoveries demonstrate a synchronized reaction to cellular stress, involving both UPR^mt and UPR^ER pathways.

Keywords: cannabinoid receptor 1; homeostasis; mitochondrial quality control; mitochondrial unfolded protein response; oxidative stress; respiratory chain supercomplexes.

Graphical abstract

FIGURE 1

CB1 deletion affects functional/structural organization of the respiratory chain. (A) Representative immunoblots of CI–CV respiratory chain complex protein levels in liver mitochondria of CB1^+/+ and CB1^−/− mice. The protein level was normalized to that of VDAC. Histograms show the results of densiometric analysis of immunoblots. BN-PAGE-based analysis of individual respiratory complexes from dodecylmaltoside-solubilized crude mitochondria from the liver of CB1^+/+ and CB1^−/− mice. (B) Representative image of a Coomassie blue stained BN-PAGE gel. Bands characteristic of individual OXPHOS complexes are recognizable. Molecular weights of standard proteins and the relative position of the respiratory complexes (I–V) are indicated. (C) Representative images of histochemical staining of complex I (I), complex IV (IV), and complex II (II) in-gel activity. (D) Densitometric quantification of the blue bands corresponding to individual complexes (arbitrary units, a.u). (E–G) Densitometric quantification of bands corresponding to individual in-gel activity of complex I (E), IV (F), and II (G) (arbitrary units, a.u). (H) Activity of Cytochrome C Oxidase in liver mitochondria of CB1^+/+ and CB1^−/− mice. Protein extracts were prepared for each animal, and each individual was assessed separately. Protein load was 15 μg/lane. All values are represented as mean ± SEM; (n = 5/group). Student’s t-test was used for statistical analysis. p < 0.05 was considered significant. *p < 0.05 vs. CB1^+/+. Abbreviations: COX, Cytochrome C Oxidase.

FIGURE 2

CB1 deletion affects the functional/structural organization of the respiratory chain. Blue native PAGE based analysis of digitonin-solubilized crude mitochondria from the liver of CB1^+/+ and CB1^−/− mice. (A) Representative image of a Coomassie blue stained BN-PAGE gel. Bands characteristic of OXPHOS supercomplexes are recognizable in all the experimental groups and highlighted in B, E (bands of obtained OXPHOS supercomplexes were numbered from 1 (top of the gel) to 16 (bottom of the gel). (C, F) Representative images of histochemical staining of in-gel activity of complex I (I) (C) and complex IV (IV) (F) supercomplexes (arrows). (D) Densitometric quantification of the blue bands corresponding to individual supercomplexes. (G, H) Densitometric quantification of bands corresponding to in-gel activity of supercomplex I (G) and IV (H). (D, G, H) x-axis: equal numbers correspond to the same bands in (A–C, E, F). Protein extracts were prepared for each animal, and each individual was assessed separately. Protein load was 15 μg/lane. Values are represented as mean ± SEM; (n = 5/group). Student’s t-test was used for statistical analysis. p < 0.05 was considered significant. *p < 0.05 vs. CB1^+/+.

FIGURE 3

CB1 deletion affects antioxidant defense status. (A) H₂O₂ levels (μmol/mg protein) in the liver of CB1^+/+ and CB1^−/− mice. (B) 4-HNE levels (pmol/g tissue) in the liver of CB1^+/+ and CB1^−/− mice. (C) Representative immunoblots of CATALASE, SOD2, and GPX1 in the liver of CB1^+/+ and CB1^−/− mice. The protein level was normalized to that of B-ACTIN. Histograms show the results of densiometric analysis of immunoblots. All values are represented as mean ± SEM; (n = 5/group). Student’s t-test was used for statistical analysis. p < 0.05 was considered significant. *p < 0.05 vs. CB1^+/+. Abbreviations: H₂O₂, hydrogen peroxide; 4-HNE, 4-Hydroxynonenal; CATALASE, Catalase; SOD2, Superoxide Dismutase 2; GPX1, Glutathione Peroxidase 1.

FIGURE 4

CB1 deletion affects biogenesis, fission, fusion, and mtDNA repair. (A), (C) and (E) mRNA expression of Nrf1, Nrf2, Pgc1α, Tfam, Opa1, Mfn1, Mfn2, Drp1, Ape1, Ogg1, Pol-γ in the liver of CB1^+/+ and CB1^−/− mice. The mRNA level was normalized to that of B-actin and Gapdh. (B), (D) and (F) Representative immunoblots of PGC1α, TFAM, NRF1, OPA1, MFN1, MFN2, DRP1, APE1, OGG1, POL-γ in the liver of CB1^+/+ and CB1^−/− mice. The protein level was normalized to that of B-ACTIN. Histograms show the results of densiometric analysis of immunoblots. All values are represented as mean ± SEM; (n = 5/group). Student’s t-test was used for statistical analysis. p < 0.05 was considered significant. *p < 0.05 vs. CB1^+/+. Abbreviations: PGC1α, Peroxisome Proliferative-Activated Receptor Gamma Coactivator 1α; NRF1, Nuclear Respiratory Factor 1; NRF2, Nuclear Respiratory Factor 2; TFAM, Mitochondrial Transcription Factor A; MFN1, Mitofusin 1; MFN2, Mitofusin 2; OPA1, Optic Atrophy 1; DRP1, Dynamin-Related Protein 1; POL-γ, DNA Polymerase γ; OGG1, 8-Oxoguanine Glycosylase 1; APE1, Apurinic/Apyrimidinic Endonuclease 1.

FIGURE 5

CB1 deletion affects autophagy/mitophagy processes. (A), (B) and (C) Representative immunoblots of LC3B, p62, PARKIN, PINK1, AMBRA1, P-ULK (Ser555), FUNDC1 in the liver of CB1^+/+ and CB1^−/− mice. Histograms show the results of densiometric analysis of immunoblots. The protein level was normalized to that of B-ACTIN and/or to the total forms for phosphorylated proteins. (D) Representative images of liver ultrastructure under TEM in CB1^+/+ and CB1^−/− mice. Scale bars: 2 μm. Red arrows pointed towards autophagosomes. All values are represented as mean ± SEM; (n = 5/group). Student’s t-test was used for statistical analysis. p < 0.05 was considered significant. *p < 0.05 vs. CB1^+/+. Abbreviations: LC3B, Microtubule-Associated Protein 1 Light Chain 3 Isoform B; SQSTM1/p62, Sequestosome 1; PARKIN, Parkin; PINK1, PTEN-Induced Kinase 1; AMBRA1, Activating Molecule in Beclin1-Regulated Autophagy; ULK, Unc-51 Like Autophagy Activating Kinase 1; FUNDC1, FUN14 Domain Containing 1.

FIGURE 6

CB1 deletion affects UPR^mt and UPR^ER processes. (A) and (B) Representative immunoblots of LONP1, CLPP, TRAP1, ATF4, ATF5, CHOP, P-PERK(Thr980), P-eiF2α(Ser51) in the liver of CB1^+/+ and CB1^−/− mice. The protein level was normalized to that of B-ACTIN and/or to the total forms for phosphorylated proteins. Histograms show the results of densiometric analysis of immunoblots. All values are represented as mean ± SEM; (n = 5/group). Student’s t-test was used for statistical analysis. p < 0.05 was considered significant. *p < 0.05 vs. CB1^+/+. Abbreviations: LONP1, Lon Peptidase 1; CLPP, Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit; TRAP1, TNF Receptor Associated Protein 1; ATF4, Activating Transcription Factor 4; ATF5, Activating Transcription Factor 5; CHOP, C/EBP Homologous Protein; PERK, Protein Kinase R (PKR)-Like Endoplasmic Reticulum Kinase; eiF2α, Eukaryotic Translation Initiation Factor 2A.