The endocannabinoid system (ECS), comprising cannabinoid type 1 receptors (CB1) and type 2 receptors (CB2), their natural ligands termed endocannabinoids, and the enzymes responsible for their synthesis and degradation, is widely distributed throughout central and peripheral tissues (Pertwee, 2015). This system plays a pivotal role in regulating various physiological processes such as energy homeostasis, synaptic plasticity, and feeding behaviors (Lu and Mackie, 2016). CB1, in particular, has been extensively studied for its involvement in modulating central and peripheral processes to regulate energy metabolism. It seems very important, especially in contemporary society where obesity and metabolic syndrome have reached epidemic proportions due to an increasingly sedentary lifestyle and a diet high in processed food. Notably, hyperactivation of CB1 has been implicated in promoting metabolic processes leading to weight gain, lipogenesis, insulin resistance, and dyslipidemia (Cota et al., 2003; Di Marzo et al., 2001; Liu et al., 2012). Pharmacological inhibition or genetic deletion of CB1 has shown promise in ameliorating metabolic abnormalities in obese animals, leading to weight loss and improving insulin sensitivity (Jourdan et al., 2010). The benefits of ECS activity are greater when food is scarce. In contrast, ECS activity promotes obesity and metabolic syndrome when food is plentiful, suggesting a reactive homeostatic mechanism to optimize survival (Mazier et al., 2015; Nunn et al., 2010). It has been proposed that the increased ECS tone during metabolic syndrome may be considered a homeostatic protective mechanism induced by oxidative stress. Thus, the increased ECS activity might start as an attempt to compensate for metabolic dysfunctions, in the long term, it may exacerbate the metabolic syndrome (Nunn et al., 2010).

In addition to their central distribution, CB1 receptors are also widely expressed in peripheral tissues, including the liver (Mackie, 2005; Tam et al., 2011). Under normal physiological conditions, CB1 is not highly expressed in the liver. However, in pathological conditions such as obesity or hepatic steatosis induced by alcohol or nonalcoholic factors, CB1 expression is significantly upregulated, playing a pivotal role in hepatic insulin resistance, fibrosis, and lipogenesis (Busquets-García et al., 2022; Liu et al., 2012; Osei-Hyiaman et al., 2008). Remarkably, animals lacking hepatic CB1 do not develop dyslipidemia, insulin/leptin resistance, or steatosis when exposed to a high-fat diet (HFD) (Wang et al., 2021).

The liver, being a principal organ responsible for regulating metabolic homeostasis, relies heavily on the quality and quantity of hepatic mitochondria to perform metabolic activities efficiently. Hepatocytes harbor a large number of mitochondria to carry out various metabolic functions, primarily dependent on the integrity of Electron Transport Chain (ETC.) complexes and their organization into supercomplexes (Avram et al., 2022; Giacomello et al., 2020; Letts and Sazanov, 2017). These supramolecular arrangements play a crucial role in metabolic adaptation, responding to changes in the metabolic source of electrons and enhancing the efficiency of respiratory chain reactions (Kobayashi et al., 2023). In addition to energy production, mitochondria are also a significant source of Reactive Oxygen Species (ROS). Mitochondrial ROS, generated by the respiratory chain, play a central role in numerous cellular signaling pathways both within and outside the mitochondria (Finkel, 2012). However, excessive ROS production can lead to oxidative stress, characterized by an imbalance between mitochondrial ROS production and removal, ultimately affecting various cellular components, including mitochondrial DNA (mtDNA) (Bolisetty and Jaimes, 2013; Valko et al., 2007).

The CB1 plays an important role in mediating the biological effects of cannabinoids, whether they are derived from plants (phytocannabinoids), synthetically produced (synthetic cannabinoids), or naturally occurring in the body (endocannabinoids) (Blebea et al., 2024; Di Marzo and Piscitelli, 2015). Phytocannabinoids are phenolic compounds and can induce metabolic effects via CB receptors, but a lot of their actions appear to be independent of CB receptors, including direct effects on mitochondria (Nunn et al., 2010). Phytocannabinoids have been shown to affect mitochondrial function in various ways. The mitochondria, being central to cellular energy production, apoptosis regulation, and ROS balance, are directly influenced by phytocannabinoids like THC (Δ9-tetrahydrocannabinol) and CBD (cannabidiol) (Pagano et al., 2023; Podinic et al., 2024; Podinić et al., 2023; Rupprecht et al., 2022). The scientific literature offers many reviews and original research discussing the relationship between phytocannabinoids and mitochondria (Athanasiou et al., 2007; Bartova and Birmingham, 1976; Chan and Duncan, 2021; Fišar et al., 2014; Mato et al., 2010; Mould et al., 2021; 2021; Nunn et al., 2023; Olivas-Aguirre et al., 2019; Ryan et al., 2009). Since 1970, several studies have analyzed the effects of phytocannabinoids on mitochondrial function. The first studies were performed in the years ‘71 and ’72. These studies have been demostrated that THC could inhibit the ATPase activity of rat liver mitochondria and strongly affected rat liver mitochondria in vitro. At concentrations of 15–60 nmoles/mg of mitochondrial protein, THC uncoupled state IV respiration. It has been also demonstrated that THC could inhibit complex I and III of the ETC (Bartova and Birmingham, 1976). Bino and colleagues demonstrated that THC could alter the mitochondrial shape in response to the dose, which correlated with their respiratory rate (Bino et al., 1972). Research performed by another group confirmed that THC could induce mitochondrial swelling in several different tissues in rats (Banerji et al., 1985). According to Devane and colleagues, the brain contains cannabinoid receptors. Consequently, the endocannabinoid system was discovered, and phytocannabinoids and their research have increased (Devane et al., 1988).

In the 2000s, interest in the study between mitochondria and phytocannabinoids increased exponentially, suggesting that both THC and CBD can modulate mitochondrial function (Athanasiou et al., 2007; Sarafian et al., 2003; Whyte et al., 2010). THC is also known to disrupt neuronal mitochondrial function, affecting complexes I, II, and III, decreasing coupling, and enhancing ROS production (Wolff et al., 2015). Later Rimmerman and colleagues studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. They showed that CBD treatment led to a biphasic increase in intracellular calcium levels and changes in mitochondrial function and morphology leading to cell death. They proposed that CBD treatment induced mitochondrial swelling in a process involving VDAC1 and mitochondrial permeability transition pore (MPTP). These effects appeared to result from direct CBD-induced inhibition of the VDAC1 channel conductance (Rimmerman et al., 2013). Olivas-Aguirre and colleagues have demonstrated that CBD directly modulates mitochondrial calcium in lymphoblastic leukemic cells (Olivas-Aguirre et al., 2019). In addition, many compounds in plants can affect mitochondrial dynamics as well as mitochondrial function. Alistar V. W. Nunn and colleagues found that phytocannabinoids can affect mitochondrial dynamics (Nunn et al., 2020). Using MCF7 cancer cells, they showed that CBD has a dose-dependent impact on mitochondrial metabolism and morphology, and there is evidence that it induces oxidative stress at higher concentrations (Mould et al., 2021). Moreover, phytocannabinoids can influence mitochondrial dynamics, which involves the processes of fission and fusion that maintain mitochondrial health and adaptability (Malheiro et al., 2023; Walker et al., 2021).

In addition to the relationship between phytocannabinoids and mitochondria, several studies in the literature have also focused their attention on the involvement of the ECS in mitochondrial function. The ECS has evolved in a living organism to ensure the survival of the animal, and because of this, it interacts and modulates other systems, such as the mitochondria (Gomez et al., 2011; Howlett et al., 2010; Moreira et al., 2012; Nunn et al., 2012; Puighermanal et al., 2009; Velasco et al., 2005). Several studies suggested that THC, AEA, and HU210 could inhibit mitochondrial function. Catanzaro et al. have also shown that AEA increased mitochondrial swelling and reduced cytochrome C release associated with reduced membrane potential and increased membrane fluidity (Athanasiou et al., 2007; Catanzaro et al., 2009). As suggested by Nunn and colleagues, all the studies reported above support the idea that ECS modulates pathways that are known to affect mitochondrial activity, reinforcing the idea that all systems are interconnected to adapt to a variable environment. The fact that the ECS regulates several pathways that act on mitochondrial activity cannot be a coincidence. The results shown above, in particular, shed light on a multiphasic stimulus-adaptation response (Nunn et al., 2012).

To maintain proper mitochondrial and cellular function, cells employ various surveillance measures, including quality control mechanisms, to counteract dysregulated mitochondrial activity (Ni et al., 2015). Mechanisms for maintaining mitochondrial quality include mitochondrial biogenesis, fusion and fission, mitophagy, mtDNA repair, and the mitochondrial Unfolded Protein Response (UPR^mt), all of which require accurate communication and coordination between the nuclear and mitochondrial genomes. Mitochondrial fusion and fission are crucial processes governing the morphology and function of the mitochondrial network. A mitochondrial fusion response may be seen as a response to the increased oxidative stress and serves several purposes (Adebayo et al., 2021; Al Ojaimi et al., 2022). While fusion promotes mitochondrial network integrity and functionality, it also creates a platform for efficient mitophagy (Shirihai et al., 2015; Twig et al., 2008). The UPR^mt functions as a signaling pathway, contributing to maintaining mitochondrial quality and ensuring the integrity of the mitochondrial proteome (Haynes and Ron, 2010). When misfolded proteins or incomplete complexes accumulate beyond the folding capacity, it disrupts proteostasis, causing damage and dysfunction within the organelle and the cell. While extensively studied in the Endoplasmic Reticulum (ER), recent findings suggest that similar signaling mechanisms operate in mitochondria, facilitating communication with the nucleus in response to proteostasis impairment (Cagin and Enriquez, 2015).

The UPR^mt is triggered by various types and levels of stress, particularly in conditions where unfolded or misfolded mitochondrial proteins accumulate and form aggregates (Haynes and Ron, 2010). It restores mitochondrial protein homeostasis and normalizes organelle function by decreasing protein levels, facilitating protein folding, or enhancing protein degradation (Fiorese and Haynes, 2017).

In mammals, the canonical UPR^mt signaling cascade comprises transcription factors such as C/EBP Homologous Protein (CHOP), Activating Transcription Factor 4 (ATF4), and Activating Transcription Factor 5 (ATF5), with ATF5 playing the central role as the main regulator (Fiorese et al., 2016; Zhao et al., 2002). Although ATF4 and CHOP have been reported to play a crucial role in UPR^mt regulation by promoting ATF5 transcription, the exact communication mechanism is not fully understood. UPR^mt activation begins with the translocation of these transcription factors into the nucleus, where they stimulate the transcription of proteases such as Caseinolytic mitochondrial matrix Peptidase Proteolytic subunit (CLPP) and Lon Peptidase 1 (LONP1), as well as Chaperones like Heat Shock Protein 60 (HSP60) and TNF Receptor-Associated Protein 1 (TRAP1), which constitute the core of restoring protein balance. They localize and operate within mitochondria in response to the accumulation of misfolded proteins or an imbalance between mitochondrial and nuclear encoded proteins within the organelle (Rd et al., 1996; Zhao et al., 2002). These components play a critical role in promoting protein folding and preventing protein aggregation across various cell types (Bukau et al., 2006).

Recent research indicates that UPR^mt can be activated under various conditions, including damage to, ETC., alterations in mitochondrial dynamics, deletion of mtDNA, and elevated levels of ROS (Muñoz-Carvajal and Sanhueza, 2020).

Similarly, to the UPR^mt, the Unfolded Protein Response of the endoplasmic reticulum (UPR^ER) maintains proteomic stability within the ER (Wang and Kaufman, 2012). Since mitochondria and the ER serve as primary centers for energy metabolism and protein synthesis in cells, it is likely that a synergistic mechanism exists between UPR^mt and UPR^ER to collectively combat stressors such as increased ROS levels. Mounting evidence suggests that the protein kinase RNA (PKR)-like Endoplasmic Reticulum Kinase (PERK) signaling pathway serves as a crucial node for coordinating UPR^mt and UPR^ER (Liu et al., 2014). The PERK pathway becomes activate in both UPR^mt and UPR^ER, with its downstream molecules, including ATF4, CHOP, and ATF5, playing crucial functions.

As a result of metabolic stimuli and other changes within mitochondria, the activation of retrograde mitochondrial to nuclear signaling can lead to changes in nuclear gene expression. The activation of this signaling is essential to facilitate the reestablishment of homeostasis by refolding or removing unfolded or misfolded proteins (Arnould et al., 2015). Furthermore, it has been suggested that mitochondria-nucleus communication plays a crucial role in mitochondrial stress responses (Haynes et al., 2007; Haynes and Ron, 2010; Yoneda et al., 2004).

In recent years, accumulating evidence has indicated that the endocannabinoid system has the capacity to regulate both the integrity and functionality of mitochondria, thereby contributing to the maintenance of cellular energy homeostasis (Lipina et al., 2014). Recent research has elucidated the involvement of CB1 and its signaling pathways in various mitochondrial activities critical for regulating energy metabolism in both the brain and peripheral organs (Busquets-García et al., 2022; Pagano Zottola et al., 2022; Paszkiewicz et al., 2020; Senese et al., 2024). Given that CB1 mRNA levels in the liver are low under normal conditions, but increase in specific liver dysfunctions such as steatosis, studies have predominantly focused on obesogenic conditions such as HFD (Liu et al., 2012, p. 1; Wang et al., 2021). Moreover, although studies on CB1-deficient mice have enhanced our comprehension of CB1 signaling’ role in regulating liver metabolic functions, there remains a gap in our knowledge regarding the modulation of mitochondria, a dynamic cell compartment. It has also been shown cannabinoids disturb calcium homeostasis via multiple mechanisms, including stimulation of CB1 (Lauckner et al., 2005). In addition, while some studies have suggested that cannabinoid-induced increases in cytosolic calcium levels are a result of extracellular Ca2+ influx, others suggest that intracellular stores, such as the endoplasmic reticulum and mitochondria, may be responsible (Drysdale et al., 2006; Mato et al., 2010; Rossi et al., 2019; Ryan et al., 2009). The mechanism underlying the regulation of mitochondrial calcium by cannabinoids does not appear to depend on plasma membrane receptors but rather occurs through the direct targeting of mitochondria (Catanzaro et al., 2009; Ma et al., 2015; Olivas-Aguirre et al., 2019). Therefore, further investigations into the role of CB1 in regulating mitochondrial processes are essential for a deeper understanding of how this regulation maintains mitochondrial homeostasis by facilitating constant interaction among various response mechanisms.

In this context, our study aims to assess the impact of CB1 absence on liver mitochondria, focusing on both functional aspects and remodeling processes, utilizing a CB1^−/− male mouse model. Specifically, we investigate changes in respiratory capacity, respiratory chain complexes and supercomplexes, mitochondrial ROS production and antioxidant defenses. In addition, we evaluate MQC mechanisms, including biogenesis, dynamics, encompassing fusion and fission processes, mitophagy, and mtDNA repair. Furthermore, given the paucity of data on the relationship between CB1 activation/blockade and UPR^mt, we explore the involvement of UPR^mt pathways in maintaining liver mitochondrial proteostasis and homeostasis in CB1^−/− mice.

Mitochondria exhibit high adaptability and dynamism, playing crucial roles in cellular metabolism and stress responses. Serving as a center for biochemical processes such as Adenosine Triphosphate (ATP) production, synthesis of fatty acids, generation of intracellular ROS, thermogenesis, and calcium homeostasis, mitochondria are recognized as the central regulators of energy metabolism. Therefore, ensuring the balance and proper functioning of mitochondria, known as mitochondrial homeostasis and proteostasis, is vital for overall cellular health. The CB1 are implicated in regulating tissue metabolism not only by influencing mitochondrial activity but also by maintaining mitochondrial integrity.

Studies indicate that CB1 agonists activate CB1 to induce intra-mitochondrial processes important for synaptic transmission (Djeungoue-Petga and Hebert-Chatelain, 2017). Additionally, research suggests that CB1 regulate mitophagy in hippocampal neurons, with CB1^−/− mice displaying altered mitochondrial dynamics and reduced mitophagy activity compared to CB1^+/+ mice (Beji et al., 2020). Furthermore, the blockade of CB1 has been linked to promoting mitochondrial biogenesis through the expression of endothelial Nitric Oxide Synthase (NOS) in white adipocytes, potentially involving 5′AMP-activated protein kinase (AMPK) in this process (Tedesco et al., 2008). On the contrary, stimulation of CB1 has been associated with impairing mitochondrial biogenesis in various tissues such as White Adipose Tissue (WAT), muscle, and liver (Tedesco et al., 2010). This suggests a dual role for CB1 in mitochondrial function, where activation and blockade can have contrasting effects on mitochondrial processes. Moreover, the presence of CB1 on mitochondrial membranes of different cell types underscores their involvement in regulating mitochondrial functions under pato-physiological conditions (Aquila et al., 2010; Bénard et al., 2012; Kamnate et al., 2022; Mendizabal-Zubiaga et al., 2016; Pagano Zottola et al., 2022).

The present research reveals that when CB1 are lacking, various adaptive response mechanisms come into play to preserve mitochondrial function, thereby seeking to ensure liver function.

Our study initially confirms that the absence of CB1 in CB1^−/− mice significantly affects body weight regulation, with these mice displaying lower body weight than their wild-type counterparts (Table 1). This result aligns with previous research illustrating the crucial role of CB1 in modulating appetite, food intake, and energy expenditure (Cota et al., 2003). Furthermore, the decrease in body weight observed in CB1^−/− mice is accompanied by a substantial reduction of the weight of sWAT, vWAT, and eWAT depots, highlighting the central role of CB1 in the modulation of body adiposity (Rossi et al., 2018). Additionally, CB1^−/− mice show a significant increase in BAT weight, suggesting activation of thermogenic mechanisms that might lead to WAT loss in such animals (Table 1). All these changes in fat distribution could be reflected in improved insulin signaling.

Our observations show that the liver, a central organ for metabolic regulation, exhibits adaptative mechanisms in response to CB1 deficiency. CB1^−/− mice display a significant increase in liver oxidative capacity, as indicated by increased COX activity (Figure 1). COX is a crucial respiratory complex involved in the mitochondrial electron transport chain, and its increased activity could imply heightened mitochondrial oxygen consumption in CB1^−/− mice. The study by Tedesco et al. linking CB1 stimulation to decreased mitochondrial oxygen consumption is in line with this result (Tedesco et al., 2010). Moreover, increased mitochondrial respiration was observed in rats treated with Rimonabant (Flamment et al., 2009), a selective CB1 antagonist with an inverse agonist profile. It was shown that the treatment with Rimonabant led to an increase in total energy expenditure in lean rats, in ob/ob mice, and in rats fed a high-fat and high-carbohydrate diet (Herling et al., 2008; Kunz et al., 2008; Liu et al., 2005). Rimonabant was able to reduce appetite thereby promoting weight loss, improved lipid profiles, and enhanced insulin sensitivity, potentially lowering the risk of type 2 diabetes (Bronander and Bloch, 2007; Thornton-Jones et al., 2006). Additionally, it increases Hypotalamus-Pituitary-Adrenal (HPA) activity and corticosteroid production in food-deprived Zucker rats, suggesting that it activates a stress response (Doyon et al., 2006; Nunn et al., 2009; Steiner et al., 2008). These results initially seemed like a promising approach for managing obesity and metabolic disease in people with poor lifestyle-induced phenotypes. Nevertheless, its severe psychiatric side effects and the broader impact on the ECS led to its withdrawal from the market. Additionally, recent research describes how the activation of mitochondrial CB1 reduces OXPHOS and hampers the metabolism of glucose in mouse astroglia (Jimenez-Blasco et al., 2020).

Increased mitochondrial respiration in CB1^−/− mice may result from the influence of CB1 deficiency on the activity and/or expression of other mitochondrial electron transport chain complexes. Our study shows, in the liver of CB1^−/− mice, in the absence of significant differences in the protein levels of individual OXPHOS complexes, reduced in-gel activity of complex I and increased in-gel activity of complex IV, paralleled by an even not significant increase of complex II activity (Figure 1). These variations suggest significant changes in the function of the mitochondrial electron transport chain. Complex I is the largest respiratory chain complex, the most sensitive to oxidative damage and one of the sites of ROS production during normal respiration (Koopman et al., 2010; Lenaz et al., 2016; Vinogradov and Grivennikova, 2016). Its inhibition has been reported to elevate oxidative stress, even if differential effects have also been described (Fato et al., 2009; Yin et al., 2021). Likely, reduced activity of complex I observed in CB1^−/− mice could be strictly associated with the increased ROS production concomitantly reported in such animals. On the other hand, the reduction of Complex I activity could be a mechanism to prevent oxidative stress induced by ROS release along, ETC. Furthermore, one might speculate that liver cellular metabolism changed in CB1^−/− mice, with a reduced use of NADH-substrates and a preference for FADH2-one oxidation. It is possible that this preference for succinate as a substrate will increase the electron transport chain’s rate to support the cell’s energy requirements. However, these speculations suggest further investigation.

Concomitant with the increased oxidative capacity, CB1^−/− mice exhibit elevated liver ROS production, mitochondrial H₂O₂ release and 4-HNE levels (Figure 3), suggesting a potential pro-oxidative effect resulting from CB1 deficiency within the liver. Furthermore, the protein levels of SOD2 and glutathione GPX1 are significantly decreased in CB1^−/− mice (Figure 3). These results are similar to those obtained by Leal et al. who demonstrated that CB1 deficiency in the skin led to an increased production of ROS and a reduction in antioxidant defenses. They postulated that a substantial decline in CB1 expression might contribute to the early aging-like changes observed in diabetes (C Leal et al., 2021). Together, these observations indicate a complex interplay between CB1 signaling and oxidative stress regulation, with potential tissue-specific variations in the response to CB1 deficiency.

Overall, our results suggest that the disruption of CB1 signaling may lead to several changes in the liver, including increased oxidative capacity, reduced activity of complex I, increased activity of complex IV, general upregulation of respiratory supercomplexes, and increased oxidative stress. These changes could have several implications for mitochondrial function and cellular metabolism, including an increased risk of ROS-related damage.

The MQC is a fundamental aspect of cellular homeostasis, ensuring proper mitochondrial function. This intricate network encompasses mechanisms that safeguard mitochondrial health, including mtDNA maintenance, biogenesis, fission, fusion, and mitophagy. Our study provides insights into the impact of CB1 deficiency on MQC within the liver.

The increase in tissue protein levels of POL-γ, OGG1, and APE1 in the livers of CB1^−/− mice indicates an enhanced capacity for mitochondrial DNA repair through the BER pathway, suggesting a role for CB1 in maintaining mtDNA integrity and stability. Additionally, the upregulation of mitochondrial biogenesis, fission, and fusion processes in CB1^−/− mice, as evidenced by increased protein levels of PGC1α, TFAM, NRF1, NRF2, OPA1, DRP1, MFN1 and MFN2, suggests a dynamic response to the absence of CB1 (Figure 4).

This adaptive mitochondrial plasticity may contribute to enhanced mitochondrial function and metabolic efficiency, potentially compensating for the loss of CB1 signaling. The trigger of compensative mechanisms in CB1^−/− hepatocytes can explain also the mainly normal appearance of hepatocyte mitochondrial ultrastructure. In CB1^−/− mice, we observe the initiation of mitophagy pathways via LC3B as an adaptive response to counteract ROS-induced damage within the liver. However, a noteworthy finding in our study is the observation of reduced PINK1 and PARKIN levels in CB1^−/− mice, key regulators of mitophagy. This intriguing observation suggests that CB1 deficiency triggers the initiation of mitophagy but may concurrently hinder its progression, potentially affecting MQC. Ultrastructural examination of liver tissue corroborated these findings by revealing the presence of phagophores around mitochondria, confirming the initiation of the mitophagic process (Figure 5). In addition, CB1 deficiency does not affect Pink1/PARKIN-independent pathway (Figure 5). Nevertheless, the inhibition of mitophagy does not lead to changes in mitochondrial morphology (Figure 5D), unlike what was noted by Kataoka et al. in hippocampal neurons of adult CB1^−/− mice. Their study reveals that diminished mitophagy correlates with the presence of elongated and thinner mitochondria (Kataoka et al., 2020). Recent evidence suggests that oxidative stress, characterized by an imbalance between the production of ROS and the antioxidant defense system, can lead to the accumulation of misfolded proteins in the mitochondria (Muñoz-Carvajal and Sanhueza, 2020). This accumulation triggers the UPR^mt, the adaptive signaling pathway that aims to restore mitochondrial proteostasis by enhancing protein folding and degrading misfolded proteins (Fiorese and Haynes, 2017). We found that in the liver of CB1^−/− mice there is an increase of UPR^mt markers.

These findings underscore the vital role of UPR^mt activation in preserving mitochondrial homeostasis, complementing its established functions in mitochondrial biogenesis, dynamics, mitophagy, and mtDNA repair. Notably, CB1^−/− mice exhibit significantly elevated levels of key proteases such as LONP1 and CLPP involved in the eradication of irreversibly ineffective, damaged and misfolded proteins, suggesting enhanced protein degradation activities essential for maintaining mitochondrial integrity and function (Figure 6). Moreover, CB1^−/− mice, compared to the control group, demonstrate increased levels of other transcripts associated with UPR^mt including the chaperone TRAP1, and the transcriptional factors ATF4, ATF5 and CHOP which upregulate the gene expression of protease and chaperones (Figure 6).

Furthermore, the interconnection between UPR^mt and UPR^ER, the two signaling pathways involved in maintaining cellular homeostasis under stress, was examined. The phosphorylation levels of P-PERK and P-eIF2α, key components of the UPR^ER signaling pathway, were significantly increased in CB1^−/− mice compared to the control group (Figure 6). This suggests that UPR^ER is activated in CB1^−/− mice, indicating a coordinated response to cellular stress involving both UPR^mt and UPR^ER signaling pathways.

In addition, PINK1/PARKIN mitophagy has been observed to be suppressed in response to oxidative stresses, whereas the UPR^mt is activated, suggesting the interplay between these pathways. Indeed, in recent years, multiple signaling pathways have been discovered to regulate mitophagy-mediated mitochondrial degradation. Interestingly, these pathways also activate the UPR^mt, indicating a potential coordinated control of these two systems responsible for maintaining mitochondrial quality. Evidence shows that under stress conditions, the UPR^mt and mitophagy are activated simultaneously and can compensate for each other when one system is insufficient or fails (Wang et al., 2023).

Our findings support a model wherein PINK1/PARKIN mitophagy serves as a final resort quality control mechanism, eliminating mitochondria irreparably damaged beyond the capacity of UPR^mt. The activation of PINK1/PARKIN mitophagy in situations where UPR^mt fails to restore proteostasis is crucial for the preservation of OXPHOS metabolism and likely plays a role in avoiding the protrusion and release of inflammatory mtDNA from severely damaged mitochondria (Uoselis et al., 2023).

In addition, it is important to mention that recent studies have shown that mildly damaged mitochondrial components are processed and disposed in Extracellular Vesicles (EVs) of mitochondrial origin (MDVs) using this alternative degradative route (Sugiura et al., 2014; Terešak et al., 2022). Ramirez et al., in 2022, showed that CBD treatment induced elevated production of MDV suggesting a PINK1-Parkin pathway dependent (Ramirez et al., 2022).

In conclusion, our study highlights the significant impact of CB1 signaling on mitochondrial function particularly in the liver and suggests that a complex adaptive response is triggered by CB1 deficiency to optimize liver function. The absence of CB1 leads to oxidative stress conditions and disrupts mitochondrial proteostasis, triggering the UPR^mt. The activation of UPR^mt in CB1^−/− mice underscores its essential role in maintaining mitochondrial homeostasis. We observed elevated levels of key proteases involved in MQC and increased expression of molecular chaperones and transcription factors related to cellular stress responses. Furthermore, our findings reveal a coordinated response to cellular stress, involving both UPR^mt and UPR^ER. In addition, the loss of CB1-mediated signaling could be mitigated by compensatory mechanisms, which allow animals without CB1 to survive. Neurotransmission and various metabolic processes primarily regulated by the CB1, in its absence can be mitigated by other mechanisms, for example, the involvement of CB2 (Bie et al., 2018; Louvet et al., 2011; Munro et al., 1993), and the activation of the G-protein coupled receptor 55 (GPR55) and Vanilloid type 1 receptor (TRPV1) (Aizpurua-Olaizola et al., 2017; Balenga et al., 2011; Chiurchiù et al., 2015; Ryberg et al., 2007; Staton et al., 2008; Yang et al., 2016).

Overall, our study emphasizes the critical importance of CB1 in preserving mitochondrial function and cellular health, shedding light on potential therapeutic targets for conditions involving mitochondrial dysfunction and oxidative stress. Further research is warranted to fully elucidate the intricate mechanisms underlying CB1-mediated mitochondrial regulation and its implications for overall health and disease. However, it is essential to acknowledge that some disparities between our study and the existing literature may arise from distinct experimental conditions. While our investigation focuses on the absence of CB1 in mice without pre-existing liver diseases, other studies assess the effects of CB1 antagonists in animal models with established liver pathologies (Liu et al., 2019; Mboumba Bouassa et al., 2022; Tam et al., 2011). In addition, discrepancies may reflect differences between the effects of short-term modulation of CB1 with the use of pharmacologic agents and the effect of a congenital deficiency of CB1 signaling with activation of compensatory mechanisms in CB1^−/− mice.